U.S. patent application number 15/243675 was filed with the patent office on 2017-03-02 for reduced risk tobacco products and methods of making same.
The applicant listed for this patent is VECTOR TOBACCO INC.. Invention is credited to Anthony P. Albino, Wendy Jin, Ellen Jorgensen.
Application Number | 20170055566 15/243675 |
Document ID | / |
Family ID | 37431845 |
Filed Date | 2017-03-02 |
United States Patent
Application |
20170055566 |
Kind Code |
A1 |
Albino; Anthony P. ; et
al. |
March 2, 2017 |
REDUCED RISK TOBACCO PRODUCTS AND METHODS OF MAKING SAME
Abstract
Embodiments provided herein concern tobacco and tobacco products
having a reduced amount of a harmful compound. More specifically,
several embodiments concern approaches to modify the expression of
a gene that is involved in the production of a harmful compound in
tobacco, tobacco products made using these approaches and methods
of determining whether the removal of said compounds using said
approaches yields a tobacco and/or a tobacco product that has a
reduced potential to contribute to a tobacco-related disease.
Inventors: |
Albino; Anthony P.; (New
York, NY) ; Jin; Wendy; (Chapel Hill, NC) ;
Jorgensen; Ellen; (South Salem, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VECTOR TOBACCO INC. |
Morrisville |
NC |
US |
|
|
Family ID: |
37431845 |
Appl. No.: |
15/243675 |
Filed: |
August 22, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14102340 |
Dec 10, 2013 |
9439452 |
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15243675 |
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11913870 |
Mar 25, 2011 |
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PCT/US06/18065 |
May 10, 2006 |
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14102340 |
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60680283 |
May 11, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A24B 15/245 20130101;
C12N 9/00 20130101; C12N 15/8243 20130101; A24B 15/243 20130101;
A24B 15/20 20130101; A24B 13/00 20130101; C12N 15/8218
20130101 |
International
Class: |
A24B 13/00 20060101
A24B013/00; C12N 15/82 20060101 C12N015/82; A24B 15/20 20060101
A24B015/20 |
Claims
1. (canceled)
2. A method of producing a transgenic tobacco plant comprising
inhibiting expression of an endogenous gene encoding A622 in a
tobacco plant, wherein said method further comprises introducing
into said tobacco plant a nucleic acid molecule comprising a
nucleic acid sequence selected from the group consisting of: a) the
nucleic acid sequence of SEQ ID NO: 5; b) a nucleic acid sequence
comprising nucleotides 1-354 of the full length A622 cDNA; c) a
nucleic acid sequence comprising nucleotides 1-814 of the full
length A622 cDNA; d) a nucleic acid sequence comprising nucleotides
1-981 of the full length A622 cDNA; e) a nucleic acid sequence
comprising nucleotides 1-1160 of the full length A622 cDNA; f) a
nucleic acid sequence comprising nucleotides 1-1179 of the full
length A622 cDNA; g) a nucleic acid sequence comprising nucleotides
354-814 of the full length A622 cDNA; h) a nucleic acid sequence
comprising nucleotides 354-981 of the full length A622 cDNA; i) a
nucleic acid sequence comprising nucleotides 354-1160 of the full
length A622 cDNA; j) a nucleic acid sequence comprising nucleotides
354-1179 of the full length A622 cDNA; k) a nucleic acid sequence
comprising nucleotides 814-981 of the full length A622 cDNA; l) a
nucleic acid sequence comprising nucleotides 814-1160 of the full
length A622 cDNA; m) a nucleic acid sequence comprising nucleotides
814-1179 of the full length A622 cDNA; n) a nucleic acid sequence
comprising nucleotides 981-1160 of the full length A622 cDNA; and
o) a nucleic acid sequence comprising nucleotides 981-1179 of the
full length A622 cDNA.
3. The method of claim 2, wherein said nucleic acid molecule is
operably linked to a heterologous nucleic acid.
4. The method of claim 3, wherein said heterologous nucleic acid is
a promoter.
5. The method of claim 2, wherein said nucleic acid molecule
confers a reduced expression of said endogenous A622 gene by
interfering RNA (RNAi), sense-suppression, antisense suppression,
targeted gene disruption, or enzymatic RNA.
6. The method of claim 2, further comprising genetically
engineering suppression of at least one additional gene encoding an
enzyme of the nicotine biosynthetic pathway, wherein said at least
one additional gene encodes an enzyme selected from the group
consisting of arginine decarboxylase (ADC), methylputrescine
oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC),
phosphoribosylanthranilate isomerase (PRAT), putrescine
N-methyltransferase (PMT), quinolate phosphoribosyl transferase
(QPT), and S-adenosyl-methionine synthetase (SAMS), or any
combination thereof.
7. A cured transgenic tobacco comprising a first heterologous
nucleic acid molecule that comprises a nucleic acid sequence
selected from the group consisting of: a) the nucleic acid sequence
of SEQ ID NO: 5; b) a nucleic acid sequence comprising nucleotides
1-354 of the full length A622 cDNA; c) a nucleic acid sequence
comprising nucleotides 1-814 of the full length A622 cDNA; d) a
nucleic acid sequence comprising nucleotides 1-981 of the full
length A622 cDNA; e) a nucleic acid sequence comprising nucleotides
1-1160 of the full length A622 cDNA; f) a nucleic acid sequence
comprising nucleotides 1-1179 of the full length A622 cDNA; g) a
nucleic acid sequence comprising nucleotides 354-814 of the full
length A622 cDNA; h) a nucleic acid sequence comprising nucleotides
354-981 of the full length A622 cDNA; i) a nucleic acid sequence
comprising nucleotides 354-1160 of the full length A622 cDNA; j) a
nucleic acid sequence comprising nucleotides 354-1179 of the full
length A622 cDNA; k) a nucleic acid sequence comprising nucleotides
814-981 of the full length A622 cDNA; l) a nucleic acid sequence
comprising nucleotides 814-1160 of the full length A622 cDNA; m) a
nucleic acid sequence comprising nucleotides 814-1179 of the full
length A622 cDNA; n) a nucleic acid sequence comprising nucleotides
981-1160 of the full length A622 cDNA; and o) a nucleic acid
sequence comprising nucleotides 981-1179 of the full length A622
cDNA.
8. The cured transgenic tobacco of claim 7, further comprising a
second heterologous nucleic acid molecule that confers suppression
of at least one additional gene, wherein said at least one
additional gene encodes an enzyme selected from the group
consisting of arginine decarboxylase (ADC), methylputrescine
oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC),
phosphoribosylanthranilate isomerase (PRAT), putrescine
N-methyltransferase (PMT), quinolate phosphoribosyl transferase
(QPT), and S-adenosyl-methionine synthetase (SAMS), or any
combination thereof.
9. The cured transgenic tobacco of claim 7, wherein said cured
transgenic tobacco comprises an amount of nicotine, nornicotine, or
a tobacco specific nitrosamine (TSNA) that is less than the amount
of nicotine, nornicotine, or said TSNA present in a cured
non-transgenic tobacco of the same variety.
10. The cured transgenic tobacco of claim 7, wherein said cured
transgenic tobacco comprises an amount of nornicotine that is less
than the amount of nornicotine present in a cured non-transgenic
tobacco of the same variety and, further wherein said cured
transgenic tobacco comprises an amount of nicotine that is
commensurate to the amount of nicotine present in a cured
non-transgenic tobacco of the same variety.
11. The cured transgenic tobacco of claim 10, wherein said cured
transgenic tobacco comprises an amount of nornicotine that is less
than or equal to 0.2 mg/g of total biomass.
12. The cured transgenic tobacco of claim 11, wherein said cured
transgenic tobacco comprises an amount of nicotine from 0.9-2.0
mg/g of total biomass.
13. The cured transgenic tobacco of claim 11, wherein said cured
transgenic tobacco comprises an amount of nornicotine that is from
0.5 ug/g-500 ug/g of total biomass.
14. The cured transgenic tobacco of claim 11, wherein said cured
transgenic tobacco comprises an amount of nicotine that is 2.17 to
3.99 mg/g of total biomass and an amount of nornicotine that is
less than or equal to 0.00 to 0.06 mg/g of total biomass.
15. The cured transgenic tobacco of claim 11, wherein said cured
transgenic tobacco comprises a collective content of TSNAs 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 of total
biomass.
16. The cured transgenic tobacco of claim 7, wherein said cured
transgenic tobacco comprises an amount of nicotine and a collective
content of TSNAs that is less than the amount of nicotine and the
collective content of TSNAs present in a cured non-transgenic
tobacco of the same variety.
17. The cured transgenic tobacco of claim 7, wherein said
transgenic tobacco comprises an amount of nicotine that is less
than or equal to 1.0 mg/g, 0.6 mg/g, 0.3 mg/g, or 0.05 mg/g of
total biomass and a collective content of TSNAs 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 of total biomass.
18. A seed, reproductive tissues, vegetative tissues, biomass,
leaves, plant parts, progeny derived from the transgenic tobacco of
claim 7.
19. A tobacco product derived from the transgenic tobacco of claim
7.
20. A tobacco product derived from the transgenic tobacco of claim
8.
21. The tobacco product of claim 19, further defined as a
cigarette.
22. The tobacco product of claim 20, further defined as a
cigarette.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 14/102,340, filed Dec. 10, 2013, which is a
continuation of U.S. application Ser. No. 11/913,870, filed Mar.
25, 2011, which is 371 entry into the U.S. of Paris Convention
Application No. PCT/US06/18065, filed May 10, 2006, which claims
the benefit of priority to U.S. Provisional Application Ser. No.
60/680,283, filed May 11, 2005. Each of the above-mentioned
priority documents is incorporated by reference in its entirety
into the present application.
FIELD OF THE INVENTION
[0002] The invention relates to reduced risk tobacco and tobacco
products and methods for detecting, identifying and evaluating such
tobacco and tobacco products to determine the potential that these
compositions have to contribute to a tobacco-related disease.
BACKGROUND
[0003] The leading preventable cause of death and disability in the
United States is the chronic use of tobacco products, in
particular, cigarettes. In addition to lung cancers, tobacco use
plays important direct and indirect roles in the etiology of a wide
range of other cancers, including those of the upper aerodigestive
tract (i.e., oral cavity, pharynx, larynx, and esophagus), bladder,
stomach, kidney, pancreas, uterine cervix, and blood (myeloid
leukemia). Exposure to tobacco carcinogens and toxins is also a
major cause of other diseases of the pulmonary system (e.g.,
bronchitis, emphysema, and chronic obstructive pulmonary disease),
the cardiovascular system (e.g., stroke, atherosclerosis, and
myocardial infarction), and the female reproductive system (e.g.,
increased risk of miscarriage, premature delivery, low birth
weight, and stillbirth). While numerous studies have elucidated
some of the biological effects of cigarette smoke that result in
its ability to induce this range of pathologies in smokers, little
is known about the nature and temporal association of molecular
events that drive specific stages in the multi-step processes that
result in clinically evident disease. This is due to the fact that
cigarette smoke is a complex chemical mixture of gases and
suspended particulate material that consists of a wide variety of
condensed organic compounds (i.e., `tar`) that collectively contain
a large number of toxins, carcinogens, co-carcinogens, mutagens,
and reactive organic and inorganic molecules. Thus, there is a
pressing need to decrease the health risk caused by tobacco
products.
SUMMARY
[0004] Embodiments described herein generally relate to tobacco
and/or tobacco products having a reduced amount of a harmful
compound, and methods of developing, screening and using such
tobacco and tobacco products. For example, several approaches are
provided to reduce the amount of one or more harmful compounds in
tobacco by, for example, modifying the expression of a gene that is
involved in the production of a harmful compound in tobacco. Also
provided are methods of determining whether the removal of a
harmful compound yields a tobacco and/or a tobacco product that has
a reduced potential to contribute to a tobacco-related disease.
Also provided are reduced-risk tobacco and tobacco products made in
accordance with the methods provided herein. Also provided are
methods of using the reduced-risk tobacco and tobacco products made
in accordance with the methods provided herein.
[0005] As described in more detail below, provided herein are
nucleic acid molecules and nucleic acid constructs that contain
sequences that can be used to inhibit expression of a gene involved
in the biosynthesis of a compound associated with a tobacco-related
disease. Also provided herein are modified tobaccos and modified
tobacco products that have been modified by composition and/or
configuration in order to deliver to the user a reduced amount of a
compound associated with a tobacco-related disease. Exemplary
modified tobaccos are tobaccos that have been genetically modified
to contain a reduced amount of a compound associated with a
tobacco-related disease. Exemplary genetically modified tobaccos
are those containing the nucleic acid molecules or constructs
provided herein. Exemplary modified tobacco products are those
containing modified tobacco or, those containing a modified filter,
where the modification results in delivery to the user of a reduced
amount of a compound associated with a tobacco-related disease.
[0006] Also provided herein are methods of analyzing tobacco
products such as the modified tobacco and modified tobacco products
described herein, so as to determine whether the tobacco product is
a reduced risk product (e.g., a product that has a reduced
propensity to modulate cellular homeostasis, or a reduced level of
induction of a cellular marker for a tobacco-related disease). Some
of these methods can be practiced, for example, by identifying a
compound that is related to a tobacco-related disease (e.g.,
nicotine or a sterol), removing the compound or a precursor for the
compound by modification to the tobacco or tobacco product, and
analyzing the ability of the modified tobacco or modified tobacco
product to contribute to a tobacco related disease by monitoring
the impact of the modified tobacco or modified tobacco product on a
marker for cellular homeostasis. In one example, a cellular marker
for a tobacco related disease is monitored. In another example, the
transcriptome and/or proteome of the cell is monitored. These
methods can be used for both in vitro and in vivo testing. That is,
the same cellular markers that have been identified in the in vitro
studies can be analyzed in smokers that consume reduced risk
cigarettes developed according to the methods above and this data
can be compared to the impact on the same cellular markers in
smokers that consume conventional cigarettes. By these approaches,
a cigarette that minimizes the disruptions of the cellular
environment of a smoker can be obtained.
[0007] Further provided herein are kits that contain the modified
tobacco or modified tobacco products provided herein, and smoking
cessation programs, which utilize the modified tobacco or modified
tobacco products provided herein.
[0008] Provided herein are methods of making a tobacco product with
a reduced potential to contribute to a tobacco related disease by
providing a genetically modified tobacco configured to deliver a
reduced amount of a compound that contributes to a tobacco related
disease, as compared to a reference tobacco or a conventional
tobacco, contacting a mammalian cell with smoke, or a smoke
condensate obtained from said genetically modified tobacco,
identifying a modulation of homeostasis of said cell, as compared
to a control cell, which has been contacted with smoke, or a smoke
condensate obtained from said reference tobacco or said
conventional tobacco, wherein a decreased modulation of homeostasis
in said cell compared to modulation of homeostasis in said control
cell indicates a reduction in the potential to contribute to a
tobacco related disease, and incorporating said identified
genetically modified tobacco into a tobacco product. In some such
methods, modulation of homeostasis in the cell is identified by
determining the presence, absence or level of a molecular marker in
the cell. In some such methods, the mammalian cell is a lung cell
or a cell of the oral cavity. In some such methods, the genetically
modified tobacco is identified as producing a reduced amount of a
compound that contributes to a tobacco related disease, as compared
to a conventional tobacco product of the same class or a reference
tobacco product of the same class. In some such methods, the
genetically modified tobacco is incorporated into a tobacco product
that contains a filter, which retains an increased amount of a
compound that contributes to a tobacco related disease, as compared
to a reference filter or a conventional filter. In some such
methods, the genetically modified tobacco comprises a heterologous
nucleic acid that inhibits expression of an enzyme in the nicotine
biosynthetic pathway. In some such methods, the heterologous
nucleic acid inhibits expression of at least two enzymes in the
nicotine biosynthetic pathway. In some such methods, the
genetically modified tobacco comprises a heterologous nucleic acid
that inhibits expression of an enzyme in the sterol biosynthetic
pathway. In some such methods, the heterologous nucleic acid
inhibits expression of at least two enzymes in the sterol
biosynthetic pathway. In some such methods, the genetically
modified tobacco comprises a heterologous nucleic acid that
inhibits expression of an enzyme in the nicotine biosynthetic
pathway and an enzyme in the sterol biosynthetic pathway. In some
such methods, the genetically modified tobacco has a reduced amount
of nornicotine and a conventional amount of nicotine. In some such
methods, genetically modified tobacco comprises a nucleic acid
construct selected from the group consisting of SEQ. ID. NOs.: 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. Also
provided herein are tobacco products made by the method provided
herein.
[0009] Also provided herein are tobacco products comprising a
genetically modified tobacco that comprises a reduced amount of
nicotine as compared to a conventional tobacco product of the same
class or a reference tobacco product of the same class and a
heterologous nucleic acid that inhibits expression of at least two
enzymes involved in nicotine biosynthesis. Also provided herein are
tobacco products comprising a genetically modified tobacco that
comprises a reduced amount of a sterol as compared to a
conventional tobacco product of the same class or a reference
tobacco product of the same class and a heterologous nucleic acid
that inhibits expression of an enzyme involved in sterol
biosynthesis. In some such tobacco products, the genetically
modified tobacco comprises a nucleic acid construct as described
herein. In some such tobacco products, the genetically modified
tobacco comprises a nucleic acid construct selected from the group
consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49 and 50.
[0010] In the methods and tobacco products provided herein, the
genetically modified tobacco comprises a reduced activity of a gene
selected from the group consisting of arginine decarboxylase (ADC),
methylputrescine oxidase (MPO), NADH dehydrogenase, omithine
decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),
putrescine N-methyltransferase (PMT), quinolate phosphoribosyl
transferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622
or comprises an inhibition of a gene that regulates the production
of sterol biosynthesis include HMG-CoA reductase, 14alpha
demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase,
A8-A7-isomerase, and C4-demethylase. In the methods and tobacco
products provided herein, the genetically modified tobacco has
reduced production of a compound that contributes to a tobacco
related disease which is stable over at least 2, 3, 4, 5, 6, 8, 10,
12, 15, 20, 25, 30, 40 or 50 generations. In the methods and
tobacco products provided herein, the genetically modified tobacco
has agronomic characteristics suitable for commercial production.
In the methods and tobacco products provided herein, the agronomic
characteristics are phenotypically different from conventional
tobacco, and said agronomic characteristics can be compensated for
by conventional agronomic methods. In the methods and tobacco
products provided herein, the conventional agronomic methods are
selected from the group consisting of irrigation, administration of
fertilizer, and administration of nutrients.
[0011] Also provided herein are genetically modified tobaccos that
produce a reduced amount of a compound that contributes to a
tobacco related disease, as compared to a conventional tobacco
product of the same class or a reference tobacco product of the
same class, comprising a heterologous nucleic acid that inhibits
expression of an enzyme in the biosynthetic pathway of a compound
that contributes to a tobacco related disease. Also provided herein
are reduced risk tobacco products comprising a genetically modified
tobacco that produces a reduced amount of a compound that
contributes to a tobacco related disease, as compared to a
conventional tobacco product of the same class or a reference
tobacco product of the same class. In some such tobaccos or tobacco
products, the modified tobacco comprises a nucleic acid construct
as described herein. In some such tobaccos or tobacco products, the
modified tobacco comprises a heterologous nucleic acid that
inhibits expression of at least two enzymes in the nicotine
biosynthetic pathway. In some such tobaccos or tobacco products,
the modified tobacco comprises a heterologous nucleic acid that
inhibits expression of at least two enzymes in the sterol
biosynthetic pathway.
[0012] Also provided herein are methods of making a reduced risk
tobacco product by providing a modified tobacco or modified tobacco
product configured to deliver to a user a reduced amount of a
compound that contributes to a tobacco related disease, as compared
to a reference tobacco or tobacco product or a conventional tobacco
or tobacco product, contacting smoke or smoke condensate obtained
from said modified tobacco or modified tobacco product with a cell,
identifying a modulation of homeostasis of said cell, as compared
to a control cell, which has been contacted with smoke or a smoke
condensate obtained from said reference tobacco or tobacco product
or said conventional tobacco or tobacco product, wherein a
decreased modulation of homeostasis in said cell compared to
modulation of homeostasis in said control cell indicates a
reduction in the potential to contribute to a tobacco related
disease, and incorporating said modified tobacco or modified
tobacco product into said reduced risk tobacco product. In some
such methods, modulation of homeostasis in the cell is identified
by determining the presence, absence or level of a molecular marker
in the cell. In some such methods, the modified tobacco is
genetically modified tobacco. In some such methods, the genetically
modified tobacco is modified according to the methods provided
herein.
[0013] Also provided are reduced risk tobaccos as substantially
described herein. Also provided are reduced risk tobacco products
as substantially described herein. Also provided are uses of the
tobaccos or tobacco products provided herein.
[0014] Also provided are isolated nulcleic acids substantially as
described herein. Also provided are isolated inhibition cassettes
substantially as described herein.
[0015] Also provided is a genetically modified tobacco having a
reduced amount of nicotine as compared to conventional tobacco,
further comprising a heterologous nucleic acid that encodes a gene
that produces a composition selected from the group consisting of a
medicinal compound, industrial oil, or dietary supplement, wherein
said composition is substantially not present in conventional or
wild-type tobacco. In some such tobaccos, the medicinal compound is
an antibody or fragment thereof or an immunogenic preparation. In
some such tobaccos, the medicinal compound is a vaccine
preparation. In some such tobaccos, the medicinal compound is a
veterinary product.
[0016] Also provided are genetically modified tobaccos that produce
a reduced amount of a compound that contributes to a tobacco
related disease, as compared to a conventional tobacco product of
the same class or a reference tobacco product of the same class,
comprising a heterologous nucleic acid that inhibits expression of
an enzyme in the biosynthetic pathway of a compound that
contributes to a tobacco related disease. Also provided are reduced
risk tobacco products comprising a genetically modified tobacco
that produces a reduced amount of a compound that contributes to a
tobacco related disease, as compared to a conventional tobacco
product of the same class or a reference tobacco product of the
same class. In some such tobaccos or tobacco products, the compound
is nicotine. In some such tobaccos or tobacco products, the
compound is a sterol. In some such tobaccos or tobacco products,
the compound is a TSNA. In some such tobaccos or tobacco products,
the compound is a PAH. In some such tobaccos or tobacco products,
the compound is nornicotine. In some such tobaccos or tobacco
products, the genetically modified tobacco has a reduced amount of
nornicotine and a conventional amount of nicotine. In some such
tobaccos or tobacco products, the genetically modified tobacco
comprises a nucleic acid construct as described herein. In some
such tobaccos or tobacco products, the genetically modified tobacco
comprises a nucleic acid construct selected from the group
consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, 48, 49, and 50. In some such tobaccos or tobacco products,
expression of two or more genes in the biosynthetic pathway of said
compound is inhibited. In some such tobaccos or tobacco products,
the genetically modified tobacco comprises two or more nucleic acid
constructs as described herein. In some such tobaccos or tobacco
products, the genetically modified tobacco comprises two or more
nucleic acid constructs selected from the group consisting of SEQ.
ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50.
Some such tobaccos or tobacco products comprise reduced activity of
a gene selected from the group consisting of arginine decarboxylase
(ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, omithine
decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI),
putrescine N-methyltransferase (PMT), quinolate phosphoribosyl
transferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622
or comprises an inhibition of a gene that regulates the production
of sterol biosynthesis include HMG-CoA reductase, 14alpha
demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase,
A8-A7-isomerase, and C4-demethylase. Some such tobaccos or tobacco
products comprise a genetically modified tobacco for which reduced
production of a compound that contributes to a tobacco related
disease is stable over at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20,
25, 30, 40 or 50 generations. Some such tobaccos or tobacco
products comprise a genetically modified tobacco having agronomic
characteristics suitable for commercial production.
[0017] Also provided herein are methods of making a reduced risk
tobacco product by providing a modified tobacco or modified tobacco
product configured to deliver to a user a reduced amount of a
compound that contributes to a tobacco related disease, as compared
to a reference tobacco or tobacco product or a conventional tobacco
or tobacco product, contacting smoke or smoke condensate obtained
from said modified tobacco or modified tobacco product with a cell
identifying a modulation of homeostasis of said cell, as compared
to a control cell, which has been contacted with smoke or a smoke
condensate obtained from said reference tobacco or tobacco product
or said conventional tobacco or tobacco product, wherein a
decreased modulation of homeostasis in said cell compared to
modulation of homeostasis in said control cell indicates a
reduction in the potential to contribute to a tobacco related
disease, and incorporating said modified tobacco or modified
tobacco product into said reduced risk tobacco product. In some
such methods, modulation of homeostasis in the cell is identified
by determining the presence, absence or level of a molecular marker
in the cell. In some such methods, the modified tobacco is
genetically modified tobacco. In some such methods, the genetically
modified tobacco is modified according to any of methods provided
herein. In some such methods, the genetically modified tobacco is
identified as producing a reduced amount of a compound that
contributes to a tobacco related disease, as compared to a
conventional tobacco product of the same class or a reference
tobacco product of the same class. In some such methods, the
modified tobacco product contains a filter that retains an
increased amount of a compound that contributes to a tobacco
related disease, as compared to a reference filter or a
conventional filter. Also provided herein are reduced risk tobacco
products made by any of the methods provided herein. Also provided
herein are methods of using a reduced risk tobacco product of any
of the methods provided herein to reduce the potential of an
individual that smokes to acquire a tobacco related disease
comprising identifying an individual in need of a reduced risk
tobacco product and providing the individual the tobacco product of
the methods provided herein.
[0018] Also provided herein are plant cells resistant to
norflurazone comprising providing said cell the nucleic acid of SEQ
ID No 10, 11, or 12; and also provided herein are method of making
the same.
[0019] Also provided herein are crops of plants comprising the
nucleic acid of SEQ ID No 10, 11, or 12. Also provided herein are
methods of cultivation of a crop of plants comprising obtaining
plants with the nucleic acid of SEQ ID No 10, 11, or 12,
cultivating said plants, and contacting said plants with
norflurazone.
[0020] Also provided herein are methods of selecting positively
transformed plant cells comprising providing the nucleic acid of
SEQ ID No 10, 11, or 12 to said plant cells and contacting said
plant cells with norflurazone, whereby the cells that survive
contact with norflurazone are positively transformed plant
cells.
[0021] Also provided herein are isolated nulcleic acids
substantially as described herein. Also provided herein are
isolated inhibition cassettes substantially as described herein.
Also provided herein are isolated selection cassettes substantially
described herein, wherein said selction cassette comprises the
sequence of of SEQ ID No 10, 11, or 12. Also provided herein are
reduced risk tobaccos substantially described herein. Also provided
herein are reduced risk tobacco products substantially described
herein.
[0022] Also provided herein are reduced risk tobacco products
comprising a transgenic tobacco that comprises a reduced expression
of a plurality of genes that regulate the production of at least
two different compounds in said tobacco that contribute to a
tobacco related disease. In some such tobacco products, the two
different compounds in said tobacco are nicotine and a sterol.
[0023] Also provided herein are kits comprising two or more
different tobaccos or tobacco products in accordance with any of
the methods provided herein. In some such kits, the different
tobaccos or tobacco products are differently labeled.
[0024] Also provided herein are uses of a tobacco or tobacco
product of any of the methods, tobaccos, tobacco products or kits
provided herein. Some such uses are tobacco-use cessation
methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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.
[0026] 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).
[0027] 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).
[0028] FIG. 4. An illustration of a A622 inhibition construct
comprising a 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).
[0029] 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).
[0030] FIG. 6. An illustration of a SMT2/A622 double inhibition
construct comprising a A622 inhibition cassette including a 628 bp
fragment of the A622 gene, an SMT2 inhibition cassette including a
779 bp fragment of the SMT2 gene and a norflurazone resistance
selection cassette including a mutant phytoene desaturase gene
(PDSM-1).
[0031] FIG. 7. 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).
[0032] FIG. 8. 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).
[0033] FIG. 9. An illustration of a PMTase inhibition construct
comprising a PMTase inhibition cassette including a 202 bp fragment
of the PMTase gene and a norflurazone resistance selection cassette
including a mutant phytoene desaturase gene (PDSM-1).
[0034] FIG. 10. An illustration of a PMTase inhibition construct
comprising a PMTase inhibition cassette including a 344 bp fragment
of the PMTase gene and a norflurazone resistance selection cassette
including a mutant phytoene desaturase gene (PDSM-1).
[0035] FIG. 11. An illustration of a QPTase inhibition construct
comprising a QPTase inhibition cassette including a 360 bp fragment
of the QPTase gene and a kanamycin resistance selection cassette
including a neomycin phosphotransferase gene (NPTII).
[0036] FIG. 12A-B. Fluorescence photomicrographs of NHBE cells
exposed to 25 .mu.g/ml of tobacco smoke condensate for 24 h. The
cells stained with DAPI and immuno-stained with .gamma.H2AX Ab were
examined under UV light- (A) or blue light- (B) fluorescence
excitation (Nikon Microphot FXA, 60.times. Objective.).
[0037] FIG. 13A-C. Bivariate (cellular DNA content vs cell
immunofluorescence) distributions (scatterplots) of A549 cells,
mock-treated (B) or exposed for 30 min to tobacco smoke (A, C),
immuno-stained either with .gamma.H2AX Ab (B,C) or with an isotype
control IgG (A). The dashed-line represents the maximal
fluorescence level (for 99% cells) of the IgG control.
[0038] FIG. 14. Plots showing the percent increase (.DELTA.) in
mean .gamma.H2AX immunofluorescence of A549 cells (per unit of DNA)
exposed to smoke for different time intervals, calculated for cells
in particular phases of the cell cycle, as described in Example 1.
The value for mock-exposed cells was subtracted from those exposed
to smoke.
[0039] FIG. 15. Plots showing percent increase (.DELTA.) in mean
.DELTA.H2AX immunofluorescence of NHBE cells treated with 10, 25 or
50 .mu.g/ml concentrations of smoke condensate for different
periods of time. As in FIG. 14, the .DELTA.H2AX value for the
mock-exposed cells was subtracted from the values of the cells
exposed to different concentrations of condensate.
[0040] FIG. 16. Percent increase (A) in mean .DELTA.H2AX
immunofluorescence of NHBE cells treated with 10 .mu.g/ml of smoke
condensate for different intervals of time, in relation to cell
cycle phase. As in FIG. 14, the .DELTA.H2AX value for the
mock-exposed cells was subtracted from the values of the cells
exposed to condensate.
[0041] FIG. 17. Plots showing the percent increase (.DELTA.) in
mean .DELTA.H2AX immunofluorescence of A549 cells (per unit of DNA)
exposed to smoke of IM16 cigarettes for different time intervals,
calculated for cells in particular phases of the cell cycle, as
described in Example 2.
[0042] FIG. 18A-D. (A) Plots showing increase (A) in mean
.DELTA.H2AX immunofluorescence of A549 cells exposed to smoke of
IM16 cigarettes for 15 minutes, relative to mock exposed cells. (B)
Scatter plots showing the increase in .DELTA.H2AX following 60 min
of recovery of the A549 cells in particular phases of the cell
cycle for mock exposed (upper plot) and for IM16 smoke exposed
(lower plot) cells. (C) Plots showing increase (.DELTA.) in mean
.DELTA.H2AX immunofluorescence of NHBE cells exposed to smoke of
IM16 cigarettes for 20 minutes, relative to mock exposed cells. (D)
Scatter plot relative increase in .DELTA.H2AX following 60 min of
recovery of the NHBE cells in particular phases of the cell cycle
for mock exposed (upper plot) and for IM16 smoke exposed (lower
plot) cells.
[0043] FIG. 19. Plots showing the increase (A) in mean .DELTA.H2AX
immunofluorescence during different time points of the recovery of
A549 cells (per unit of DNA) after exposure to smoke of IM16, Quest
3.RTM., and Omni.RTM. cigarettes for 20 minutes, calculated for
cells in particular phases of the cell cycle.
[0044] FIG. 20. Bar plots showing the increase (A) in mean
.DELTA.H2AX immunofluorescence of A549 cells (top) and NHBE cells
(bottom) exposed to smoke of IM16 cigarettes for 20 minutes,
followed by a 1 hour recovery, for cells treated with
phosphate-buffered saline (PBS) or N-acetyl-L-cysteine (NAC) during
exposure (first value) and during recovery (second value).
[0045] FIG. 21. Bar plot showing the increase (A) in mean
.DELTA.H2AX immunofluorescence of A549 cells exposed to smoke from
IM16, Omni.RTM. and Quest 3.RTM. in the presence of PBS or NAC.
[0046] FIG. 22. Plot of the relative amount of mean .DELTA.H2AX
immunofluorescence of A549 cells exposed to smoke from IM16 as a
function of different concentrations of NAC, calculated for cells
in particular phases of the cell cycle. Horizontal dashed line
indicates 50% reduction in .DELTA.H2AX immunofluorescence. Vertical
dashed lines indicate the estimated NAC concentration for each cell
type at 50% reduction.
[0047] FIG. 23. Bar plots showing the increase (A) in mean
.DELTA.H2AX immunofluorescence of A549 cells (upper plot) and NHBE
cells (lower plot) exposed to the vapor phase of smoke from IM16,
Quest 1.RTM. and Quest 3.RTM., and smoke from IM16 in the presence
of PBS or NAC.
[0048] FIG. 24. Bar plots showing the increase (.DELTA.) in mean
.DELTA.H2AX immunofluorescence of G.sub.1, S and G.sub.2M phase
A549 cells (left plots) and G.sub.1, S and G.sub.2M phase NHBE
cells (right plots) exposed to the vapor phase of smoke from IM16,
Quest 1.RTM. and Quest 3.RTM. in the presence of PBS or NAC.
[0049] FIG. 25. Bar plot showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke from IM16
or Marlboro.RTM. for 10, 15 or 20 minutes.
[0050] FIG. 26. Bar plots showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke from IM16,
Quest 1.RTM. or Quest 3.RTM. for 10, 20 or 30 minutes (top two
plots), or 6 days after (bottom plot) exposure to smoke from IM16,
Marlboro.RTM. or Omni.RTM., for 10, 15 or 20 minutes.
[0051] FIG. 27. Bar plot showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke from IM16
for 20 minutes in the presence of PBS or 1 mM, 5 mM, 10 mM or 25 mM
NAC.
[0052] FIG. 28. Bar plot showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke from IM16,
Omni.RTM. or Quest 3.RTM. for 20 minutes in the presence of PBS or
25 mM NAC.
[0053] FIG. 29. Bar plot showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to vapor phase of
smoke from IM16, Quest 1.RTM. or Quest 3.RTM., or smoke of IM16 for
20 minutes in the presence of PBS or 25 mM NAC.
[0054] FIG. 30. Bar plot of results from Example 2 showing the
increase (A) in mean .DELTA.H2AX immunofluorescence of A549 cells
exposed to smoke from IM16, Omni.RTM. and Quest 3.RTM. in the
presence of PBS or NAC, calculated for cells in particular phases
of the cell cycle.
[0055] FIG. 31. Plot depicting .DELTA.H2AX associated fluorescence
(.gamma.H2AX; X-axis) and the number of cells having the
corresponding .DELTA.H2AX fluorescence level (Y axis), for buccal
cells of a subject subsequent to smoking a cigarette (smoker) or a
subject who did not smoke a cigarette (non-smoker).
[0056] FIG. 32. Bar plot of results from Example 2 showing the
increase (A) in mean .DELTA.H2AX immunofluorescence of A549 cells
exposed to smoke from IM16, Marlboro.RTM., Marlboro Light.RTM., and
Quest 3.RTM., calculated for cells in particular phases of the cell
cycle.
[0057] FIG. 33. Bar plot of results from FIG. 32 showing the
increase (A) in mean .DELTA.H2AX immunofluorescence of A549 cells
exposed to smoke from IM16, Marlboro.RTM., Marlboro Light.RTM., and
Quest 3.RTM., averaged for all cell cycles.
[0058] FIG. 34A is a Venn diagram comparing gene expression
modulations induced by cigarette smoke condensates of two different
tobacco products (e.g., cigarettes) CSC-A (3665) and CSC-B (3668).
The number of genes uniquely affected by exposure to each product
CSC-A (1226) and CSC-B (1229) is given in each sector. The
intersections between sectors reflect the number of genes that are
affected by both CSCs (2439).
[0059] FIG. 34B is a Venn diagram comparing gene expression
modulations induced by CSC-A (3665), CSC-B (3668), and S9 metabolic
fraction (1680). The number of unique genes affected by each
treatment is given, CSC-A (992), CSC-B (1039), and S9 (383) and the
intersections between sectors reflect the number of genes that are
affected by more than one treatment (e.g., a common set of 873
genes is affected by CSC-A, CSC-B and S9).
[0060] FIG. 35A-C illustrate gene expression profiles between 0 and
12 hours, which are expressed a percent of highest expression value
for each gene. F-cluster numbers are given at the top of each
cluster of profiles. The number of member genes in each cluster (n)
is shown for each cluster. FIG. 35A shows Clusters that contain 50
or more genes in CSC-A-treated cells. FIG. 35B shows Clusters
containing 50 or more genes in CSC-B-treated cells. FIG. 35C shows
Clusters containing 50 or more genes in S9-treated cells.
[0061] FIG. 36 illustrates a cluster analysis of genes that were
hypervariable (HV) in all three treatment groups (A: CSC-A, B:
CSC-B, and S9) in the form of a Dendrogram that depicts the
hierarchical relationship between the three treatments based on
their gene expression patterns at all time points from 0-12
hours.
[0062] FIG. 37 shows correlation mosaics of the genes listed in
Table 2. Correlation coefficients were generated for each of the 40
genes in Table 2, comparing the set to itself in each of the three
conditions. The same gene order runs across the x and y axes of the
mosaics. Correlation mosaics for HV genes highly correlated in
response to CSC-A and CSC-B, and not correlated with responses to
S9. Each pixel in the plot represents a correlation coefficient of
gene expression. Genes highly positively correlated are denoted in
gray and those highly negatively correlated are in black. The same
order of the genes along axis is used for all three mosaics. Genes
highly correlated in CSC-A and CSC-B, but not in S9-treated cells
are denoted as a gray cluster in the lower left hand corner of
CSC-A and the CSC-B mosaic. This cluster is disrupted in the S9
mosaic demonstrating the variance in gene regulation that occurred
in S9-treated cells.
[0063] FIG. 38 shows the functional associations of HV genes
specific for CSC-A and CSC-B treatment. The expression patterns of
this set of genes are highly correlated in CSC-treated NHBE cells
and not correlated with those seen in cells treated with S9 alone.
Cross-hatched ovals indicate genes from Table 2 (i.e., HV genes
specific for CSC-A and CSC-B treatment). Ovals with slanted lines
(indicating additional proteins not in Table 2) were added to
better define the regulatory networks of the genes identified in
this analysis. Ovals with dashed lines indicate classes of
functional peptides. Rectangles indicate cellular processes in
which these genes participate. Each line indicates a regulatory
relationship (binding, regulation, etc.) based upon a literature
reference. Regulatory relationships are denoted in a box on the
line with positive regulation represented as a plus sign, negative
regulation as a minus sign, and unknown relationships by no
sign.
[0064] FIG. 39 shows the functional associations of genes, which
are highly correlated in all three treatment groups (CSC-A, CSC-B,
and S9). The genes, pathways, and functional interconnections among
these elements for genes correlated in all three treatment groups
are represented. Gene and pathway symbols are described in FIG. 38.
Cross-hatched ovals indicate genes from Table 3 (i.e., genes
specific for S9 treatment). Ovals with slanted lines (indicate
additional proteins not in Table 3), cross-hatched oval (cell
object--DNA) and white triangle (indicating small
molecule-estrogen) were added to better define the regulatory
networks of the genes identified in this analysis. Ovals with
dashed lines indicate classes of functional peptides. White
rectangles indicate cellular processes in which these genes
participate. Each line indicates a regulatory relationship
(binding, regulation, etc.) based upon a literature reference.
Regulatory relationships are denoted in a box on the line with
positive regulation represented as a plus sign, negative regulation
as a minus sign, and unknown relationships by no sign.
[0065] FIG. 40 shows the results of a discriminant function
analysis (DFA), which identified genes having high discriminatory
capabilities. Values of the roots obtained by DFA analysis were
used to graphically depict the differences of the gene expression
values obtained for the three treatments (CSC-A, CSC-B, and S9).
Root values for the 2-12 h time points for each treatment are
represented by filled circles (CSC-A), open circles (CSC-B), and
filled triangles (S9).
[0066] FIG. 41 shows the functional associations of genes, which
are provided in Table 3. The genes, pathways, and functional
interconnections among these elements for genes having the highest
discriminatory potential among all three treatment groups are
represented. Gene and pathway symbols are described in previous
figures.
[0067] FIGS. 42A and B show a comparison of expression behavior of
heat shock protein family members DNAJA1 and DNAJB1 in Experiment 1
(FIG. 42A) and 2 (FIG. 42B). Each time point represents the average
of 2 or 3 replicates per condition.
[0068] FIG. 43 is a hierarchical clustering of samples using 105
genes that were both over-expressed upon treatment of NHBE cells
with CS in two separate experiments, and encoded protein products
that modulate one of the 4 major CS-affected GO-defined cellular
functions identified. Samples a-b are from Experiment 1, samples
c-e are from Experiment 2. A bar indicates heat shock and heat
shock-associated genes showing greatly increased expression
exclusively at 4 h. Markings indicate genes whose expression is
known to be regulated by transcription factor NRF2.
[0069] FIG. 44 shows a plot of .DELTA.H2AX immunofluorescence in
A549 cells exposed to smoke of different combinations of tobaccos
and filters from IM16, Omni.RTM. and Quest 3.RTM. cigarettes,
corrected according to the .DELTA.H2AX immunofluorescence for
mock-exposed cells. FIG. 44A depicts .DELTA.H2AX immunofluorescence
for the unmodified cigarettes. FIG. 44B depicts .DELTA.H2AX
immunofluorescence for cigarettes containing IM16 tobacco and IM16,
Omni.RTM. and Quest 3.RTM. filters. FIG. 44C depicts .DELTA.H2AX
immunofluorescence for cigarettes containing Omni.RTM. tobacco and
either an IM16 or Omni.RTM. filter. FIG. 44D depicts .DELTA.H2AX
immunofluorescence for cigarettes containing Quest 3.RTM. tobacco
and either an IM16 or Quest 3.RTM. filter.
[0070] FIG. 45 shows bar plots showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke of
different combinations of tobaccos and filters from IM16, Omni.RTM.
or Quest 3.RTM. cigarettes, relative to mock cloning
efficiency.
[0071] FIG. 46 shows bar plots showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke of
different combinations of tobaccos and filters from IM16, Quest
1.RTM. or Quest 3.RTM., relative to mock cloning efficiency.
[0072] FIG. 47 shows bar plots showing the relative percent cloning
efficiency of A549 cells 5 days after exposure to smoke of
different combinations of tobaccos and filters from IM16, Omni.RTM.
or Quest 3.RTM. cigarettes, relative to mock cloning
efficiency.
DETAILED DESCRIPTION
I. Introduction
[0073] The health consequences of tobacco consumption are known but
many people continue to use tobacco products. The addictive
properties of tobacco products are largely attributable to the
presence of nicotine. In addition to being one of the most
addictive substances known, nicotine is also a precursor for a
large number of carcinogenic compounds present in tobacco and the
body. Many other harmful compounds in addition to nicotine are
present in conventional tobacco, however.
[0074] There is currently a great interest in developing approaches
to decrease the levels of noxious, carcinogenic, or addictive
substances including tar, TSNAs, and nicotine in tobacco. Although
researchers have developed several approaches to reduce some of
these harmful compounds, many conventional techniques result in a
product that has poor taste, fragrance, or smoking properties. Some
processes, for example, reduce the nicotine content of tobacco by
microbial enzymatic degradation, chemical extraction, or high
pressure extraction. (See e.g., U.S. Pat. Nos. 4,557,280;
4,561,452; 4,848,373; 4,183,364; and 4,215,706, all of which are
hereby expressly incorporated by reference in their entireties).
More recently, techniques in genetic engineering and
chemically-induced gene suppression have been employed to make
reduced nicotine and/or reduced tobacco specific nitrosamine (TSNA)
tobacco. (See e.g., 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; Timko et al., WO 00/67558, which designated the United
States and was published in English, Nakatani et al., U.S. Pat.
Nos. 5,684,241; 5,369,023; 5,260,205; and Roberts et al. U.S. Pat.
No. 6,700,040, all of which are hereby expressly incorporated by
reference in their entireties). In view of the foregoing, and
notwithstanding the various efforts exemplified in the above
reports, there remains a need for tobacco that has a reduced
potential to contribute to a tobacco-related disease and methods of
producing such tobacco.
[0075] Embodiments provided herein relate to tobacco and/or tobacco
products having a reduced amount of a harmful compound, and methods
of developing, screening and using such tobacco and tobacco
products. Several approaches are provided to reduce the amount of
one or more harmful compounds in tobacco by, for example, modifying
the expression of a gene that is involved in the production of a
harmful compound in tobacco. Also provided are methods of
determining whether the removal of a harmful compound yields a
tobacco and/or a tobacco product that has a reduced potential to
contribute to a tobacco-related disease. Also provided are
reduced-risk tobacco and tobacco products made in accordance with
the methods provided herein. Also provided are methods of using the
reduced-risk tobacco and tobacco products made in accordance with
the methods provided herein.
II. Modified Tobacco
[0076] Several approaches to create a reduced risk tobacco product
having a reduced amount of a harmful compound are described. At
least some of the reduced risk tobacco products provided herein
contain modified tobacco. As used herein, "modified tobacco" refers
to a tobacco that has been subjected to one or more genetic,
chemical or processing steps that is different than the
conventional treatment or processing of traditional "wild-type"
tobacco products. In one example, a tobacco product can be
genetically modified, by, for example, administering to a tobacco
plant a nucleic acid molecule that modulates expression of one or
more genes in the tobacco plant that produce a compound.
Genetically modified tobacco and methods of preparing same are
provided elsewhere herein. In another example, a tobacco product
can be chemically modified, by, for example, extracting or
chemically altering one or more components of tobacco, according to
methods known in the art as exemplified in U.S. Pat. Nos.
6,789,548, 4,557,280; 4,561,452; 4,848,373; 4,183,364; 4,215,706;
4,257,430; 4,248,251; 4,235,251; 4,216,784; 4,177,822; 4,055,191
(all of which are herein expressly incorporated by reference in
their entireties) or by adding one or more compounds to a tobacco
plant prior to harvesting the tobacco, as known in the art and
exemplified in U.S. Pat. Pub. No. 20050072047, herein expressly
incorporated by reference in its entirety. Additional modified
tobaccos contemplated herein include reconstituted tobacco,
extracted tobacco, and expanded or puffed tobacco. In some
embodiments, the tobacco is modified to have a reduced amount of a
compound that contributes to a tobacco-related disease, including,
but not limited to, a compound associated with a tobacco-related
disease or a metabolite thereof (e.g., tobacco sterols, nicotine, a
TSNA, and a gene product that is involved in the production of a
compound associated with a tobacco-related disease or a metabolite
thereof).
[0077] The modified tobacco described herein is 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 modified tobacco (e.g., reduced nicotine/TSNA and/or
sterol 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, TSNAs, and/or
sterols.
[0078] In some embodiments, the modified tobacco has reduced levels
of nicotine, nornicotine, and/or sterols in tobacco. Alkaloids such
as nicotine and nornicotine are precursors for a number of harmful
compounds that contribute to tobacco-related disease (e.g., the
tobacco specific nitrosamines (TSNAs): N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),
4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal
(NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL),
4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or
4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC) and
acrolein). Sterols are precursors for a number of harmful
compounds, which are generated by pyrolysis of tobacco, that also
contribute to tobacco-related disease (e.g., polycyclic aromatic
hydrocarbons (PAHs), such as benz[a]pyrene (BAP), heterocyclic
hydrocarbons, terpenes, paraffins, aromatic amines, and aldehydes).
Because the presence of these harmful compounds in tobacco
contributes to tobacco-related disease, a modified tobacco that
comprises a reduced amount of any one of these compounds, as
compared to a reference tobacco (e.g., the industry standard
reference tobacco IM16 (Philip Morris.RTM. USA) or the low tar
reference cigarette 2R4F or the ultra low tar cigarette 1R5F, which
are Kentucky reference cigarettes that can be obtained from the
Tobacco and Health Institute at the University of Kentucky), a
conventional tobacco (e.g., a commercially available tobacco of the
same class (e.g., "full-flavor" or "light" or "ultra-light")) or a
non-transgenic tobacco (e.g., a tobacco of the same variety, such
as Burley, Virginia Flue-cured, or Oriental, or strain, such as LA
Burley 21, K326, Tn90, Djebel174, as the transgenic tobacco prior
to genetic modification) has a reduced potential to contribute to a
tobacco-related disease. Tobacco products comprising the modified
tobacco can also be analyzed by various approaches to confirm that
the tobacco is "reduced risk," as compared to a parental strain or
a reference tobacco using one or more of the assays described
herein or otherwise known in the art. This "reduced risk" modified
tobacco can then be processed, optionally, sterilized or otherwise
made substantially-free of microbes, and said tobacco can be
incorporated into tobacco products, preferably, cigarettes,
optionally, by an aseptic approach so as to not introduce microbes
(e.g., bacteria, mold, yeast, and fungi) into the products. Tobacco
products comprising the modified tobacco can then be packaged,
optionally, by an aseptic approach in air-tight or microbe-free
packaging so as to not introduce microbes into the products.
[0079] In this manner, the conversion of alkaloid to TSNA, which
results from microbial growth on the tobacco when microbes are
introduced during processing, packaging, and storage, is
significantly reduced. By using the embodied tobacco preparative
methods, which may include several aseptic processing,
manufacturing, and packaging procedures, one can maintain an amount
of total TSNA (e.g., the collective content of NNN, NAT, NAB, and
NNK) in or delivered by (e.g., as measured by FTC or ISO
methodologies) a commercially available tobacco product of less
than or equal to 0.5 .mu.m/g (e.g., 0.05 .mu.m/g, 0.1 .mu.g, 0.2
.mu.g/g, 0.3 .mu.g/g, 0.4 .mu.g/g, or 0.5 .mu.g/g) for a period of
at least 1 week, 1 month, or 1-5 years after packaging or
incorporation of the tobacco into a tobacco product (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).
[0080] In some embodiments, a modified 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 transgenic
tobacco in a controlled fashion. By this approach, nicotine levels
in transgenic tobacco that comprises a reduced amount of endogenous
nicotine (i.e., nicotine that is produced by the transgenic plant
from which the transgenic 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, modified 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, modified tobacco comprising a reduced amount of
endogenous nicotine and/or TSNAs can be contacted with an amount of
exogenous nicotine that is or delivers (e.g., as measured by FTC or
ISO methodologies) 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, 1.0 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, the modified tobacco
contacted with the exogenous nicotine is a transgenic tobacco
comprising, for example, one or more of the isolated nucleic acids,
isolated nucleic acid cassettes, or isolated nucleic acid
constructs described herein.
[0081] 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 modified tobacco that provides virtually any amount of nicotine
can be obtained.
[0082] 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 modified
tobacco (e.g., a transgenic tobacco comprising a reduced amount of
nicotine and/or TSNA as prepared as described herein) after the
modified tobacco has been made substantially free of microbes
(e.g., bacteria, yeast, mold, or fungi). The reduced alkaloid
modified 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 (e.g., the resultant concentration of or the amount of
delivered or provided total TSNA (e.g., NNN, NNK, NAT, and NAB) in
or delivered by a tobacco or tobacco product is equal to or below
0.5 .mu.g/g (e.g., 0.05 .mu.g/g, 0.1 .mu.g, 0.2 .mu.g/g, 0.3
.mu.g/g, 0.4 .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 modified
tobacco described herein, the term "substantially-free of bacteria"
can refer to tobacco or a tobacco product containing the modified
tobacco that has less than or equal to 20% of the bacteria normally
present on the strain of tobacco prior to 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%).
[0083] Once the exogenous nicotine has been contacted with the
microbe-free modified 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
or delivered by a commercially available tobacco product, which
comprises exogenous nicotine, of less than or equal to 0.5 .mu.g/g
(e.g., 0.05 .mu.g/g, 0.1 .mu.g, 0.2 .mu.g/g, 0.3 .mu.g/g, 0.4
.mu.g/g, or 0.5 .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 modified tobacco and a collective content of
NNN, NAT, NAB, and NNN that is present or delivered by the tobacco
is less than or equal to 0.5 .mu.g/g (e.g., 0.05 .mu.g/g, 0.1
.mu.g, 0.2 .mu.g/g, 0.3 .mu.g/g, 0.4 .mu.g/g, or 0.5 .mu.g/g). In
some embodiments, a collective content of NNN, NAT, NAB, and NNN of
less than or equal to 0.5 .mu.g/g (e.g., 0.05 .mu.g/g, 0.1 .mu.g,
0.2 .mu.g/g, 0.3 .mu.g/g, 0.4 .mu.g/g, or 0.5 .mu.g/g) in or
delivered by a tobacco product containing said transgenic 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). An exemplary modified tobacco is
transgenic tobacco comprising, for example, one of the nucleic acid
constructs described herein. 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 modified 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.
[0084] Tobacco products that comprise a modified tobacco described
herein include "full-flavor," "lights," and "ultra light"
cigarettes typically having both reduced levels of alkaloids and
levels of alkaloids commensurate with a level of alkaloid common to
the particular class of cigarette (i.e., a conventional amount of
nicotine). The term "tobacco products" includes, but is not limited
to, smoking materials (e.g., cigarettes, cigars, pipe tobacco),
snuff, chewing tobacco, gum, and lozenges.
[0085] The term "reduced risk tobacco product" or "reduced risk
tobacco" includes, but is not limited to, a tobacco product or
tobacco comprising a modified tobacco that has a reduced amount of
a compound that contributes to a tobacco-related disease, or
increased amounts of a compound that reduces the harmful effects of
of a compound that contributes to a tobacco-related disease such as
nicotine, nornicotine, a sterol, or the metabolites thereof
including, but not limited to, a TSNA, an acrolein, an aldehyde, or
harmful compounds generated upon pyrolysis of tobacco, including
but not limited to, PAH, BAP, a heterocyclic hydrocarbon, or an
aromatic amine, as compared to the amount of these compounds in or
generated by a reference tobacco or reference tobacco product
(e.g., IM16, 2R4F or 1R5F), a commercially available tobacco
product of the same class (e.g., full-flavor, lights, and
ultra-lights), or, preferably, a tobacco of the same variety (e.g.,
Burley, Virginia Flue-cured, or Oriental) or strain (e.g., LA
Burley 21, K326, Tn90, Djebel174) as the transgenic tobacco prior
to genetic modification). For example, a reduced risk tobacco or a
reduced risk tobacco product can include a transgenic tobacco or a
tobacco product comprising transgenic tobacco that up-regulates
fewer genes associated with a tobacco-related disease as compared
to a reference tobacco or reference tobacco product (e.g., IM16,
2R4F or 1R5F), a commercially available tobacco product of the same
class (e.g., full-flavor, lights, and ultra-lights), or,
preferably, a tobacco of the same variety (e.g., Burley, Virginia
Flue-cured, or Oriental) or strain (e.g., LA Burley 21, K326, Tn90,
Djebel174) as the transgenic tobacco prior to genetic
modification).
[0086] Nitrosamines and Tobacco-Specific Nitrosamines
[0087] The term nitrosamine generally refers to any of a class of
organic compounds with the general formula R.sub.2NNO or RNHNO
(where R denotes an amine-containing group). Nitrosamines are
present in numerous foods and have been found to be carcinogenic in
laboratory animals. These compounds are formed by nitrosation
reactions of amines such as amino acids and alkaloids with nitrites
and/or nitrous oxides. By themselves, nitrosamines are not
carcinogenic substances, but in mammals nitrosamines undergo
decomposition by enzymatic activation to form alkylating
metabolites which appear to react with biopolymers to initiate
their tumorogenic effect. Thus, by reducing the amount of
nitrosamine intake, one has effectively reduced the carcinogenic
potential in humans.
[0088] Nitrosamines have been identified in tobacco, tobacco
products, and tobacco smoke by the use of techniques such as gas
chromatography-thermal energy analysis (GC-TEA). Some of these
nitrosamines have been identified as tobacco-specific nitrosamines
(TSNAs). TSNAs are primarily formed by reactions between the two
most abundant alkaloids, nicotine and nornicotine, with nitrous
oxides (NOx), and they account proportionately for the highest
concentration of nitrosamines in both tobacco products and in
mainstream smoke. Of the TSNAs identified, and the subset that have
been found to be present in cigarette smoke, the most characterized
is N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(N-nitrosamine-ketone), or NNK. When injected at relatively high
doses, NNK is carcinogenic in rodents. Minimal amounts of TSNAs are
found in green tobacco, indicating that TSNA formation may occur
during processing steps such as curing, drying, fermentation,
burning or storage of tobacco.
[0089] TSNA formation is attributed to chemical, enzymatic and
bacterial influences during tobacco processing, particularly during
curing, fermentation and aging. Nitrosation of nornicotine,
anatabine, and anabasine gives the corresponding nitrosamines:
N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT) and
N'-nitrosoanabasine (NAB). Nitrosation of nicotine in aqueous
solution affords a mixture of
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), NNN, and
4-(N-nitro so methylamino)-4-(3-pyridyl)-1-butanal (NNA). Less
commonly encountered TSNAs include NNAL
(4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol), iso-NNAL
(4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol, 11) and iso-NNAC
(4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid, 12). See,
U.S. Pat. No. 6,135,121, the entire disclosure of which is hereby
expressly incorporated by reference in its entirety.
[0090] TSNA levels are particularly high in chewing tobaccos and
snuff. The partially anaerobic processes that occur during
fermentation promote the formation of TSNAs from tobacco alkaloids
by promoting increased nitrite levels; in particular,
over-fermentation can increase TSNA levels in snuff by its effects
on nitrate levels and microbial enzymatic activity. The reduction
of the TSNA level in snuff in recent years has been achieved by
maintaining a better control over the bacterial content in these
products.
[0091] Since the nitrate level of tobacco is important for TSNA
formation in cigarette smoke, a significant reduction of TSNAs in
smoke can be achieved by low-nitrate leaf and stem blends. However,
these methods may negatively impact the smokability or the taste of
the tobacco. The TSNA content of mainstream smoke can be reduced by
as much as 80% by cellulose acetate filters, and it can be reduced
still further by filter ventilation.
[0092] Air-cured tobaccos such as Burley and dark-fired may have
higher levels of TSNAs than certain types of Flue-cured bright,
Burley, or dark tobaccos apparently because the high temperatures
associated with flue-curing can kill the micro-organisms that
transform the alkaloids into TSNAs. In air-cured types, nitrate
(N--NO.sub.3) is more abundant in the leaf (particularly in the
leaf and stems) than in Flue-cured tobacco and the alkaloid content
is also much higher. This N--NO.sub.3 is reduced to nitrite
(NO.sub.2.sup.-) by microbes during curing and the NO.sub.2.sup.-
can be further reduced to NOx or react directly with alkaloids to
form TSNAs.
[0093] It is contemplated that, in addition to the techniques
described above, nitrate levels in tobacco (especially in the leaf)
can be reduced by limiting exposure to nitrosating agents or
conditions. Air-curing experiments at a higher temperature have
shown that considerably higher levels of N-nitrosamines are formed
at a curing temperature of 32.degree. C. than at 16.degree. C.,
which is associated with a rise of the nitrite level in the
tobacco, and may also be associated with a rise in microbial
enzymatic activity. Modified curing that involves faster drying
from wider spacing or from more open curing structures has been
shown to reduce TSNA levels in Burley tobacco. The climatic
conditions prevailing during curing exert a major influence on
N-nitrosamine formation, and the relative humidity during
air-curing can be of importance. Stalk curing results in higher
TSNA levels in the smoke than primed-leaf curing. Sun-cured
Oriental tobaccos have lower TSNA levels than flue- and air-cured
dark tobaccos. Accelerated curing of crude tobaccos such as
homogenized leaf curing limits the ability of bacteria to carry out
the nitrosation reactions. However, many of the methods described
above for reducing TSNAs in Burley tobacco can have undesirable
effects on tobacco taste.
[0094] TSNA formation in Flue-cured tobacco also results from
exposure of the tobacco to combustion gases during curing, where
nearly all of the TSNAs in Flue-cured tobacco (e.g., Virginia
Flue-cured) result from a reaction involving NOx and nicotine. The
predominant source of NOx is the mixture of combustion gases in
direct-fired barns. At present, Flue-cured tobacco is predominantly
cured in commercial bulk barns. As a result of energy pressures in
the U.S. during the 1960's, farmer-built "stick barns" with
heat-exchanged flue systems were gradually replaced with more
energy efficient bulk barns using direct-fired liquid propane gas
(LPG) burners. These LPG direct-fired burner systems exhaust
combustion gases and combustion by-products directly into the barn
where contact is made with the curing tobacco. Studies indicate
that LPG combustion by-products react with naturally occurring
tobacco alkaloids to form TSNA.
[0095] In contrast to direct-fired curing, heat-exchange burner
configurations completely vent combustion gases and combustion
by-products to the external atmosphere rather than into the barn.
The heat-exchange process precludes exposure of the tobacco to LPG
combustion by-products, thereby eliminating an important source of
nitrosating agent for TSNA formation, without degrading leaf
quality or smoking quality. The use of heat exchangers reduces TSNA
levels by about 90%. Steps are being taken to reduce TSNA levels in
US tobacco by converting barns to indirect heat through the use of
a heat exchanger, but these methods are very expensive. Although
many of the approaches described in this section have significant
drawbacks, it should be understood that any or all of these
techniques can be used with other techniques, as described herein,
to make tobacco and tobacco products having reduced TSNAs. The
section below provides more detail on nicotine and approaches to
reduce nicotine in tobacco.
[0096] Nicotine
[0097] Nicotine is formed primarily in the roots of the tobacco
plant and is subsequently transported to the leaves, where it is
stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp.
233-34, Dowden, Hutchinson & Ross, Stroudsburg, Pa. (1972)).
Classical crop breeding techniques have produced tobacco with lower
levels of nicotine, including varieties with as low as 8% of the
amount of nicotine found in wild-type tobacco. The many methods
described herein can be used with virtually any tobacco variety but
are preferably used with Burley, Oriental or Flue-cured (e.g.,
Virginia Flue-cured) varieties.
[0098] 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. Enzyme analyses of root tissue from single and double
Nic mutants show that the activities of two enzymes, quinolate
phosphoribosyl transferase ("QPTase") and putrescene methyl
transferase (PMTase), are directly proportional to levels of
nicotine biosynthesis. An obligatory step in nicotine biosynthesis
is the formation of nicotinic acid from quinolinic acid, a step
that is catalyzed by QPTase. QPTase appears to be a rate-limiting
enzyme in the pathway supplying nicotinic acid for nicotine
synthesis in tobacco. (See, 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 level of
nicotine in the leaves.
[0099] The modification of nicotine levels in tobacco plants by
antisense regulation of putrescene methyl transferase expression
has been proposed in U.S. Pat. Nos. 5,369,023 and 5,260,205, to
Nakatani and Malik, and in PCT application WO 94/28142 and U.S.
Pat. No. 5,668,295 to Wahad and Malik, which describe DNA encoding
PMT and the use of sense and antisense PMT constructs, the entire
disclosures of each of which are hereby expressly incorporated by
reference in their entireties. Other genetic modifications proposed
to reduce nicotine levels are described in PCT application WO
00/67558, to Timko, and WO 93/05646, to Davis and Marcum; the
entire contents of each are hereby expressly incorporated by
reference in their entireties. Although these investigators made
significant contributions, there were significant drawbacks to
their experimental design.
[0100] Provided herein are tobacco and tobacco products in which a
plurality of genes involved in nicotine biosynthesis are inhibited.
Most notably, it is presently revealed that there are several
different PMT genes and each may play a role in nicotine
biosynthesis. Knocking-out only one PMT gene may create a leaky
system allowing the other genes to compensate for the reduction in
nicotine biosynthesis. Accordingly, the PMT constructs described
herein were designed to inhibit a plurality of different PMT genes.
That is, in some embodiments, 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 with a single construct. Although many of the
approaches described in this section have significant drawbacks, it
should be understood that any or all of these techniques can be
used with other techniques, as described herein, to make tobacco
and tobacco products having reduced nicotine. The section below
explains several approaches to reduce the amount of nicotine and
sterols in tobacco and tobacco products.
[0101] Reducing the Amount of Nicotine and Sterols in Tobacco
[0102] As discussed above, TSNAs, nicotine, nornicotine, and
sterols contribute significantly to tobacco-related disease, most
notably the carcinogenic potential of tobacco and tobacco products.
Thus, tobacco and tobacco products that have or produce reduced
amounts of these compounds are reduced risk compositions (e.g.,
products that have a reduced potential to contribute to a
tobacco-related disease). Without wishing to be bound by any
particular theory, it is contemplated that the creation of tobacco
plants, tobacco and tobacco products that have a reduced amount of
nicotine will also have reduced amounts of TSNAs. That is, by
removing nicotine from tobacco plants, tobacco and tobacco
products, one effectively removes the most significant alkaloid
substrate for TSNA formation. It was found that the reduction of
nicotine in tobacco was directly related to the reduction of TSNAs.
Similarly, it is contemplated that by removing sterols from
tobacco, one can reduce the amount of PAHs generated from pyrolysis
of the tobacco. Unexpectedly, the methods described herein not only
produce tobacco with a reduced addictive potential but,
concomitantly, produce a tobacco that has a reduced potential to
contribute to a tobacco related disease.
[0103] It should be emphasized that the phrase "a reduced amount"
as applied to nicotine and/or TSNAs is intended to refer to an
amount of nicotine and/or TSNAs in a treated or transgenic tobacco
plant, tobacco or a tobacco product that is less than what would be
found in a tobacco plant, tobacco or a tobacco product from the
same variety of tobacco, processed in the same manner, which has
not been treated or was not made transgenic for reduced nicotine
and/or TSNAs. Thus, in some contexts, wild-type tobacco of the same
variety that has been processed in the same manner is used as a
control by which to measure whether a reduction in nicotine,
nornicotine, a sterol and/or TSNAs or PAHs has been obtained by the
inventive methods described herein.
[0104] The amount of TSNAs (e.g., collective content of NNN, NAT,
NAB, and NNK) and nicotine in wild-type tobacco varies
significantly depending on the variety and the manner it is grown,
harvested and cured. For example, a cured Burley tobacco leaf can
have approximately 30,000 parts per million (ppm) nicotine and
8,000 parts per billion (ppb) TSNA (e.g., collective content of
NNN, NAT, NAB, and NNK); a Flue-cured leaf can have approximately
20,000 ppm nicotine and 300 ppb TSNA (e.g., collective content of
NNN, NAT, NAB, and NNK); and an Oriental cured leaf can have
approximately 10,000 ppm nicotine and 100 ppb TSNA (e.g.,
collective content of NNN, NAT, NAB, and NNK). Tobacco having a
reduced amount of nicotine and/or TSNA, can have no detectable
nicotine and/or TSNA (e.g., collective content of NNN, NAT, NAB,
and NNK), or may contain some detectable amounts of one or more of
the TSNAs and/or nicotine, so long as the amount of nicotine and/or
TSNA is less than that found in tobacco of the same variety, grown
under similar conditions, and cured and/or processed in the same
manner. That is, cured Burley tobacco, as described herein, having
a reduced amount of nicotine can have between 0 and 30,000 ppm
nicotine and 0 and 8,000 ppb TSNA, desirably between 0 and 20,000
ppm nicotine and 0 and 6,000 ppb TSNA, more desirably between 0 and
10,000 ppm nicotine and 0 and 5,000 ppb TSNA, preferably between 0
and 5,000 ppm nicotine and 0 and 4,000 ppb TSNA, more preferably
between 0 and 2,500 ppm nicotine and 0 and 2,000 ppb TSNA and most
preferably between 0 and 1,000 ppm nicotine and 0 and 1,000 ppb
TSNA. Embodiments of cured Burley leaf prepared by the methods
described herein can also have between 0 and 1000 ppm nicotine and
0 and 500 ppb TSNA, 0 and 500 ppm nicotine and 0 and 250 ppb TSNA,
0 and 250 ppm nicotine and 0 and 100 ppb TSNA, 0 and 100 ppm
nicotine and 0 and 50 ppb TSNA, 0 and 50 ppm nicotine and 0 and 5
ppb TSNA and some embodiments of cured Burley leaf described herein
have virtually no detectable amount of nicotine or TSNA. In some
embodiments above, the amount of TSNA refers to the collective
content of NNN, NAT, NAB, and NNK.
[0105] Similarly, a Flue-cured tobacco embodiment having a reduced
amount of nicotine can have between 0 and 20,000 ppm nicotine and 0
and 300 ppb TSNA, desirably between 0 and 15,000 ppm nicotine and 0
and 250 ppb TSNA, more desirably between 0 and 10,000 ppm nicotine
and 0 and 200 ppb TSNA, preferably between 0 and 5,000 ppm nicotine
and 0 and 150 ppb TSNA, more preferably between 0 and 2,500 ppm
nicotine and 0 and 100 ppb TSNA and most preferably between 0 and
1,000 ppm nicotine and 0 and 50 ppb TSNA. Embodiments of Flue-cured
tobacco, as described herein, can also have between 0 and 500 ppm
nicotine and 0 and 25 ppb TSNA, 0 and 200 ppm nicotine and 0 and 10
ppb TSNA, 0 and 100 ppm nicotine and 0 and 5 ppb TSNA and some
embodiments of Flue-cured tobacco have virtually no detectable
amount of nicotine or TSNA. In some embodiments above, the amount
of TSNA refers to the collective content of NNN, NAT, NAB, and
NNK.
[0106] Further, a cured Oriental tobacco embodiment having a
reduced amount of nicotine can have between 0 and 10,000 ppm
nicotine and 0 and 100 ppb TSNA, desirably between 0 and 7,000 ppm
nicotine and 0 and 75 ppb TSNA, more desirably between 0 and 5,000
ppm nicotine and 0 and 50 ppb TSNA, preferably between 0 and 3,000
ppm nicotine and 0 and 25 ppb TSNA, more preferably between 0 and
1,500 ppm nicotine and 0 and 10 ppb TSNA and most preferably
between 0 and 500 ppm nicotine and no detectable TSNA. Embodiments
of cured Oriental tobacco can also have between 0 and 250 ppm
nicotine and no detectable TSNA and some embodiments of cured
Oriental tobacco have virtually no detectable amount of nicotine or
TSNA. In some embodiments above, the amount of TSNA refers to the
collective content of NNN, NAT, NAB, and NNK.
[0107] Some embodiments comprise cured tobaccos (e.g., Burley,
Flue-cured, or Oriental) with reduced amounts of nicotine as
compared to control varieties, wherein the amount of nicotine in or
delivered by the product (e.g., as measured by FTC or ISO
methodologies) is less than about 2 mg/g, 1 mg/g, 0.75 mg/g, 0.5
mg/g or desirably less than about 0.1 mg/g, and preferably less
than 0.08 mg/g, 0.07 mg/g, 0.06 mg/g, 0.05 mg/g, 0.04 mg/g, 0.03
mg/g, 0.02 mg/g, 0.01 mg/g. Tobacco products made from these
reduced nicotine and TSNA tobaccos are also embodiments. The term
"tobacco products" include, but are not limited to, smoking
materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing
tobacco, gum, and lozenges. As mentioned above, these reduced
nicotine and TSNA tobaccos can be treated with exogenous nicotine
so as to incrementally increase the amount of nicotine in the
product and by employing aseptic processing and packaging
techniques, the amounts of total TSNAs in the product can be kept
at or below 0.5 .mu.g/g for prolonged periods of time.
[0108] In some contexts, the phrase "reduced amount of nicotine
and/or TSNAs" refers to the tobacco plants, cured tobacco, and
tobacco products, as described herein, which have less nicotine
and/or TSNAs (e.g., the collective content of NNN, NAT, NAB, and
NNK) by weight than the same variety of tobacco grown, processed,
and cured in the same way. For example, wild type cured tobacco can
have has approximately 1-4% dry weight nicotine and approximately
0.2%-0.8% dry weight TSNA depending on the manner it was grown,
harvested and cured. A typical cigarette has between 2-11 mg of
nicotine and approximately 5.0 .mu.g of TSNAs. Thus, the tobacco
plants, tobacco and tobacco products provided herein can have or
deliver, in dry weight for example, less than 0.01%, 0.015%, 0.02%,
0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%,
0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%,
0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.35%, 0.375%, 0.4%,
0.425%, 0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%,
0.85%, 0.9%, 0.95%, and 1.0% nicotine and less than 0.01%, 0.015%,
0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%,
0.065%, 0.07%, 0.075%, and 0.08% TSNA (e.g., collective content of
NNN, NAT, NAB, and NNK).
[0109] Alternatively, a cigarette provided herein can have or
deliver, for example, less than 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg,
0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg,
0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1.0 mg, 1.1 mg,
1.15 mg, 1.2 mg, 1.25 mg, 1.3 mg, 1.35 mg, 1.4 mg, 1.45 mg, 1.5 mg,
1.55 mg, 1.6 mg, 1.65 mg, 1.7 mg, 1.75 mg, 1.8 mg, 1.85 mg, 1.9 mg,
1.95 mg, 2.0 mg, 2.1 mg, 2.15 mg, 2.2 mg, 2.25 mg, 2.3 mg, 2.35 mg,
2.4 mg, 2.45 mg, 2.5 mg, 2.55 mg, 2.6 mg, 2.65 mg, 2.7 mg, 2.75 mg,
2.8 mg, 2.85 mg, 2.9 mg, 2.95 mg, 3.0 mg, 3.1 mg, 3.15 mg, 3.2 mg,
3.25 mg, 3.3 mg, 3.35 mg, 3.4 mg, 3.45 mg, 3.5 mg, 3.55 mg, 3.6 mg,
3.65 mg, 3.7 mg, 3.75 mg, 3.8 mg, 3.85 mg, 3.9 mg, 3.95 mg, 4.0 mg,
4.1 mg, 4.15 mg, 4.2 mg, 4.25 mg, 4.3 mg, 4.35 mg, 4.4 mg, 4.45 mg,
4.4 mg, 4.45 mg, 4.5 mg, 4.55 mg, 4.6 mg, 4.65 mg, 4.7 mg, 4.75 mg,
4.8 mg, 4.85 mg, 4.9 mg, 4.95 mg, 5.0 mg, 5.5 mg, 5.7 mg, 6.0 mg,
6.5 mgmg, 6.7 mg, 7.0 mg, 7.5 mg, 7.7 mg, 8.0 mg, 8.5 mg, 8.7 mg,
9.0 mg, 9.5 mg, 9.7 mg, 10.0 mg, 10.5 mg, 10.7 mg, and 11.0 mg
nicotine and less than 0.001 .mu.g, 0.002 .mu.g, 0.003 .mu.g, 0.004
.mu.g, 0.005 .mu.g, 0.006 .mu.g, 0.007 .mu.g, 0.008 .mu.g, 0.009
.mu.g, 0.01 .mu.g, 0.02 .mu.g, 0.03 .mu.g, 0.04 .mu.g, 0.05 .mu.g,
0.06 .mu.g, 0.07 .mu.g, 0.08 .mu.g, 0.09 .mu.g, 0.1 .mu.g, 0.15
.mu.g, 0.24 .mu.g, 0.25 .mu.g, 0.3 .mu.g, 0.336 .mu.g, 0.339 .mu.g,
0.345 .mu.g, 0.35 .mu.g, 0.375 .mu.g, 0.4 .mu.g, 0.414 .mu.g, 0.45
.mu.g, 0.5 .mu.g, 0.515 .mu.g, 0.55 .mu.g, 0.555 .mu.g, 0.56 .mu.g,
0.578 .mu.g, 0.58 .mu.g, 0.6 .mu.g, 0.611 .mu.g, 0.624 .mu.g, 0.65
.mu.g, 0.7 .mu.g, 0.75 .mu.g, 0.8 .mu.g, 0.85 .mu.g, 0.9 .mu.g,
0.95 .mu.g, 1.0 .mu.g, 1.1 .mu.g, 1.114 .mu.g, 1.15 .mu.g, 1.24
.mu.g, 1.25 .mu.g, 1.3 .mu.g, 1.35 .mu.g, 1.4 .mu.g, 1.45 .mu.g,
1.5 .mu.g, 1.55 .mu.g, 1.6 .mu.g, 1.65 .mu.g, 1.7 .mu.g, 1.75
.mu.g, 1.8 .mu.g, 1.85 .mu.g, 1.9 .mu.g, 1.95 .mu.g, 2.0 .mu.g, 2.1
.mu.g, 2.15 .mu.g, 2.24 .mu.g TSNA (e.g., collective content of
NNN, NAT, NAB, and NNK).
[0110] Unexpectedly, it was discovered that several methods for
reducing endogenous levels of nicotine in a plant are suitable for
producing tobacco that is substantially free of nitrosamines,
especially TSNAs. Any method that reduces levels of other
alkaloids, including norniticotine, is likewise suitable for
producing tobacco substantially free of nitrosamines, especially
TSNAs. As described, embodiments comprise methods of reducing the
carcinogenic potential of a tobacco product comprising providing a
cured tobacco as described herein and preparing a tobacco product
from said cured tobacco, whereby the carcinogenic potential of said
tobacco product is thereby reduced.
[0111] In some embodiments that employed the A622 inhibition
construct, it was found that transgenic tobacco that had
conventional levels of nicotine but significantly reduced levels of
nornicotine were produced. This particular line of tobacco is
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.
[0112] Other embodiments include the use of the cured tobacco
described herein for the preparation of a tobacco product that
contains reduced amounts of carcinogens as compared to control
varieties and/or that reduces the amount of a TSNA or TSNA
metobolite in a human that uses tobacco. In some embodiments, for
example, the tobacco smoking products described herein reduce the
carcinogenic potential of side stream or main stream tobacco smoke
in humans exposed to said side stream or main stream tobacco smoke.
By providing the modified cured tobacco described herein in a
product that undergoes pyrolysis, for example, the side stream
and/or main stream smoke produced by said product comprises a
reduced amount of TSNAs and/or nicotine. Thus, the cured tobacco
described herein can be used to prepare a tobacco smoking product
that comprises a reduced amount of TSNAs in side stream and/or
mainstream smoke.
[0113] 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.
[0114] 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.
[0115] 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).
[0116] The FTC and ISO smoking methods differ in the following
eight areas. [0117] 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. [0118] 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.
[0119] ISO defines the position of the ashtray at 20-60 millimeters
below the cigarettes in the smoking machine. FTC does not specify a
position. [0120] 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. [0121] The ISO method specifies airflow across the
cigarettes at the cigarette level. FTC specifies the use of a
monitor cigarette to adjust airflow. [0122] 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. [0123] 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. [0124] ISO defines
the internal standards for the gas chromatographic determination of
nicotine and water. The FTC procedure does not specify the internal
standards.
[0125] 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.
[0126] In some embodiments, for example, the collective content of
NNN, NAT, NAB, and NNK in the mainstream or side stream smoke from
a tobacco product comprising the modified tobacco, including
genetically modified tobacco, described herein is between about
0-5.0 .mu.g/g, 0-4.0 .mu.g/g, 0-3.0 .mu.g/g, 0-2.0 .mu.g/g, 0-1.5
.mu.g/g, 0-1.0 .mu.g/g, 0-0.75 .mu.g/g, 0-0.5 .mu.g/g, 0-0.25
.mu.g/g, 0-0.15 .mu.g/g, 0-0.1 .mu.g/g, 0-0.05 .mu.g/g, 0-0.02
.mu.g/g, 0-0.015 .mu.g/g, 0-0.01 .mu.g/g, 0-0.005 .mu.g/g, 0-0.002
.mu.g/g, or 0-0.001 .mu.g/g. That is, some embodiments are
genetically modified Burley tobacco, wherein the side stream or
mainstream smoke produced from a tobacco product comprising said
Burley tobacco has a collective content of NNN, NAT, NAB, and NNK
in the mainstream or side stream smoke between about 0-5.0 .mu.g/g,
0-4.0 .mu.g/g, 0-3.0 .mu.g/g, 0-2.0 .mu.g/g, 0-1.5 .mu.g/g, 0-1.0
.mu.g/g, 0-0.75 .mu.g/g, 0-0.5 .mu.g/g, 0-0.25 .mu.g/g, 0-0.15
.mu.g/g, 0-0.1 .mu.g/g, 0-0.05 .mu.g/g, 0-0.02 .mu.g/g, 0-0.015
.mu.g/g, 0-0.01 .mu.g/g, 0-0.005 .mu.g/g, 0-0.002 .mu.g/g, or
0-0.001 .mu.g/g.
[0127] Other embodiments concern modified Flue-cured tobacco, such
as genetically modified Flue-cured tobacco, wherein the sidestream
or mainstream smoke produced from a tobacco product comprising said
Flue-cured tobacco has a collective content of NNN, NAT, NAB, and
NNK in the mainstream or side stream smoke between about 0-5.0
.mu.g/g, 0-4.0 .mu.g/g, 0-3.0 .mu.g/g, 0-2.014/g, 0-1.5 .mu.g/g,
0-1.0 .mu.g/g, 0-0.75 .mu.g/g, 0-0.5 .mu.g/g, 0-0.25 .mu.g/g,
0-0.15 .mu.g/g, 0-0.1 .mu.g/g, 0-0.05 .mu.g/g, 0-0.02 .mu.g/g,
0-0.015 .mu.g/g, 0-0.01 .mu.g/g, 0-0.005 .mu.g/g, 0-0.002 .mu.g/g,
or 0-0.001 .mu.g/g.
[0128] More embodiments concern modified Oriental tobacco, wherein
the sidestream or mainstream smoke produced from a tobacco product
comprising said Oriental tobacco has a collective content of NNN,
NAT, NAB, and NNK in the mainstream or side stream smoke between
about 0-5.0 .mu.g/g, 0-4.0 .mu.g/g, 0-3.0 .mu.g/g, 0-2.0 .mu.g/g,
0-1.5 .mu.g/g, 0-1.0 .mu.g/g, 0-0.75 .mu.g/g, 0-0.5 .mu.g/g, 0-0.25
.mu.g/g, 0-0.15 .mu.g/g, 0-0.1 .mu.g/g, 0-0.05 .mu.g/g, 0-0.02
.mu.g/g, 0-0.015 .mu.g/g, 0-0.01 .mu.g/g, 0-0.005 .mu.g/g, 0-0.002
.mu.g/g, or 0-0.001 .mu.g/g.
[0129] Additional Tobacco Modifications
[0130] Additional modified tobaccos that can be used in the methods
and tobacco products provided herein include, but are not limited
to, chemically modified tobacco, expanded, extracted, or puffed
tobacco, and reconstituted tobacco.
[0131] Any of a variety of chemically modified tobaccos can be
included in the methods and tobacco products provided herein. For
example, the chemical modification can include palladium, or can
include an auxin, auxin analog, or jasmonate antagonist (see e.g.,
U.S. Pat. No. 6,789,548 and U.S. Pat. App. Pub. No. 20050072047,
both of which are hereby expressly incorporated by reference in
their entirety).
[0132] By one approach, a chemically modified tobacco is made as
follows. A tobacco is provided and a casing solution is applied
thereto. Thereafter, a plurality of metallic or carbonaceous
catalytic particles having a mean average or a mode average
particle size of less than about 20 microns is applied to the
tobacco in a form separate from the casing solution. Next, a
nitrate or nitrite source in a form separate from the casing
solution and in a form separate from the plurality of metallic or
carbonaceous catalytic particles is applied to the tobacco, before,
after or simultaneously with applying the plurality of particles
but after applying the casing solution, whereby a smoking
composition is obtained. In some embodiments of this modified
tobacco, a polyaromatic hydrocarbon, azaarene, carbazole, or a
phenolic compound is reduced. Using this approach, the Omni.RTM.
tobacco product was developed.
[0133] By another approach, a chemically modified tobacco is made
by identifying a tobacco plant in a field for nicotine reduction;
and contacting said tobacco plant with a composition selected from
the group consisting of an auxin, auxin analog, and jasmonate
antagonist from between about 21 days before topping to about 21
days after topping said tobacco plant, whereby the amount of
nicotine in said topped tobacco plant contacted with said
composition is below that of a topped tobacco plant of the same
variety, grown under the same conditions, which has not been
contacted with said composition.
[0134] In another example, the chemically modified tobacco can be
extracted tobacco. By some approaches the chemically modified
tobacco is extracted with an organic solvent and other processes
use super-critical fluid extraction or carbon dioxide. In another
example, the chemical modification can be a biotic modification.
Microbes that ingest nitrates and alkaloids can be applied to
tobacco so as to obtain a reduced nicotine tobacco; for example
such a biotic modification can include bacteria. In another
example, the tobacco is processed to remove the presence of a
microbe. In another example the chemically modified tobacco can be
sterilized, pasteurized, or radiated.
[0135] In another example, the chemically modified tobacco can have
added thereto an exogenous component of tobacco or analog thereof.
Tobacco can be modified to increase or decrease one or more
compounds such as proteins, metabolites, nicotine-related compounds
and sterols. In some methods provided herein, a tobacco which has
been modified to produce lower levels of one or more compounds such
as nicotine or a nicotine metabolite, or a sterol, can have
exogenously added thereto, one of these lower-level compounds, one
or more but not all lower-level compounds, or all lower-level
compounds or an analog of the compound(s).
[0136] Such tobaccos with one or more exogenously added compounds
can be compared in accordance with the methods provided herein to
the same tobacco to which no exogenous compound has been added, to
which a different exogenous compound has been added, or to which a
different level of the same exogenous compound has been added. For
example, the methods provided herein can be used to compare a
tobacco that has been genetically modified to produce reduced
nicotine levels with the same tobacco to which exogenous nicotine
or a nicotine analog has been added thereto. By performing such
methods, the role of the exogenously added compound on cell damage
or other response determined according to the methods provided
herein (e.g., apoptosis or cell proliferation), can be
determined.
[0137] In another example, the chemically modified tobacco has had
added thereto a compound or composition containing antioxidants.
Tobacco at any stage of its processing can have added thereto an
antioxidant compound or a composition with antioxidant properties.
Any of a variety of known antioxidant compounds can be added to the
tobacco, including, but not limited to, lycopene, tocopherol,
tocopherol metabolites, ascorbic acid, unsaturated fatty acids,
N-acetyl cysteine, and other antioxidants known in the art. A
composition with antioxidant properties can include a biological
composition or extract that can neutralize oxidants, such as milk
or milk proteins, tumeric or tumeric extracts, barley or barley
extracts, alfalfa or alfalfa extracts. Other compounds that can be
added to the tobacco include thiol-containing proteins, plant
extracts, aromatic compounds (e.g., caffeine or pentoxyfyllen,
which are contemplated to scavenge carcinogens).
[0138] Another form of modified tobacco is expanded or puffed
tobacco. Included herein are methods to produce reduced-exposure
tobacco products by utilizing the tobacco provided herein,
deproteinized tobacco fiber, and freeze dried tobacco in any
combination and in conjunction with expanded or puffed tobacco.
More than 150 patents have been issued related to tobacco expansion
(e.g., U.S. Pat. No. 3,991,772, herein expressly incorporated by
reference in its entirety). "Expanded tobacco" is an important part
of tobacco filler which is processed through expansion of suitable
gases so that the tobacco is "puffed" resulting in reduced density
and greater filling capacity. It reduces the weight of tobacco used
in cigarettes. Advantageously, expanded tobacco reduces tar,
nicotine and carbon monoxide deliveries and finds use, for example,
in making low tar, low nicotine, and low carbon monoxide delivery
cigarettes. Expanded tobacco is particularly useful in making
low-tar delivery cigarettes. Carlton cigarettes, which have had
claims of being the lowest tar and nicotine delivery cigarette, are
reportedly made with a very large percentage of expanded tobacco.
However, use of expanded tobacco also results in reduced nicotine
delivery, which can result in compensation.
[0139] Any method for expansion of tobacco known in the art can be
used in the methods provided herein. The most common method used
today incorporates liquid carbon dioxide (U.S. Pat. Nos. 4,340,073
and 4,336,814, herein expressly incorporated by reference in its
entirety). Liquid propane has also been used for making commercial
cigarettes, predominantly in Europe (U.S. Pat. No. 4,531,529,
herein expressly incorporated by reference in its entirety). Liquid
propane offers advantages over carbon dioxide since higher 3Q
degrees of expansion are possible, in the range of 200%. Under
pressure, the liquid carbon dioxide (or liquid propane) permeates
the tobacco cell structure. When the tobacco is rapidly heated the
carbon dioxide (or liquid propane) expands the cell back to its
pre-cured size.
[0140] Another form of modified tobacco is reconstituted tobacco.
Included herein are methods to produce reduced-exposure tobacco
products by utilizing the tobacco provided herein, deproteinized
tobacco fiber, and freeze dried tobacco in any combination and in
conjunction with reconstituted tobacco. "Reconstituted tobacco"
("Recon") is an important part of tobacco filler made from tobacco
dust and other tobacco scrap material, processed into sheet form
and cut into strips to resemble tobacco. In addition to the cost
savings, reconstituted tobacco is very important for its
contribution to cigarette taste from processing flavor development
using reactions between ammonia and sugars.
[0141] The process to produce sheets of Recon began during the
1950s. U.S. patents that describe such processes include: U.S. Pat.
Nos. 3,499,454, 4,182,349 4,962,774, and 6,761,175, herein
expressly incorporated by reference in their entirety. Recon is
traditionally produced from tobacco stems and/or smaller leaf
particles in a process that closely resembles a typical paper
making process. The tar and nicotine yields of reconstituted
tobacco are lower than those from equivalent quantities of whole
tobacco leaf. This process entails processing the various tobacco
portions that are to be made into Recon. After the Recon sheets are
produced they are cut into a size and shape that resembles cut rag
tobacco made from whole leaf tobacco. This cut Recon then gets
mixed with cut-rag tobacco and is ready for cigarette making.
Cigarettes can be manufactured with all Recon, no Recon, or any
combination thereof. Most major brands have at least 10% of Recon
in the Filler.
[0142] In another embodiment nicotine can be added, or nicotine
salts, to produce Recon, which is made from reduced-nicotine
transgenic tobacco or any non-tobacco plant material including but
not limited to herbal blends so that when the Recon is burned it
yields substantially less tobacco-specific nitrosamines and other
carcinogens produced from conventional cigarettes, yet satisfactory
amounts are nicotine are present.
[0143] Processes of removing proteins from tobacco, thereby
creating "deproteinized tobacco fiber" are known in the art, as
exemplified in U.S. Pat. Nos. 4,289,147 and 4,347,324, herein
expressly incorporated by reference in its entirety. Tobacco fiber
is a major byproduct after removing protein. The fibrous remains
from deproteinized tobacco can be included in any percentage as an
ingredient of Recon. Cigarettes made from deproteinized tobacco
have a different taste than conventional cigarettes. However,
appropriate amounts of additives, including flavorings and
nicotine, can be added to help alleviate this taste deficiency.
[0144] Cigarettes containing deproteinized tobacco have a
significant advantage over conventional cigarettes since they
produce reduced levels of carcinogens and harmful combustion
products. "A 71% reduction in protein content of a Flue-cured
tobacco sheet resulted in an 81% reduction in the TA98 Ames
mutagenicity" of the pyrolytic condensate (Clapp, W. L., et al.,
"Reduction in Ames Salmonella mutagenicity of cigarette mainstream
smoke condensate by tobacco protein removal", Mutation Research,
446, pg 167-174, 1999). Previous research in this area had
determined that tobacco leaf protein might be the principal
precursor of mutagens in TSC (Matsumoto, et aI., "Mutagenicities of
the pyrolysis of peptides and proteins", Mutation Research, 56, pg
281-288, 1978).
[0145] Extracting tobacco fiber from genetically modified
reduced-nicotine tobacco effectively eliminates virtually all
carcinogenic TSNAs in such tobacco, since nitrosamines require
relatively high concentrations of nicotine and other alkaloids to
form at detectable levels. Therefore, it can be advantageous to
utilize reduced-nicotine tobacco in reduced-exposure cigarettes or
other tobacco products to further reduce TSNAs. Nicotine can be
either left out or introduced later in the process, which can also
be in the form of nicotine salts.
[0146] PAHs are formed from high temperature pyrolysis of amino
acids, sugars, paraffins, terpenes, phytosterols, celluloses and
other components of tobacco. Most of these components are greatly
reduced in tobacco fiber, effectively reducing formation of PAHs.
Catechols and phenols, recognized carcinogenic co-factors in CS,
would also be reduced since low levels of soluble sugar are present
in tobacco fiber.
[0147] Harmful gas phase compounds such as hydrogen cyanide,
nitrogen oxides, and carbon monoxide are also reduced when
cigarette containing only tobacco fiber is smoked compared to
cigarettes made with whole-leaf tobacco. Hydrogen cyanide is formed
from burning proteins and chlorophyll. Nitrogen oxides are formed
from burning soluble protein, chlorophyll, nitrates, and alkaloids.
These components would not be present in significant amounts in
deproteinized tobacco. Tobacco fiber has approximately 85 percent
less starches and cellulosic material thus reducing the major
pyrolytic precursors of carbon monoxide.
[0148] In another embodiment, methods are provided to produce
reconstituted tobacco that includes extracted tobacco fiber derived
from conventional tobacco, reduced-nicotine transgenic tobacco, or
increased-nicotine transgenic tobacco.
[0149] If the tobacco curing process is circumvented, virtually no
TSNAs will be present in traditional tobacco products such as
cigarettes, cigar filler or wrapper, roll-your-own tobacco for
cigarettes, pipe tobacco, chewing tobacco, snuff, reconstituted
tobacco and other preparations made with freeze-dried tobacco would
contain virtually no TSNAs since traditional curing processes are
eliminated.
[0150] In another embodiment TSNAs can be virtually eliminated
through processing freshly harvested tobacco using lyophilization.
This is accomplished by processing freshly harvested tobacco
through freeze-drying units located near tobacco farms. Tobacco
processed in this manner can be grown in a traditional fashion with
spacing of plants or in a biomass setting. In addition to the
economic advantages of eliminating the costs associated with the
curing process, the tobacco can now be grown in a biomass fashion
that can create hundreds of thousands of pounds of fresh tobacco
per acre.
[0151] By growing tobacco in a biomass setting and immediately
freeze drying the fresh tobacco for cigarettes,
roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper,
chewing tobacco, snuff, and other versions of smokeless tobacco,
labor is reduced not only by eliminating the transplant of each
plant from greenhouse to the field but also by eliminating
traditional harvesting and curing of the tobacco. Also, farmland
needed for this purpose is greatly reduced. The yield of tobacco
from one acre of tobacco grown in biomass is equivalent to
approximately 100 acres of tobacco grown in a traditional
manner.
[0152] "Tobacco biomass" is achieved by directly sowing an acre of
land with copious quantities of tobacco seed within a few inches of
each other in the field. Unlike tobacco planted with traditional
spacing, individual plants can no longer be differentiated when
tobacco is planted in a biomass fashion. An acre of tobacco biomass
has the appearance of a continuous, dense, green carpet. U.S. Pub.
Pat. App. No. 20020197688, herein expressly incorporated by
reference in its entirety, describes such methods.
[0153] Lyophilization removes most of the water (-80%) from the
weight of fresh harvested tobacco biomass. The result is Freeze
Dried Tobacco ("FDT"). FDT is easily pulverized into fine particles
suitable for processing into Recon. This Recon can be cut and made
into any type of tobacco product such as filler for cigarettes,
roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper,
chewing tobacco, snuff, and other forms of smokeless tobacco.
Flavorings and additives, including sugars, can be incorporated
into the recon process.
[0154] Such Recon can be made from 100 percent FDT or in any
proportion that consumers prefer. The lyophilization process can
have adverse affects on the taste of such tobacco products.
Therefore, FDT can even be mixed in any percentage with traditional
pulverized, cured tobacco so that the mixture can be made into
Recon. Alternatively, FDT can be mixed in any percentages with any
forms of traditional tobacco conducive for manufacturing
cigarettes, roll-your-own tobacco, pipe tobacco, and cigar filler
or wrapper, chewing tobacco, snuff and other versions of smokeless
tobacco in order to satisfy the tastes of the mass market.
[0155] In another embodiment, genetically modified reduced-nicotine
tobacco can be used for reducing TSNAs as described elsewhere
herein, thereby creating an additional benefit of such cigarettes,
roll-your-own-tobacco, pipe tobacco, cigar filler or wrapper,
chewing tobacco, snuff and other versions of smokeless tobacco
being non-addictive and without any TSNAs.
[0156] In another embodiment, nicotine can be added, in amounts
that deliver the desired physiological response, back to the FDT
for uses in cigarettes, cigar filler or wrapper, roll-your-own
tobacco for cigarettes, pipe tobacco, chewing tobacco, snuff, and
other versions of smokeless tobacco so that they will contain
virtually no TSNAs. Cigarettes produced from tobacco fiber obtained
from green leaf cured tobacco.
[0157] In another embodiment, Nicotiana rustica and/or
increased-nicotine transgenic Nicotiana tabacum are freeze dried
after harvest and are incorporated into recon. The benefits are
that the high alkaloid content is preserved for low TNR cigarettes
and that the tobacco curing step is saved. Also, the associated
increase in TSNAs with high alkaloid tobaccos will not materialize.
Preferred tobaccos for use with the methods described herein
include genetically modified tobaccos as described in the following
sections.
[0158] Curing
[0159] 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. No. 4,499,911 to
Johnson; U.S. Pat. No. 5,685,710 to Martinez Sagrera; U.S. Pat. No.
3,905,123 to Fowler; U.S. Pat. No. 3,840,025 to Fowler; and U.S.
Pat. No. 4,192,323 to Horne describe aspects of the tobacco curing
process which may be used for some embodiments provided herein.
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 TSNA tobacco
product.
[0160] "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 TSNAs.
[0161] 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.
[0162] "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.
[0163] 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.
[0164] The curing process, and most particularly the flue-curing
process, is generally divided into the following four stages:
[0165] A) Firing Up: During this step, the tobacco leaves turn
bright lemon-orange in color. This is achieved by a gradual
increase in temperature.
[0166] 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.
[0167] 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.
[0168] D) Stem Drying: The drying process continues, as the stem of
the tobacco leaf becomes dried.
[0169] 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 a tobacco product provided
herein.
[0170] Tobacco Blending
[0171] 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 below. In some
embodiments, blending of the transgenic tobacco is conducted to
prepare the tobacco so that it will contain specific amounts of
nicotine, nornicotine, sterol and/or TSNA in specific products.
Preferably, the blending is conducted so that tobacco products of
varying amounts of nicotine are made in specific products.
[0172] 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.
[0173] 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.
[0174] 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% to about
5%. The dried, expanded tobacco blend is then in a suitable mode to
be processed into the tobacco-use cessation product as described
below.
[0175] Some blending approaches begin with tobacco prepared from
varieties that have extremely low amounts of nicotine, nornicotine,
sterols 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-cured, and genetically modified
Oriental tobaccos that contain reduced amounts of nicotine and/or
TSNAs) and/or low sterol tobaccos (e.g., Burley, Flue-cured, and
Oriental). 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.
[0176] 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. By another
approach, a step 1 tobacco product is comprised of approximately
25% low sterol/PAH tobacco and 75% conventional tobacco; a step 2
tobacco product can be comprised of approximately 50% low
sterol/PAH tobacco and 50% conventional tobacco; a step 3 tobacco
product can be comprised of approximately 75% low sterol/PAH
tobacco and 25% conventional tobacco; and a step 4 tobacco product
can be comprised of approximately 100% low sterol/PAH tobacco and
0% conventional tobacco. By another approach, a step 1 tobacco
product is comprised of approximately 25% low sterol/PAH and low
nicotine/TSNA tobacco and 75% conventional tobacco; a step 2
tobacco product can be comprised of approximately 50% low
sterol/PAH and low nicotine/TSNA tobacco and 50% conventional
tobacco; a step 3 tobacco product can be comprised of approximately
75% low sterol/PAH and low nicotine/TSNA tobacco and 25%
conventional tobacco; and a step 4 tobacco product can be comprised
of approximately 100% low sterol/PAH and low nicotine/TSNA tobacco
and 0% conventional tobacco. A tobacco-use cessation kit can
comprise an amount of tobacco product from any combination 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, TSNA, sterol and/or
PAH 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, TSNA, sterol and/or PAH they individually desire. There
are many ways to obtain various low nicotine/low TSNA and/or low
sterol/low PAH tobacco blends using the tobaccos and teachings
described herein and the following is intended merely to guide one
of skill in the art to one possible approach.
[0177] To obtain a step 1 tobacco product, which is a 25% low
nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA tobacco can be mixed with conventional Burley,
Flue-cured, or Oriental in a 25%/75% ratio respectively to obtain a
Burly tobacco product having 22,500 ppm nicotine and 6,000 ppb
TSNA, a Flue-cured product having 15,000 ppm nicotine and 225 ppb
TSNA, and an Oriental product having 7,500 ppm nicotine and 75 ppb
TSNA. Similarly, to obtain a step 2 product, which is 50% low
nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA tobacco can be mixed with conventional Burley,
Flue-cured, or Oriental in a 50%/50% ratio respectively to obtain a
Burly tobacco product having 15,000 ppm nicotine and 4,000 ppb
TSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb
TSNA, and an Oriental product having 5000 ppm nicotine and 50 ppb
TSNA. Further, a step 3 product, which is a 75%/25% low
nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA tobacco can be mixed with conventional Burley,
Flue-cured, or Oriental in a 75%/25% ratio respectively to obtain a
Burly tobacco product having 7,500 ppm nicotine and 2,000 ppb TSNA,
a Flue-cured product having 5,000 ppm nicotine and 75 ppb TSNA, and
an Oriental product having 2,500 ppm nicotine and 25 ppb TSNA.
[0178] By a preferred method, conventional Virginia Flue-cured
tobacco was blended with genetically modified Burley (i.e., Burley
containing a significantly reduced amount of nicotine and TSNA) to
yield a blended tobacco that was incorporated into three levels of
reduced nicotine cigarettes: 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.
[0179] In some cigarettes, approximately, 28% of the blend was
Virginia Flue-cured 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
[0180] Recon. The amount of total TSNAs in cigarettes containing
this blend was approximately 1.5 .mu.g/g.
[0181] 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, TSNAs, sterols and PAHs will differ
from crop to crop. Nevertheless, by using conventional techniques
one can easily determine an average amount of nicotine, TSNA,
sterol and PAH 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
modified tobacco described herein, such as, for example 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, TSNA, sterol and/or PAH with other
considerations such as appearance, flavor, and smokability. In this
manner, a variety of types of tobacco products having varying level
of nicotine, TSNA, sterol and/or PAH, as well as, appearance,
flavor and smokability can be created.
[0182] A. Genetically Modified Tobacco
[0183] In some embodiments, the modified tobacco is a genetically
modified tobacco. Several approaches to create genetically modified
tobacco having a reduced amount of a harmful compound are
described. Many embodiments concern nucleic acid constructs that
inhibit the expression of a gene, which regulates production of a
compound that is associated with a tobacco-related disease. Since
these nucleic acid constructs efficiently reduce the presence of a
compound that contributes to a tobacco-related disease, the
genetically modified tobacco, prepared as described herein, can be
used to create a tobacco product, such as a cigarette, snuff or
pipe tobacco, which has a reduced potential to contribute to a
tobacco-related disease. That is, embodiments provided herein
concern reduced risk tobacco products made from reduced risk
transgenic tobacco created using the nucleic acid constructs
described herein.
[0184] More specifically, embodiments provided herein concern
nucleic acid constructs that inhibit the expression of a number of
genes involved in the synthesis and regulation of the production of
nicotine, nornicotine, and/or sterols in tobacco. Alkaloids such as
nicotine and nornicotine are precursors for a number of harmful
compounds that contribute to tobacco-related disease (e.g., the
tobacco specific nitrosamines (TSNAs): N'-nitrosonornicotine (NNN),
N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),
4-(N-nitrosomethylamino)-4-(3-pyridyl)-1-butanal
(NNA)-4-N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL),
4-N-nitrosomethylamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and/or
4-(N-nitrosomethylamino)-4-(3-pyridyl)-butanoic acid (iso-NNAC) and
acrolein). Sterols are precursors for a number of harmful
compounds, which are generated by pyrolysis of tobacco, that also
contribute to tobacco-related disease (e.g., polyaromatic
hydrocarbons (PAHs), such as benz[a]pyrene (BAP), heterocyclic
hydrocarbons, terpenes, paraffins and aromatic amines). Because the
presence of these harmful compounds in tobacco contributes to
tobacco-related disease, a transgenic or genetically modified
tobacco that comprises a reduced amount of any one of these
compounds, as compared to a reference tobacco has a reduced
potential to contribute to a tobacco-related disease.
[0185] Other embodiments concern nucleic acid constructs for
heterologous expression of a gene that reduces, or is related to
production of a compound that reduces, the harmful effect of one or
more compounds associated with a tobacco-related disease. Since
these nucleic acid constructs introduce or increase the presence of
a compound that results in reduction of the harmful effect of a
compound associated with a tobacco-related disease, the genetically
modified tobacco, prepared as described herein, can be used to
create a tobacco product, such as a cigarette, snuff or pipe
tobacco, which has a reduced potential to contribute to a
tobacco-related disease. That is, embodiments provided herein
concern reduced risk tobacco products made from reduced risk
transgenic tobacco created using the nucleic acid constructs
described herein.
[0186] Other embodiments are directed to genetically modified
tobacco in which expression of two or more genes in the
biosynthetic pathway of a compound associated with a
tobacco-related disease is inhibited. Inhibition of two or more
genes in the biosynthetic pathway of a compound associated with a
tobacco-related disease can be attained by inhibition of two or
more genes that act on a substrate at the same step in the
biosynthetic pathway (e.g., inhibition of two or more isoforms of a
biosynthetic gene) or inhibition of two or more genes that act on a
substrate at different steps in the biosynthetic pathway. In such
embodiments, the genetically modified tobacco can contain one or
more heterologous nucleic acids such as the nucleic acids and
constructs provided herein, where the heterologous nucleic acids
can contain one or more sequences that can inhibit expression of
two or more genes in the biosynthetic pathway of a compound
associated with a tobacco-related disease.
[0187] Other embodiments are directed to genetically modified
tobacco in which the active form of a gene in the biosynthetic
pathway of a compound associated with a tobacco-related disease is
inhibited. The active form of a gene in the biosynthetic pathway of
a compound associated with a tobacco-related disease can be
inhibited by any of a variety of methods for inhibiting protein
activity, including, but not limited to: knocking out part or all
of a gene encoding the endogenous protein using, for example,
homologous recombination; and heterologous expression of a dominant
negative protein that inhibits the activity of the endogenous
protein.
[0188] By using the constructs described herein, the amount of
harmful compounds in tobacco or the harmful effects thereof, such
as alkaloids and sterols, can be reduced or removed and a tobacco
product comprising this genetically modified tobacco, with or
without exogenous nicotine, will have a reduced potential to
contribute to a tobacco-related disease. That is, genetically
modified tobacco comprising the constructs described herein can be
used to manufacture "reduced risk" tobacco products (e.g., a
tobacco product comprising a reduced endogenous nicotine, reduced
endogenous nornicotine, and/or reduced sterol tobacco), such as a
cigarette, snuff or pipe tobacco, which may have exogenous nicotine
incorporated therein.
[0189] Accordingly, embodiments provided herein concern genetically
modified tobacco and tobacco products containing a tobacco that
comprises a genetic modification, which have a reduced amount or
are substantially free of a harmful compound including, but not
limited to, nicotine, nornicotine, a sterol, an acrolein, an
aldehyde, a TSNA selected from the group consisting of
N'-nitrosonornicotine (NNN),
4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK),
N'-nitrosoanatabine (NAT), and/or N'-nitrosoanabasine (NAB) or
generate a reduced amount of a PAH, a BAP, a heterocyclic
hydrocarbon, an aromatic amine upon pyrolysis, wherein this reduced
risk genetically modified tobacco is made by lowering the
expression of a gene in said tobaccos with one of the constructs
described herein. Preferred embodiments include a transgenic
tobacco and a tobacco product (e.g., cigarette) that comprises a
cured tobacco comprising a genetic modification and comprising or
delivering by FTC method a reduced amount of nicotine or total
alkaloid (e.g., below a conventional level of nicotine or total
alkaloid typical for the strain of plant, preferably, less than or
equal to 3,000 ppm, 2000 ppm, 1000 ppm, or 500 ppm), wherein said
genetic modification comprises an inhibition of a gene that
regulates the production of nicotine and/or nornicotine, such as
arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH
dehydrogenase, omithine decarboxylase (ODC),
phosphoribosylanthranilate isomerase (PRAI), putrescine
N-methyltransferase (PMT), quinolate phosphoribosyl transferase
(QPT), S-adenosyl-methionine synthetase (SAMS), or A622 or
comprises an inhibition of a gene that regulates the production of
sterol biosynthesis include HMG-CoA reductase, 14alpha demethylase,
squalene synthase, SMT2, SMT1, C14 sterol reductase,
A8-A7-isomerase, or C4-demethylase, using one or more of the
constructs described herein.
[0190] Preferred embodiments also include a transgenic tobacco and
a tobacco product (e.g., cigarette, snuff or pipe tobacco) that
comprises a cured tobacco comprising a genetic modification and a
reduced amount of a sterol (e.g., comprises an amount of sterol or
delivers and amount of sterol that is below a conventional level of
said sterol typical for the strain of plant) wherein said genetic
modification comprises an inhibition of a gene that regulates the
production of a sterol in tobacco using one or more of the
constructs described herein. Related embodiments include a
transgenic tobacco and tobacco product made therefrom (e.g., a
cigarette, snuff or pipe tobacco) that upon pyrolysis generates a
reduced amount of a PAH, BAP, a heterocyclic hydrocarbon, or an
aromatic amine, as compared to that generated by a reference
tobacco or reference tobacco product (e.g., IM16, 2R4F or 1R5F), a
commercially available tobacco product of the same class (e.g.,
full-flavor, lights, and ultra-lights), or, preferably, a tobacco
of the same variety (e.g., Burley, Virginia Flue-cured, or
Oriental) or strain (e.g., LA Burley 21, K326, Tn90, Djebel174) as
the transgenic tobacco prior to genetic modification).
[0191] Preferred embodiments also include a transgenic tobacco and
a tobacco product (e.g., cigarette, snuff or pipe tobacco) that
comprises a cured tobacco comprising a genetic modification and a
reduced amount of nicotine or total alkaloid and a sterol (e.g.,
comprise or provides an amount of nicotine or total alkaloid an/or
sterols that is below a conventional level of nicotine, total
alkaloid, or sterol typical for the strain of plant) wherein said
genetic modification comprises an inhibition of a gene that
regulates the production of both nicotine and sterols in tobacco.
That is, embodiments provided herein concern isolated nucleic
acids, isolated nucleic acid cassettes, and isolated nucleic acid
constructs that inhibit the expression of a plurality of genes that
regulate the production of nicotine and TSNAs, isolated nucleic
acids, isolated nucleic acid cassettes, and isolated nucleic acid
constructs that inhibit the expression of a plurality of genes that
regulate the production of sterols and, thus PAHs, and isolated
nucleic acids, isolated nucleic acid cassettes, and isolated
nucleic acid constructs that inhibit the expression of a plurality
of genes that regulate the production of nicotine and TSNAs and
sterols and, thus, PAHs (e.g., a double knock-out of at least two
different genes that regulate the production of at least two
different harmful compounds in tobacco).
[0192] In some embodiments, the tobacco that is substantially free
or comprises a reduced amount of nicotine, nornicotine, TSNAs,
sterols, and/or produces a reduced amount of PAHs upon pyrolysis is
made by exposing at least one tobacco cell of a selected variety
(e.g., Burley, Virginia Flue-cured, 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 or sterol 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, TSNAs and/or a
sterol 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 or
sterol biosynthesis, and a second strand that is complementary or
substantially complementary to the first strand, are
contemplated.
[0193] In some embodiments, the genetically modified tobacco
provided herein will be genetically stable for at least 2, 3, 4, 5,
6, 8, 10, 12, 15, 20, 25, 30, 40 or 50, or more, generations. For
example, the genetically modified tobacco produces a reduced amount
of a compound associated with a tobacco related disease for at
least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50, or more,
generations. It is contemplated, for example, that crossings of
multiple tobaccos each having different genetic modifications that
are stable over many generations can be performed so as to obtain a
genetically modified tobacco having a reduced level of expression
of a plurality of genes that encode precursors for various tobacco
related diseases.
[0194] In some embodiments, the genetically modified tobacco
provided herein will have agronomic characteristics suitable for
commercial production. Although in some instances genetically
modified tobacco can have agronomic characteristics that are
different from conventional tobacco, such a tobacco can be suitable
for commercial production because these different agronomic
characteristics can be compensated for by employing techniques
common to those of skill in the art. That is, although the
agronomic characteristics for a genetically modified tobacco
created as described herein may differ from those of conventional
tobacco, such alterations may not necessarily yield a plant that is
no longer suitable for commercial production. For example, a
genetically modified tobacco may have a reduced root mass, but
tobacco plants having reduced root mass can nevertheless be
suitable for commercial production when such tobaccos are raised
under conditions in which the plants are thoroughly irrigated
and/or not subjected to drought conditions. Additional nutritional
requirements (e.g., nitrogen) may be required. Any of a variety of
conventional agronomic methods can be used to produce commercial
quantities of a genetically modified tobacco, where such methods
include, but are not limited to, irrigation, fertilization,
providing nutrients for plant growth, and use of pesticides. As
referred to herein, a genetically modified tobacco that is suitable
for commercial production is a genetically modified tobacco that,
under appropriate agronomic conditions will produce at least 25%,
30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more,
tobacco useful for creation of a tobacco product relative to an
unmodified or conventional tobacco, grown under its standard
growing conditions.
[0195] 1. Genes to Modify
[0196] In some embodiments, the gene product is one that is
involved in nicotine biosynthesis. 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,
PMTase, and A622. In some embodiments, the tobacco that is made
substantially free of nicotine and/or TSNAs (e.g., comprises or
delivers 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
or Tn90), Oriental tobacco (Djebal 174), or Virginia Flue-cured
(K326) tobacco. It should be understood, however, that most tobacco
varieties 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 are used as the host for the genetic
engineering that results in the reduction of nicotine and/or TSNAs
so that the resultant transgenic plants are a Burley 21 variety
that has a reduced amount of nicotine and/or TSNAs.
[0197] Accordingly, some embodiments concern a tobacco that
comprises a genetic modification comprising a reduced amount or a
reduced level of expression of QPTase, PMTase, or A622, comprising
or delivering 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 0.5 .mu.g/g (e.g., 0.05 .mu.g/g, 0.1 .mu.g, 0.2
.mu.g/g, 0.3 .mu.g/g, 0.4 .mu.g/g, or 0.5 .mu.g/g). More
embodiments concern a tobacco that comprises or delivers a reduced
amount or a reduced level of expression of A622, a normal or
conventional amount of nicotine (e.g., comprising or delivering by
FTC methodology an amount of nicotine equal to, less than, or
greater than 0.9 mg/g, 1.0 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, and 2.0
mg/g), and a reduced amount of nornicotine (e.g., comprising or
delivering by FTC methodology an amount of nornicotine less than or
equal to 0.5 .mu.g/g), and/or a reduced amount of NNN (e.g.,
comprising or delivering by FTC methodology an amount of total
TSNAs equal to or less than 0.05 .mu.g/g, 0.1 .mu.g, 0.2 .mu.g/g,
0.3 .mu.g/g, 0.4 .mu.g/g, or 0.5 .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).
[0198] Some of the nucleic acid constructs provided herein 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 or
sterol 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 or sterol biosynthesis and a
second RNA stand that is complementary to said first strand. Some
interfering RNAs provided herein 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.
[0199] A preferred method of producing tobacco having a reduced
amount of nicotine and TSNAs, involves genetic engineering directed
at reducing the levels of nicotine and/or nornicotine or other
alkaloids. Any enzyme involved in the nicotine synthesis pathway
can be a suitable target for genetic engineering to reduce levels
of nicotine and, optionally, levels of other alkaloids including
nornicotine. Suitable targets for genetic engineering to produce
tobacco having a reduced amount of nicotine and/or nitrosamines,
especially TSNAs, include but are not limited to putrescene
N-methyltransferase, N-methylputrescene oxidase, ornithine
decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase, quinolate phosphoribosyl
transferase (QPTase) or a combination of any of the above targets.
Additionally, enzymes that regulate the flow of precursors into the
nicotine or sterol synthesis pathway are suitable targets for
genetic engineering to produce tobacco with a reduced amount of
nicotine and nitrosamines, especially TSNAs, and tobaccos with
reduced amounts of sterols, which produce a reduced amount of PAHs
upon pyrolysis. Suitable methods of genetic engineering are known
in the art and include, for example, the use of antisense and sense
suppression technology to reduce or eliminate the production of
enzymes, the use of interfering RNA molecules (gene silencing) as
described herein to reduce or eliminate the expression of gene
products, and the use of random or targeted mutagenesis to disrupt
gene function, for example, using T-DNA insertion or EMS
mutagenesis. The next section provides more description of these
techniques.
[0200] 2. Modification Methods
[0201] a) Knockouts
[0202] Inhibition of Gene Expression Using Nucleic Acids
[0203] Inhibition of gene expression refers to the absence or
observable reduction in the level of polypeptide and/or mRNA gene
product. Some embodiments provided herein relate to inhibiting the
expression of one or more genes involved in the biosynthesis of
nicotine, nornicotine, and/or sterols 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 or sterol biosynthesis.
Inhibitory nucleic acids include, but are not limited to,
interfering RNAs, antisense nucleic acids and catalytic RNAs. Some
preferred embodiments provided herein relate to interfering RNAs
(RNAi).
[0204] 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 be of any
length, however, short dsRNA constructs are commonly used. Such
constructs are known as small interfering RNAs (siRNA), and are
typically 21-23 bp in length.
[0205] 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.
[0206] 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.
[0207] 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 provided herein, a
portion of a nucleotide sequence includes the full-length coding
sequence of the gene or the target mRNA.
[0208] 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 or sterol
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 be 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.
[0209] In other embodiments provided herein, 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.
[0210] The modification of nicotine levels in tobacco plants by
antisense regulation of putrescene methyl transferase (PMTase)
expression has been proposed in U.S. Pat. Nos. 5,369,023 and
5,260,205, to Nakatani and Malik, and in PCT application WO
94/28142 and U.S. Pat. No. 5,668,295 to Wahad and Malik, which
describe DNA encoding PMT and the use of sense and antisense PMT
constructs, the entire disclosures of each of which are hereby
expressly incorporated by reference in their entireties. Other
genetic modifications proposed to reduce nicotine levels are
described in PCT application WO 00/67558, to Timko, and WO
93/05646, to Davis and Marcum; the entire contents of each are
hereby expressly incorporated by reference in their entireties.
Although these investigators made significant contributions, there
were significant drawbacks to their experimental design.
[0211] Provided herein are tobacco and tobacco products in which a
plurality of genes involved in nicotine biosynthesis are inhibited.
Most notably, it is presently revealed that there are several
different PMT genes and each may play a role in nicotine
biosynthesis. Knocking-out only one PMT gene can create a leaky
system allowing the other PMT genes to compensate for the
reduction. Accordingly, each of the PMT constructs described herein
were designed to inhibit a plurality of different PMT genes with a
single construct. 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
with one inhibitory fragment. 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.
[0212] In some embodiments that employed the A622 inhibition
construct, it was found that transgenic tobacco that had
conventional levels of nicotine but significantly reduced levels of
nornicotine were produced. This particular line of tobacco is
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.
[0213] 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 quinolate phosphoribosyltransferase
(QTPase) gene, the putrescene N-methyltransferase (PMTase) gene,
and the A622 gene. However, it will be appreciated that interfering
RNAs specific for other gene products or combinations of gene
products involved in nicotine and nornicotine biosynthesis and/or
sterol biosynthesis are contemplated. For example, additional gene
products involved in nicotine biosynthesis include, but are not
limited to, N-methylputrescene oxidase, ornithine decarboxylase,
S-adenosylmethionine synthetase, NADH dehydrogenase, and
phosphoribosylanthranilate isomerase. Additionally, it will be
appreciated that interfering RNAs specific for other gene products
or combinations of gene products involved in sterol biosynthesis
include HMG-CoA reductase, 14alpha demethylase, squalene synthase,
SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, and
C4-demethylase.
[0214] 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 sterol 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 and/or sterol 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,
nornicotine, or sterols. For example, as described below, it was
found that the A622 inhibitory fragment and inhibition cassette
(SEQ. ID. Nos. 5 and 26) 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 25) 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.
[0215] In still more embodiments, it is contemplated that a single
T-DNA containing construct be used to overexpress one gene and, in
the same construct, inhibiting expression of a second gene. That
is, some embodiments concern constructs, tobacco containing said
constructs, and tobacco products containing said tobacco, wherein
said constructs comprise an overexpression cassette that comprises
a gene that regulates the production of a compound that improves
the composition of the tobacco (e.g., overexpression of a gene
encoding an antioxidant) and, on the same construct, an inhibition
cassette that comprises an inhibitory sequence that reduces the
production of a compound that contributes to a tobacco related
disease (e.g., nicotine, nornicotine, or a sterol).
[0216] 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
provided herein, 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] Other embodiments provided herein 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] In other embodiments provided herein, 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 hairpin structure, or two antiparallel
RNA strands that form a double-stranded RNA, wherein both ends form
a hairpin structure. In some embodiments, synthetic interfering
RNAs comprise a plurality of RNA duplexes.
[0225] 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. 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 sterol
biosynthesis.
[0226] Some embodiments provided herein 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.
[0227] 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 the 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.
[0228] Specific examples of interfering nucleic acids useful in
certain embodiments of provided herein 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.
[0229] 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.
[0230] 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.
[0231] 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
incorporated by reference in its entirety. Further teaching of PNA
compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0232] In still other embodiments provided herein, 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)--OCH.sub.3,
O(CH.sub.2)--NH.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).
[0233] An embodiment provided herein includes the use of Locked
Nucleic Acids (LNAs) to 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--)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.
[0234] 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.
[0235] 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]benzoxazi-n-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 0-6
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.
[0236] 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).
[0237] 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 a 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.
[0238] 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.
[0239] 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.
[0240] 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.
[0241] Some embodiments provided herein relate to methods of
reducing or eliminating the expression of one or more target genes
involved in nicotine, nornicotine, and/or sterol biosynthesis.
Target genes that are involved in nicotine, nornicotine, and/or
sterol 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 polpypeptides. Target polypeptides involved in nicotine and
nornicotine biosynthesis include, for example, putrescene
N-methyltransferase, N-methylputrescene oxidase, ornithine
decarboxylase, S-adenosylmethionine synthetase, NADH dehydrogenase,
phosphoribosylanthranilate isomerase, and quinolate phosphoribosyl
transferase (QPTase). In a preferred embodiment, the expression of
the QPTase, PMTase, and A622 product is inhibited. Target
polypeptides involved in sterol biosynthesis include, for example,
HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2,
SMT1, C14 sterol reductase, A8-A7-isomerase, and
C4-demethylase.
[0242] Reduction of the expression of one or more target genes
and/or target gene products that are involved in nicotine,
nornicotine, and/or sterol biosynthesis leads to a reduction in the
amount of nicotine, sterols, and TSNAs produced in tobacco and PAHs
upon pyrolysis of the tobacco. In certain embodiments, the
expression of one or more target gene products involved in
nicotine, nornicotine, and/or sterol biosynthesis is eliminated.
Elimination of such target gene products can result in the
elimination of nicotine, nornicotine, and/or sterol biosynthesis,
thereby reducing the amount of nicotine, nornicotine, and/or sterol
present in tobacco to levels below the detection limit of methods
commonly used. Reduction of the amount of nicotine and 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. Similarly, the reduction
in the amount of sterol present in tobacco can lead to a reduction
in the amount of PAH generated from the tobacco upon pyrolysis. In
some embodiments, the amount of PAH present in tobacco is reduced
to levels below the detection limit of methods commonly used to
detect PAH.
[0243] The reduction in or elimination of the expression of target
genes or target gene products involved in nicotine, nornicotine,
and/or sterol 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.
[0244] 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 provided herein, 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. In some
embodiments provided herein, 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 sterol biosynthesis and a second strand complementary or
substantially complementary to the first strand.
[0245] The reduction in or elimination of the expression of genes
and/or gene products involved in nicotine, nornicotine, and/or
sterol biosynthesis can be characterized by comparing the amount of
nicotine, nornicotine, and/or sterol produced in genetically
modified cells, with the amount of nicotine, nornicotine, and/or
sterol 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%, 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 in 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 provided herein.
[0246] b) Transgenics
[0247] Transgenic Plant Cells and Plants
[0248] Embodiments provided herein 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, nornicotine,
and/or sterol 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, nornicotine, and/or sterol
biosynthesis. The RNA duplex also comprises a second strand that is
complementary or substantially complementary to the first
strand.
[0249] 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 embodiments
provided herein 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, sterol and/or TSNA and/or generate
a reduced amount of PAHs upon pyrolysis, as compared to unmodified
or control tobacco cells and plants.
[0250] 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 TSNAs. These blended tobacco products can be used in tobacco
product cessation programs so as to slowly move a consumer from a
high nicotine and/or sterol product to a low nicotine and/or sterol
product. Some embodiments provided herein comprise a tobacco use
cessation kit, comprising two or more tobacco products with
different levels of nicotine. For example, a smoker can begin the
program smoking blended cigarettes having or delivering 0.6 mg of
nicotine, gradually move to smoking cigarettes having or delivering
0.3 mg of nicotine, followed by cigarettes having or delivering
less than 0.1 mg nicotine until the consumer decides to quit
smoking altogether. Accordingly, the blended cigarettes described
herein provide the basis for an approach to reduce the exposure of
a tobacco consumer to a tobacco related disease in a step-wise
fashion. The components of the tobacco use cessation kit described
herein may include other tobacco products, including but not
limited to, smoking materials (e.g., cigarettes, cigars, pipe
tobacco), snuff, chewing tobacco, gum, and lozenges.
[0251] 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.
[0252] 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.
[0253] In some embodiments, the heterologous sequence utilized in
the methods provided 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 QTPase mRNA
sequence, or to a portion thereof, and a second strand that is
complementary to the entire QPTase 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.
[0254] 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 provided herein 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 provided herein. 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.
[0255] 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.
[0256] 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.
[0257] 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.
[0258] Nucleic acid constructs, or "transcription cassettes,"
encoding the interfering RNAs that are used to produce the
transgenic cells and plants provided herein 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).
[0259] The term "operatively associated," as used herein, refers to
nucleotide sequences on a single nucleic acid molecule that are
associated so that the function of one sequence 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 nucleic acid 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.
[0260] In some embodiments, 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), provide
complementation by imparting prototrophy to an auxotrophic host
and/or provide a visible phenotype through the production of a
novel compound in the plant.
[0261] The various fragments comprising the various constructs,
transcription cassettes, markers and the like may be introduced
consecutively by restriction enzyme cleavage of an appropriate
replication system and insertion of the particular construct or
fragment into the available site. After ligation and cloning, the
DNA construct may be isolated for further manipulation. All of
these techniques are amply exemplified in the literature as
demonstrated by J. Sambrook et al., Molecular Cloning, A Laboratory
Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).
[0262] Vectors that may be used to transform plant tissue with
nucleic acid constructs provided herein include Agrobacterium and
Transbacter vectors and ballistic vectors, as well as vectors
suitable for DNA-mediated transformation. In this particular
embodiment, the promoter is a region of a DNA sequence that
incorporates the necessary signals for the efficient expression of
the coding sequence. This region may include sequences to which an
RNA polymerase binds, but is not limited to such sequences, and may
include sequences to which other regulatory proteins bind along
with sequences involved in the control of protein translation. Such
regions may also include coding sequences.
[0263] 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.
[0264] 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)).
[0265] Use of the CaMV 35S promoter for expression of interfering
RNAs in the transformed tobacco cells and plants provided herein 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 provided herein. 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.
[0266] The recombinant interfering nucleic acid molecules and
vectors used to produce the transformed tobacco cells and plants
provided herein 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.
[0267] Transformed tobacco cells are selected out of the
surrounding population of non-transformed cells by placing the
mixed population of cells into a culture medium containing an
appropriate concentration of the antibiotic (or other compound
normally toxic to tobacco cells) against which the chosen dominant
selectable marker gene product confers resistance. Thus, only those
tobacco cells that have been transformed will survive and multiply.
Additionally, the positive selection techniques described by
Jefferson (e.g., WO 00055333; WO 09913085; U.S. Pat. Nos.
5,599,670; 5,432,081; and 5,268,463, hereby expressly incorporated
by reference in their entireties) can be used.
[0268] Methods of making recombinant plants provided herein, 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 an
interfering RNA or a nucleic acid construct encoding an interfering
RNA comprising a transcription cassette provided herein (as
described above) 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.
[0269] Numerous Agrobacterium vector systems useful in carrying out
the present invention are known. For example, U.S. Pat. No.
4,459,355 discloses a method for transforming susceptible plants,
including dicots, with an Agrobacterium strain containing the Ti
plasmid. The transformation of woody plants with an Agrobacterium
vector is disclosed in U.S. Pat. No. 4,795,855. Further, U.S. Pat.
No. 4,940,838 to Schilperoort et al. discloses a binary
Agrobacterium vector (i.e., one in which the Agrobacterium contains
one plasmid having the vir region of a Ti plasmid but no T region,
and a second plasmid having a T region but no vir region) useful in
carrying out the present invention, all references are hereby
expressly incorporated by reference in their entireties.
[0270] Microparticles suitable for the ballistic transformation of
a plant cell, carrying a nucleic acid construct provided herein,
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.
[0271] Plant species may be transformed with the interfering RNA or
nucleic acid construct encoding an interfering RNA provided herein
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)).
[0272] 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).
[0273] 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.
[0274] 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.
[0275] 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. 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.
[0276] 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.
[0277] 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.
Media and Solutions for Tobacco Transformation:
TABLE-US-00001 [0278] 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
[0279] Keep poured plates for 2 days at room temperature to
visualize any contamination, then store at 4.degree. C.
RMOP+RMOP-TCH Media
[0280] (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).
1 L Final Conc.
TABLE-US-00002 [0281] Sucrose 30 g (3%) Myo-inositol 100 mg (0.1%)
MS Macro 10 .times. 100 mL (1x) MS Micro 1000 .times. 1 mL (1x)
Fe2EDTA Iron 100 .times. 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)
[0282] 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)
[0283] Dissolve 100 mg NAA in 1 mL absolute ethanol. Add 3 mL 1N
KOH. Make up to 80 mL with Milli-Q H2O. Adjust pH to 6.0 with 1N
HCl, make up to 100 mL with Milli-Q H2O, and store at 4.degree.
C.
Cefotaxamine (250 mg/ml)
[0284] Add 8 ml sterile Milli-Q H2O to 2 g Claforan and store at
4.degree. C. in dark
Timentin (200 mg/ml)
[0285] Add 15 ml sterile Milli-Q H2O to 3 g Timentin and store at
4.degree. C.
MST+MST-TCH Media
[0286] (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).
1 L Final Concentration
TABLE-US-00003 [0287] Sucrose 30 g (3%) MS Macro 10 .times. 100 mL
(1x) MS Micro 1000 .times. 1 mL (1x) Fe2EDTA Iron 100 .times. 10mL
(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)
MS Macro 10.times. ((Murashige and Skoog., Phys. Plant. 15: 473-497
(1962), herein expressly incorporated by reference in its
entirety)).
Final Concentration
TABLE-US-00004 [0288] 10x (g/L) KNO3 19.0 NH4 N03 16.5
CaCl2.cndot.2H2O 4.4 MgS04.cndot.7H2O 3.7 KH2PO4 1.7 Store
4.degree. C.
Substituting Chemicals:
CaCl2 3.3 g/L
MgSO4 1.8 g/L
MS Micro 1000.times.
[0289] (Murashige and Skoog., Phys. Plant. 15: 473-497 (1962),
herein expressly incorporated by reference in its entirety).
Final Concentration
TABLE-US-00005 [0290] 1000x (g/L) MnS04.cndot.4H20 22.3
ZnS04.cndot.7H20 8.6 H3BO3 6.2 KI 0.83 Na2MoO4.cndot.2H2O 0.25
CuSO4.cndot.5H2O 25 mg CoCl2.cndot.6H2O 25 mg Store 4.degree.
C.
Substituting Chemicals:
MnSO4.H20 16.9/L
FeSO4EDTA Iron 100.times.
TABLE-US-00006 [0291] (g/1 L ) FeS04.cndot.7H20 2.78 Na2EDTA 3.72
Store 4.degree. C. in dark bottle
[0292] 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.
[0293] 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.
[0294] Any plant tissue capable of subsequent clonal propagation,
whether by organogenesis or embryogenesis, may be transformed with
a nucleic acid embodiment provided herein. Preferred plants for
introduction of a nucleic acid embodiment, described herein,
include Nicotiana. Preferred varieties of Nicotiana 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 Flu One Newest Varieties
Varieties Cured Other Virginia Hybrid Sucker Varieties Oriental KT
200 BLACK K 149 CU BROWN NBH OS400 GL 350 D174 LC MAMMOTH 748 LEAF
98 KT 204 DF 485 K 326 GL LIZARD MS KY Izmir LC 737 TAIL 21xKY 160
ORNOCO 10 KY DF 911 K346 OX LIZARD MS 207 TAIL 14xKY TURTLE L8 FOOT
KY 10 DT 508 K 394 PVH M and N TN 97 03 KY 14 DT 518 K730 PVH
SHIREY KT 200 09 KY 17 DT 592 Coker PVH WALKER 371 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 K358 RS 1410 MS KY 171 K399 Speight Burley 168 21
x KY 10 MS LITTLE NC 102 Speight KY14 x CRITTENDEN 179 L8 N126
LITTLE NC 291 Speight WOOD 190 N 777 NARROW NC 297 Speight LEAF 196
MADOLE 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 TN D950 NC
72 Speight LC 220 TN 90 TR MADOLE NC 810 Speight H-20 TN 90 VA 309
RGH 4 Speight LC H-6 TN 97 VA 312 RGH 51 Speight LC NF-3 VA 509 VA
355 VA 119 LA21 VA 359 NC 37 NF OX 414 NF Sp. G- 172
[0295] 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).
[0296] Plants provided herein 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.
[0297] As used herein, a crop comprises a plurality of plants
provided herein, and of the same genus, planted together in an
agricultural field. By "agricultural field" is meant a common plot
of soil or a greenhouse. Thus, the present invention provides a
method of producing a crop of plants having reduced amounts of
nicotine, nornicotine, and/or sterol, as compared to a similar crop
of non-transformed plants of the same species and variety.
[0298] 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.
[0299] 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).
[0300] 3. Exemplary Constructs
[0301] Several embodiments concern isolated nucleic acids that
comprise, consist, or consist essentially of the nucleic acids
described in the sequence listing (SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49 or 50) and fragments thereof at
least 30 consecutive nucleotides in length. That is, embodiments
provided herein include an isolated nucleic acid comprising,
consisting of, consisting essentially of, any one or more of the
sequences of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49 or 50, 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.: 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49 or 50.
[0302] In preferred embodiments, the target gene or target mRNA
encodes QTPase, 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 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 NOS: 2, 3, 4, 5, 39 or 40, and inhibits the
production of QTPase, 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: 5, and inhibits production of nornicotine but not nicotine in a
tobacco. In still more 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: 6, 7, 8, or 9,
and inhibits production of at least one sterol (e.g., squalene
synthase, HMG-CoA reductase, SMT2, or 14alpha demethylase) in a
tobacco and at least one PAH upon pyrolysis of said tobacco.
[0303] 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). Some of these
nucleic acids concern fragments of genes involved in sterol
biosynthesis (e.g., squalene synthase, HMG-CoA reductase, SMT2, or
14alpha demethylase) and these fragments are particularly useful
for inhibiting production of sterols in tobacco and PAHs when said
tobacco undergoes pyrolysis.
[0304] 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.: 10, 11, or 12) 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.:10, 11, or 12 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.
[0305] 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 the RD2 promotor (SEQ. ID NO.
37 or SEQ. ID NO. 50), truncated RD2 promoter (SEQ. ID NO. 13) or
the Putrescene methyl transferase promoter (PMT-1) (SEQ. ID NO.
14). Constitutive promoters that can be used with embodiments
described herein include the GapC promoter (SEQ. ID. NO.: 15),
Actin 2 promoter (Act2P) (SEQ. ID NO. 16), the tobacco alcohol
dehydrogenase promoter (ADP) (SEQ. ID NO. 17), the Arabidopsis
ribosomal protein L2 promoter (RPL2P) (SEQ. ID NO. 18), and the
nopaline synthase promoter (NOS P) (SEQ ID NO. 46). Developmentally
regulated promoters that can be used with the nucleic acid
sequences described herein include the cinnamyl alcohol
dehydrogenase promoter (SEQ. ID NO. 19) and the metallothionein I
promoter (SEQ. ID NO. 20). Additional embodiments also include
isolated nucleic acids that comprise, consist, or consist
essentially of the GAD2 terminator (SEQ. ID NO. 21), nopaline
synthase terminator (NOS T) (SEQ ID NO 38), a FAD2 intron (provided
by (SEQ. ID NO. 22), ACT11 intron 3 (SEQ ID NO 41), 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. 33). 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, embodiments
provided herein 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, nornicotine, or a sterol).
[0306] Embodiments provided herein also concern isolated nucleic
acids that comprise, consist, or consist essentially of the
inhibition and selection cassettes identified as SEQ. ID. Nos. 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 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. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50).
[0307] Embodiments provided herein 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. 27). Another double knock-out construct
comprises, consists of or consists essentially of a first isolated
nucleic acid that inhibits nicotine biosynthesis (e.g., A622) and a
second isolated nucleic acid that inhibits synthesis of at least
one sterol (e.g., SMT2). (See (SEQ. ID. No. 33)). Accordingly,
embodiments provided herein concern an isolated nucleic acid
construct that inhibits the expression of a plurality of genes that
regulate the production of more than one harmful compound in
tobacco. In some aspects of these embodiments, said isolated
nucleic acid construct inhibits the expression of at least two
nicotine biosynthesis genes, a nicotine biosynthesis gene and a
sterol biosynthesis gene, or two sterol biosynthesis genes. It
should also be understood that embodiments provided herein 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
and a transgenic tobacco having a reduced amount of a sterol.
Crossings of the transgenic tobacco described herein and wild-type
tobacco are also embodiments provided herein.
[0308] 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 provided herein 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.
[0309] Still other embodiments provided herein 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 or sterol 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.
[0310] Certain embodiments provided herein relate to tobacco plants
and cured tobacco products having a reduced amount or nicotine,
nornicotine, TSNAs, and/or sterols. In such embodiments, reduction
in nicotine, nornicotine, TSNAs, and/or sterol 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 provided
herein.
[0311] 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 provided herein
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.
[0312] Some embodiments provided herein relate to methods of
preparing a tobacco cell having a reduced nicotine and/or sterol
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 sterol
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 QTPase, PMTase, or A622 and the target gene product
involved in sterol biosynthesis is squalene synthase, HMG-CoA
reductase, SMT2, or 14alpha demethylase.
[0313] Other embodiments provided herein relate to methods of
preparing a tobacco plant having a reduced nicotine and/or sterol
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 and/or sterol 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 and/or sterol 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 and/or produce a reduced amount of PAHs upon pyrolysis, 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.
[0314] As mentioned above, additional embodiments include tobacco
products that have been carefully blended so that desired levels of
nicotine, TSNAs, and/or sterols are obtained. For example, tobacco
having a reduced level of nicotine and/or TSNAs, prepared as
described above, can be blended with conventional tobacco so as to
obtain virtually any amount of nicotine and/or sterols.
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 in or delivered by the product (e.g.,
as measured by FTC methodology) at less than 7,000 ppm, 5,000 ppm,
3000 ppm, 2000 ppm, 1000 ppm, or 500 ppm and TSNA levels at 0.5
.mu.g/g or less. Similarly, two or more varieties of transgenic
tobacco having a reduced amount of sterols can be blended, as
above, or varieties of sterol-reduced transgenic tobacco can be
blended with varieties of nicotine reduced transgenic tobacco. In
this manner, differences in variety, flavor, as well as amounts of
nicotine and/or sterols can be incrementally adjusted. These
blended tobaccos can be processed into tobacco products, which can
be incorporated into tobacco use cessation kits (e.g., a multiple
step nicotine reduction program, whereby a consumer's exposure to
nicotine, TSNA, or PAH is gradually reduced over time by
consumption of tobacco products that have increasingly smaller
quantities of these compounds). Such kits and programs, are
designed to reduce or eliminate nicotine dependence and reduce the
potential to contribute to a tobacco related disease.
[0315] More embodiments concern methods to reduce the carcinogenic
potential of tobacco products, including cigarettes, cigars,
chewing tobacco, snuff and tobacco-containing gum and lozenges.
Some methods, for example involve the use of the constructs
described herein to obtain transgenic tobacco that comprises a
reduced amount of nicotine, TSNAs, and/or sterols and the
manufacture of tobacco products containing said tobacco.
Accordingly, the transgenic tobacco plants, described above, are
harvested, cured, and processed into tobacco products. These
tobacco products have a reduced carcinogenic potential because they
are prepared from tobacco that has a reduced amount of nicotine,
TSNAs, and sterols. Smoke or smoke condensate generated from these
tobaccos and tobacco products can also be evaluated using the
assays provided herein so as to confirm that said tobaccos and
tobacco products have a reduced potential to contribute to a
tobacco-related disease and that said tobaccos and tobacco products
are reduced risk compositions.
[0316] Yet another aspect provided herein concerns the reduction of
the amount of TSNAs, preferably NNN and NNK, and polyaromatic
hydrocarbons (PAHs), preferably, benz[a]pyrene and metabolites
thereof in humans who smoke, consume or otherwise ingest tobacco.
This method is practiced by providing a tobacco product comprising
a transgenic tobacco that comprises a reduced amount of nicotine
and/or a sterol to said humans, thereby lowering the amount of
TSNAs and/or PAHs in said humans exposed to said tobacco product.
By one approach, for example, the carcinogenic potential of side
stream or main stream tobacco smoke in a human exposed to said side
stream or main stream tobacco smoke is reduced by providing the
cured tobacco as described above in a product that undergoes
pyrolysis, wherein pyrolysis of said product results in side stream
or main stream smoke comprising a reduced amount of TSNAs and/or
PAHs. The section below describes several preferred approaches to
develop genetically modified tobaccos and tobacco products
containing genetically modified tobacco that have a reduced amount
of a compound that contributes to a tobacco related disease.
[0317] Preparation of Preferred Transgenic Tobaccos
[0318] 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. 2) 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)).
[0319] 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.
[0320] 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.
[0321] 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 (-70 ppm), and this transformant was
designated as "Vector 21-41."
[0322] 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.
[0323] 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.
[0324] 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.
[0325] 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).
[0326] 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. 23). 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. 34). 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 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.
Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco
plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or
Oriental comprising this RNAi construct are embodiments provided
herein.
[0327] FIG. 2 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a QTPase inhibition cassette (SEQ. ID. No. 24)
and a norflurazone selection cassette (SEQ. ID. No. 35). 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 QTPase gene,
joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense
fragment of the QTPase 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).
Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco
plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or
Oriental comprising this RNAi construct are embodiments provided
herein.
[0328] 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.
[0329] 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.
[0330] Oriental tobacco will be transformed with the construct
shown in FIG. 2 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. 2 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.
[0331] 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. 25) and a
norflurazone selection cassette (SEQ. ID. No. 35). 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). Accordingly,
tobacco products (e.g., cigarettes), tobacco, tobacco plants,
tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental
comprising this RNAi construct are embodiments provided herein.
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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. 26) and a
norflurazone selection cassette (SEQ. ID. No. 35). 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). Accordingly,
tobacco products (e.g., cigarettes), tobacco, tobacco plants,
tobacco cells, tobacco seeds, in Burley, Flue-cured or Oriental
comprising this RNAi construct are embodiments provided herein.
[0336] 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.
[0337] 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) VDG 2486.53 0.00
2.30 020 VDG 4683.01 0.00 3.48 032 VDG 4490.79 0.00 3.94 045 VDG
2855.58 0.00 2.61 052 VDG 2291.89 0.00 2.17 054 VDG 4857.86 0.06
3.99 077 VDG 3072.40 0.00 2.58 097 VDG 4921.31 0.03 3.59 107 VDG
4960.64 0.00 3.56 116 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.
[0338] Tobacco products containing the selectively reduced
nornicotine transgenic tobacco described above are also embodiments
provided herein. That is, tobacco products comprising a transgenic
tobacco that comprises a conventional amount of nicotine (e.g.,
comprise or delivers according to FTC methodology 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 provided herein.
Particularly preferred are transgenic tobacco and tobacco products
made therefrom, which comprise a conventional amount of nicotine
(e.g., comprises or delivers by FTC methodology 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. 5, or the
cassette of SEQ. ID. No. 26.
[0339] 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.
[0340] 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.
[0341] 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.27) and a norflurazone selection
cassette (SEQ. ID. No. 35). 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). Accordingly, tobacco products (e.g.,
cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds,
in Burley, Flue-cured or Oriental comprising this RNAi construct
are embodiments provided herein.
[0342] Flue-cured 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
Flue-cured 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.
[0343] 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.
[0344] 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.
[0345] More embodiments concern an RNAi construct designed to
reduce the amount of sterols in tobacco and thereby reduce
production of a PAH upon pyrolysis of said transgenic tobacco. A
first sterol-reducing RNAi construct has a 14alpha demethylase
inhibition cassette (SEQ. ID. No. 28). The 14alpha demethylase
inhibition cassette comprises a double (two promoters in tandem)
35S promoter (Bp 1-618) operably linked to an antisense 14alpha
demethylase nucleic acid (Bp 619-1503), which is joined to a FAD2
intron (Bp 1504-2634), which is joined to a sense nucleic acid of
the 14alpha demethylase gene (Bp 2635-3519), which is joined to the
Nos terminator (Bp 3520-3773). Accordingly, tobacco products (e.g.,
cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds,
in Burley, Flue-cured or Oriental comprising this RNAi construct
are embodiments provided herein.
[0346] Flue-cured tobacco will be transformed with the 14alpha
demethylase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Flue-cured tobacco that will be
created using the construct above will have significantly reduced
levels of sterol and will generate significantly less PAHs upon
pyrolysis, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
[0347] Burley tobacco will be transformed with the 14alpha
demethylase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0348] Oriental tobacco will be transformed with the 14alpha
demethylase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Oriental tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0349] More embodiments concern another RNAi construct designed to
reduce the amount of a sterol in tobacco and thereby reduce
production of a PAH upon pyrolysis of said transgenic tobacco. A
second sterol-reducing RNAi construct has a SMT2 inhibition
cassette (SEQ. ID. No. 29). The SMT2 inhibition cassette comprises
a double (two promoters in tandem) 35S promoter (Bp 1-618) operably
linked to an antisense SMT2 nucleic acid (Bp 619-1398), which is
joined to a FAD2 intron (Bp 1399-2529), which is joined to a sense
nucleic acid of the SMT2 gene (Bp 2530-3309), which is joined to
the Nos terminator (Bp 3310-3563). Accordingly, tobacco products
(e.g., cigarettes), tobacco, tobacco plants, tobacco cells, tobacco
seeds, in Burley, Flue-cured or Oriental comprising this RNAi
construct are embodiments provided herein.
[0350] Flue-cured tobacco will be transformed with the SMT2
inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Flue-cured tobacco that will be
created using the construct above will have significantly reduced
levels of sterol and will generate significantly less PAHs upon
pyrolysis, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
[0351] Burley tobacco will be transformed with the SMT2 inhibition
cassette 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 sterol content. It is expected that approximately 50% of
the lines tested will have significantly less sterol than the
parent strain of tobacco. Accordingly, it is expected that the
transgenic Burley tobacco that will be created using the construct
above will have significantly reduced levels of sterol and will
generate significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0352] Oriental tobacco will be transformed with the SMT2
inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Oriental tobacco that will be created
using the construct above will have significantly reduced levels of
sterols and will generate significantly less PAHs upon pyrolysis,
as compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0353] More embodiments concern another RNAi construct designed to
reduce the amount of a sterol in tobacco and thereby reduce
production of a PAH upon pyrolysis of said transgenic tobacco. A
third sterol-reducing RNAi construct has a squalene synthase
inhibition cassette (SEQ. ID. No. 30). The squalene synthase
inhibition cassette comprises a double (two promoters in tandem)
35S promoter (Bp 1-618) operably linked to an antisense squalene
synthase nucleic acid (Bp 619-1057), which is joined to a FAD2
intron (Bp 1058-2188), which is joined to a sense nucleic acid of
the squalene synthase gene (Bp 2189-2627), which is joined to the
Nos terminator (Bp 2628-2881). Accordingly, tobacco products (e.g.,
cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds,
in Burley, Flue-cured or Oriental comprising this RNAi construct
are embodiments provided herein.
[0354] Flue-cured tobacco will be transformed with the squalene
synthase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Flue-cured tobacco that will be
created using the construct above will have significantly reduced
levels of sterol and will generate significantly less PAHs upon
pyrolysis, as compared to a conventional tobacco, a reference
tobacco, or the parental strain of tobacco prior to genetic
modification.
[0355] Burley tobacco will be transformed with the squalene
synthase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0356] Oriental tobacco will be transformed with the squalene
synthase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Oriental tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0357] More embodiments concern yet another RNAi construct designed
to reduce the amount of a sterol in tobacco and thereby reduce
production of a PAH upon pyrolysis of said transgenic tobacco. A
fourth sterol-reducing RNAi construct has a HMG-CoA reductase
inhibition cassette (SEQ. ID. No. 31). The HMG-CoA reductase
inhibition cassette comprises a double (two promoters in tandem)
35S promoter (Bp 1-618) operably linked to an antisense HMG-CoA
reductase nucleic acid (Bp 619-1468), which is joined to a FAD2
intron (Bp 1469-2599), which is joined to a sense nucleic acid of
the HMG-CoA reductase gene (Bp 2600-3449), which is joined to the
Nos terminator (Bp 3450-3703). Accordingly, tobacco products (e.g.,
cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds,
in Burley, Flue-cured or Oriental comprising this RNAi construct
are embodiments provided herein.
[0358] Flue-cured tobacco (K326) was transformed with the HMG-CoA
reductase inhibition cassette using Agrobacterium-mediated
transformation and independent lines were selected, regenerated,
and transplanted in the greenhouse. Several independent lines grown
in the greenhouse were harvested and tested for the presence of
various sterols (see Table 3). As shown in the table, several lines
(e.g., HMGIR 1, HMGIR 2, HMGIR 3-2, HMGIR 4, HMGIR 7, HMGIR 11,
HMGIR 13, HMGIR 16, HMGIR 18, HMGIR 19) were found to have
significantly reduced levels of sterols, as compared to the
parental strain of tobacco (i.e., tobacco of the same variety prior
to genetic modification). Accordingly, embodiments include
transgenic tobacco and tobacco products made therefrom comprising a
reduced amount of sterols, as compared to a tobacco of the same
variety, parental strain or a tobacco that has not been genetically
modified. It is expected that the transgenic Flue-cured tobacco
that was created using the construct above will generate
significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
TABLE-US-00009 TABLE 3 HmgCoa Reductase inhibition K326 HMGIR HMGIR
HMGIR HMGIR HMGIR HMGIR HMGIR HMGIR HMGIR HMGIR cont 1 2 3-2 4 7 11
13 16 18 19 Squalene 1 1.47 0.90 1.96 2.64 1.00 1.25 1.21 0.72 0.90
0.75 Squalene 1 1.48 0.88 2.13 2.78 0.94 1.14 1.12 0.97 0.73 0.96
Tocopherol 1 1.67 2.02 1.15 1.40 1.13 1.69 1.15 1.36 1.48 1.13
Tocopherol 1 1.73 2.08 1.33 1.34 0.84 1.54 0.88 1.05 1.11 0.87
Campesterol 1 0.74 1.13 0.47 0.60 0.76 0.75 0.83 0.90 1.20 1.21
Stigmasterol 1 0.45 1.00 0.34 0.30 0.50 0.55 0.65 0.85 1.42 1.27
Sitosterol 1 0.84 0.59 0.69 0.92 0.86 0.93 1.01 0.76 0.83 0.84
*Highlighted entries indicate transgenic tobacco lines having a
reduction in sterols
[0359] Burley tobacco will be transformed with the HMG-CoA
reductase cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0360] Oriental tobacco will be transformed with the HMG-CoA
reductase inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Oriental tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0361] More embodiments concern still another RNAi construct
designed to reduce the amount of a sterol in tobacco and thereby
reduce production of a PAH upon pyrolysis of said transgenic
tobacco. A fifth sterol-reducing RNAi construct has a
developmentally regulated SMT2 inhibition cassette (SEQ. ID. No.
32). The developmentally regulated SMT2 inhibition cassette
comprises a cinnamyl alcohol dehydrogenase promoter (Bp 1-995)
operably linked to an antisense SMT2 nucleic acid (Bp 996-1775),
which is joined to a PAP 1 intron (Bp 1776-2955), which is joined
to a sense nucleic acid of the SMT2 gene (Bp 2956-3735), which is
joined to the RuBisCo small subunit terminator (Bp 3736-4286).
Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco
plants, tobacco cells, tobacco seeds, in barley, Flue-cured or
Oriental comprising this RNAi construct are embodiments provided
herein.
[0362] Flue-cured tobacco will be transformed with the
developmentally regulated SMT2 inhibition cassette 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 sterol
content. It is expected that approximately 50% of the lines tested
will have significantly less sterol than the parent strain of
tobacco. Accordingly, it is expected that the transgenic Flue-cured
tobacco that will be created using the construct above will have
significantly reduced levels of sterol and will generate
significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0363] Burley tobacco will be transformed with the developmentally
regulated SMT2 inhibition cassette 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 sterol content. It is expected
that approximately 50% of the lines tested will have significantly
less sterol than the parent strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct above will have significantly reduced levels of
sterol and will generate significantly less PAHs upon pyrolysis, as
compared to a conventional tobacco, a reference tobacco, or the
parental strain of tobacco prior to genetic modification.
[0364] Oriental tobacco will be transformed with the
developmentally regulated SMT2 inhibition cassette 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 sterol
content. It is expected that approximately 50% of the lines tested
will have significantly less sterol than the parent strain of
tobacco. Accordingly, it is expected that the transgenic Oriental
tobacco that will be created using the construct above will have
significantly reduced levels of sterol and will generate
significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0365] FIG. 6 illustrates a double-knock-out RNAi construct that
can be used to create a reduced nicotine, TSNA, sterol transgenic
tobacco that generates a reduced amount of PAH upon pyrolysis. This
double-knock-out RNAi construct has a A622/SMT2 inhibition cassette
(SEQ. ID. No. 33) and a norflurazone selection cassette (SEQ. ID.
No. 35). Starting from the right border (RB), the A622/SMT2
inhibition cassette comprises an RD2 promoter (Bp 1-2010) operably
linked to a A622 antisense nucleic acid (628 bp) (Bp 2011-2638) of
a A622 gene, which is joined to a FAD2 intron (Bp 2639-3769), which
is joined to a sense nucleic acid of the A622 gene (628 bp)
(Bp3770-4397), which is joined to the GAD2 terminator (Bp
4398-4670); which is joined to a cinnamyl alcohol dehydrogenase
promoter (Bp 4671-5665) operably linked to an antisense SMT2
nucleic acid (Bp 5666-6445), which is joined to a PAP 1 intron (Bp
6446-7625), which is joined to a sense nucleic acid of the SMT2
gene (Bp 7626-8405), which is joined to the RuBisCo small subunit
terminator (Bp 8406-8956). Accordingly, tobacco products (e.g.,
cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds,
in Burley, Flue-cured or Oriental comprising this RNAi construct
are embodiments provided herein.
[0366] Flue-cured tobacco will be transformed with the construct
shown in FIG. 6 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 and sterol content. It is expected that
approximately 50% of the lines tested will have less than 1,000 ppm
total alkaloid and a reduced amount of sterols, as compared to the
parental strain of tobacco, and approximately 10% of the lines
tested will have less than 500 ppm total alkaloid and a reduced
amount of sterols, as compared to the parental strain of tobacco.
Accordingly, it is expected that the transgenic Flue-cured tobacco
that will be created using the construct shown in FIG. 6 will have
significantly reduced levels of nicotine, TSNA, sterol, and will
generate significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0367] Burley tobacco will be transformed with the construct shown
in FIG. 6 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 and sterol content. It is expected that
approximately 50% of the lines tested will have less than 1,000 ppm
total alkaloid and a reduced amount of sterols, as compared to the
parental strain of tobacco, and approximately 10% of the lines
tested will have less than 500 ppm total alkaloid and a reduced
amount of sterols, as compared to the parental strain of tobacco.
Accordingly, it is expected that the transgenic Burley tobacco that
will be created using the construct shown in FIG. 6 will have
significantly reduced levels of nicotine, TSNA, sterol, and will
generate significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0368] Oriental tobacco will be transformed with the construct
shown in FIG. 6 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 and sterol content. It is expected that
approximately 50% of the lines tested will have less than 1,000 ppm
total alkaloid and a reduced amount of sterols, as compared to the
parental strain of tobacco, and approximately 10% of the lines
tested will have less than 500 ppm total alkaloid and a reduced
amount of sterols, as compared to the parental strain of tobacco.
Accordingly, it is expected that the transgenic Oriental tobacco
that will be created using the construct shown in FIG. 6 will have
significantly reduced levels of nicotine, TSNA, sterol, and will
generate significantly less PAHs upon pyrolysis, as compared to a
conventional tobacco, a reference tobacco, or the parental strain
of tobacco prior to genetic modification.
[0369] FIG. 7 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a QTPase inhibition cassette (SEQ. ID. No. 42)
and a norflurazone selection cassette (SEQ. ID. No. 35). 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 QTPase gene,
joined to a FAD2 intron (Bp 2371-3501), which is joined to a sense
fragment of the QTPase gene (360 bp) (Bp 3502-3861), which is
joined to the nopaline synthase (NOS) terminator (Bp 3862-4115).
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). Accordingly, tobacco products (e.g., cigarettes),
tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley,
Flue-cured or Oriental comprising this RNAi construct are
embodiments provided herein.
[0370] Flue-cured tobacco will be transformed with the construct
shown in FIG. 7 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,
as compared to the parental strain of tobacco, and approximately
10% of the lines tested will have less than 500 ppm total alkaloid,
as compared to the parental strain of tobacco. Accordingly, it is
expected that the transgenic Flue-cured tobacco that will be
created using the construct shown in FIG. 7 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.
[0371] Burley tobacco will be transformed with the construct shown
in FIG. 7 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,
as compared to the parental strain of tobacco, and approximately
10% of the lines tested will have less than 500 ppm total alkaloid,
as compared to the parental strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct shown in FIG. 7 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.
[0372] Oriental tobacco will be transformed with the construct
shown in FIG. 7 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. 7 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.
[0373] FIG. 8 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a QTPase inhibition cassette (SEQ. ID. No. 43)
and a norflurazone selection cassette (SEQ. ID. No. 35). Starting
from the right border (RB), the QPTase inhibition cassette
comprises a PMTase1 promoter (Bp 1-711) operably linked to an
antisense fragment (360 bp) (Bp 712-1071) of the QTPase gene,
joined to a FAD2 intron (Bp 1072-2202), which is joined to a sense
fragment of the QTPase gene (360 bp) (Bp 2203-2562), which is
joined to the Gad2 terminator (Bp 2563-2835). 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).
Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco
plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or
Oriental comprising this RNAi construct are embodiments provided
herein.
[0374] Flue-cured tobacco was transformed with the construct shown
in FIG. 8 using Agrobacterium-mediated transformation and more than
about 98% of putative transformants were successfully transformed.
Of the independent lines, 200 plants were regenerated, transplanted
in the greenhouse, harvested and tested for alkaloid content. A
total of 75 lines were identified as having less than 1,000 ppm
total alkaloid and no lines were identified as having less than 500
ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco
created using the construct shown in FIG. 8 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.
[0375] Burley tobacco was also transformed with the construct shown
in FIG. 8 using Agrobacterium-mediated transformation and more than
about 98% of putative transformants were successfully transformed.
Of the independent lines, 201 plants were regenerated, transplanted
in the greenhouse, harvested and tested for alkaloid content. A
total of 86 lines were identified as having less than 3,000 ppm
total alkaloid and 12 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 Burley tobacco created using
the construct shown in FIG. 8 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.
[0376] Oriental tobacco will be transformed with the construct
shown in FIG. 8 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. 8 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.
[0377] FIG. 9 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 44)
and a norflurazone selection cassette (SEQ. ID. No. 35). Starting
from the right border (RB), the PMTase inhibition cassette
comprises a truncated RD2 promoter (Bp 1-1061) operably linked to
an antisense fragment (202 bp) (Bp 1062-1263) of the PMTase gene,
joined to an Act11 intron (Bp 1264-1418), which is joined to a
sense fragment of the PMTase gene (262 bp) (Bp 1419-1620), which is
joined to the Gad2 terminator (Bp 1621-1893). 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).
Accordingly, tobacco products (e.g., cigarettes), tobacco, tobacco
plants, tobacco cells, tobacco seeds, in Burley, Flue-cured or
Oriental comprising this RNAi construct are embodiments provided
herein.
[0378] Flue-cured tobacco was transformed with the construct shown
in FIG. 9 using Agrobacterium-mediated transformation and more than
about 98% of putative transformants were successfully transformed.
Of the independent lines, 100 plants were regenerated, transplanted
in the greenhouse, harvested and tested for alkaloid content. A
total of 86 lines were identified as having less than 1,000 ppm
total alkaloid and 12 lines were identified as having less than 500
ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco
created using the construct shown in FIG. 9 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.
[0379] Burley tobacco was also transformed with the construct shown
in FIG. 9 using Agrobacterium-mediated transformation and more than
about 98% of putative transformants were successfully transformed.
Of the independent lines, 99 plants were regenerated, transplanted
in the greenhouse, harvested and tested for alkaloid content. A
total of 29 lines were identified as having less than 3,000 ppm
total alkaloid and no 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 Burley tobacco created using
the construct shown in FIG. 9 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.
[0380] Oriental tobacco will be transformed with the construct
shown in FIG. 9 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. 9 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.
[0381] FIG. 10 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct has a PMTase inhibition cassette (SEQ. ID. No. 45)
and a norflurazone selection cassette (SEQ. ID. No. 35). Starting
from the right border (RB), the PMTase inhibition cassette
comprises a RD2 promoter (Bp 1-2006) operably linked to an
antisense fragment (344 bp) (Bp 2007-2350) of the PMTase gene,
joined to an Fad2 intron (Bp 2351-3481), which is joined to a sense
fragment of the PMTase gene (344 bp) (Bp 3482-3825), which is
joined to the Gad2 terminator (Bp 3826-4098) at the left border
(LB). 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). Accordingly, tobacco products (e.g.,
cigarettes), tobacco, tobacco plants, tobacco cells, tobacco seeds,
in Burley, Flue-cured or Oriental comprising this RNAi construct
are embodiments provided herein.
[0382] Flue-cured tobacco was transformed with the construct shown
in FIG. 10 using Agrobacterium-mediated transformation and more
than about 98% of putative transformants were successfully
transformed. Of the independent lines, 66 plants were regenerated,
transplanted in the greenhouse, harvested and tested for alkaloid
content. A total of 44 lines were identified as having less than
1,000 ppm total alkaloid and 17 lines were identified as having
less than 500 ppm total alkaloid. Accordingly, the transgenic
Flue-cured tobacco created using the construct shown in FIG. 10 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.
[0383] Burley tobacco will be transformed with the construct shown
in FIG. 10 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,
as compared to the parental strain of tobacco, and approximately
10% of the lines tested will have less than 500 ppm total alkaloid,
as compared to the parental strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct shown in FIG. 10 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.
[0384] Oriental tobacco will be transformed with the construct
shown in FIG. 10 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. 10 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.
[0385] FIG. 11 shows another RNAi construct that was used to
generate several lines of reduced nicotine and TSNA tobacco. This
RNAi construct (SEQ ID No 49) has a QTPase inhibition cassette
(SEQ. ID. No. 42) and a kanamycin selection cassette (SEQ. ID. No.
48). 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 QTPase 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 NOS terminator (Bp 3862-4115). The selection cassette
comprises the nopaline synthase (NOS) promoter (Bp 4116-4422)
operably linked to a neomycin phosphotransferase (NPTII) gene (Bp
4435-5229) joined to the NOS terminator (Bp 5619-5872) at the left
border (LB). Accordingly, tobacco products (e.g., cigarettes),
tobacco, tobacco plants, tobacco cells, tobacco seeds, in Burley,
Flue-cured or Oriental comprising this RNAi construct are
embodiments provided herein.
[0386] Flue-cured tobacco was transformed with the construct shown
in FIG. 11 using Agrobacterium-mediated transformation and more
than about 98% of putative transformants were successfully
transformed. Of the independent lines, 99 plants were regenerated,
transplanted in the greenhouse, harvested and tested for alkaloid
content. A total of 43 lines were identified as having less than
1,000 ppm total alkaloid and 15 lines were identified as having
less than 500 ppm total alkaloid. Accordingly, the transgenic
Flue-cured tobacco created using the construct shown in FIG. 11 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.
[0387] Burley tobacco will be transformed with the construct shown
in FIG. 11 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,
as compared to the parental strain of tobacco, and approximately
10% of the lines tested will have less than 500 ppm total alkaloid,
as compared to the parental strain of tobacco. Accordingly, it is
expected that the transgenic Burley tobacco that will be created
using the construct shown in FIG. 11 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.
[0388] Oriental tobacco was transformed with the construct shown in
FIG. 11 using Agrobacterium-mediated transformation and more than
about 98% of putative transformants were successfully transformed.
Of the independent lines, 122 plants were regenerated, transplanted
in the greenhouse, harvested and tested for alkaloid content. A
total of 22 lines were identified as having less than 1,000 ppm
total alkaloid and 6 lines were identified as having less than 500
ppm total alkaloid. Accordingly, the transgenic Flue-cured tobacco
created using the construct shown in FIG. 11 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.
[0389] It should be emphasized that other promoters and terminators
can be used with the nucleic acids provided herein interchangeably.
Although RD2 (SEQ. ID. No. 13, 37, or 50) 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. 14) 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. 16) is preferred for driving expression of a
norflurazone resistance gene, other constitutive promoters such as
the GapC promoter (SEQ. ID. No. 15), the tobacco alcohol
dehydrogenase (ADP) (SEQ. ID. No. 17) and the Arabidopsis ribosomal
protein L2 (RPL2P) (SEQ. ID. No. 18) can be used to drive
expression of the norflurazone resistance gene. Additionally,
developmentally regulated promoters such as, cinnamyl alcohol
dehydrogenase (SEQ. ID. No. 19) and metallothionein I promoter
(SEQ. ID. No. 20) can be used interchangeable with the cassettes
described herein.
[0390] 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 QTPase
inhibition cassette, the PMTase inhibition cassette, the A622
inhibition cassette, a sterol 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. 21), NOS terminator (SEQ ID No 38) and the
FAD 2 (SEQ. ID. No. 22) or PAP1 introns can be used
interchangeably, as well.
[0391] Other embodiments provided herein concern the discovery of
several mutants of the phytoene desaturase gene that confer
resistance to the herbicide norflurazone (e.g., SEQ. ID. Nos.10,
11, and 12). 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 uM-0.1 uMconc.). 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 uM. Preferably, less than or equal to 0.05 uM
concentration of norflurazone is used when selecting transformants
with Flue-cured tobacco and less than or equal to 0.0125 uM
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.
[0392] 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. 10, 11, and 12,
were identified as sequences that confer resistance to
norflurazone. Accordingly, embodiments provided herein 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.
[0393] In a first selection construct, the Arabidopsis phytoene
desaturase gene (PDS) (SEQ. ID. No. 36) 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. 10). 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.11 and 12.
[0394] That is, in a second selection construct, the Arabidopsis
phytoene desaturase gene (PDS) (SEQ. ID. No. 36) 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
[0395] Further, in a third selection construct, the Arabidopsis
phytoene desaturase gene (PDS) (SEQ. ID. No. 36) 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
[0396] Accordingly, embodiments provided herein 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. 36, 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. 36) is mutated one residue at a time and each mutant
is screened for resistance to the herbicide. Accordingly,
embodiments provided herein 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. 10, 11, or
12 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. Embodiments provided herein 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. 10, 11, or 12) that confer
resistance to an herbicide, in particular norflurazone.
[0397] 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 provided herein. 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 provided herein.
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.
[0398] 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, nornicotine, or sterol 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.
[0399] 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, nornicotine, or
sterol 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,
nornicotine, or sterol production or confer resistance to
norflurazone).
[0400] Accordingly embodiments provided herein also include
compositions comprising, consisting of, or consisting essentially
of: (a) the nucleic acid sequences shown in the sequence listing
(SEQ. ID. NOS. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or
50); (b) nucleotide sequences encoding the amino acid sequences
encoded by the nucleic acids of the sequence listing (SEQ. ID. NOS.
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50); (c) any
nucleotide sequences that hybridizes to the complement of the
sequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49 or 50) under stringent
conditions, e.g., hybridization to filter-bound DNA in 0.5 M
NaHPO4, 7.0% sodium dodecyl sulfate (SDS), 1 mM EDTA at 50 degrees
C. and washing in 0.2.times.SSC/0.2% SDS at 50 degrees C.; and (d)
any nucleotide sequence that hybridizes to the complement of the
sequences shown in the sequence listing (SEQ. ID. NOS. 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49 or 50) under less stringent
conditions (e.g., hybridization in 0.5 M NaHPO4, 7.0% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 37 degrees C. and washing in
0.2.times.SSC/0.2% SDS at 37 degrees C. Preferably these sequences
also perform the functions of the particular nucleic acid
embodiment (e.g., inhibition of nicotine, nornicotine, or sterol
production or confer resistance to norflurazone). Embodiments
provided herein also include peptides encoded by the nucleic acid
sequences of (a), (b), (c), or (d), above.
[0401] The examples described herein demonstrate that several
different RNAi constructs can be used to effectively reduce the
levels of nicotine, nornicotine, and sterols 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. Additionally, the norflurazone resistance nucleic acids
described herein can be used in a general sense (e.g., in plants
other than tobacco) to efficiently select positively transformed
plant cells from plant cells that do not contain a construct
comprising the norflurazone resistance gene. Thus, the norflurazone
selection cassette or the norflurazone resistance gene described
herein can be used to confer resistance to norflurazone in plants
including, but not limited to, corn (Zea mays), canola (Brassica
napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Orya
sativa), rape (Brassica napus), rye (Secale cereale), sorghum
(Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus),
wheat (Triticum aestivum), soybean (Glycine max), tobacco
(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis
hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea
batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut
(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus
spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana
(Musa spp.), avocado (Persea americana), fig (Ficus casica), guava
(Psidium guajava), mango (Mangifera indica), olive (Olea europaea),
papaya (Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), apple (Malus pumila), blackberry (Rubus),
strawberry (Fragaria), walnut (Juglans regia), grape (Vitis
vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach
(Prunus persica), plum (Prunus domestica), pear (Pyrus communis),
watermelon (Citrullus vulgaris), duckweed (Lemna), oats, barley,
vegetables, ornamentals, conifers, and turfgrasses (e.g., for
ornamental, recreational or forage purposes). Vegetables include
Solanaceous species (e.g., tomatoes; Lycopersicon esculentum),
lettuce (e.g., Lactuea sativa), carrots (Caucuis carota),
cauliflower (Brassica oleracea), celery (apium graveolens),
eggplant (Solanum melongena), asparagus (Asparagus officinalis),
ochra (Abelmoschus esculentus), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of
the genus Cucurbita such as Hubbard squash (C. Hubbard), Butternut
squash (C. moschtata), Zucchini (C. pepo), Crookneck squash (C.
crookneck), C. argyrosperma, C. argyrosperma ssp, C. digitata, C.
ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii,
and members of the genus Cucumis such as cucumber (Cucumis
sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
Ornamental plants include azalea (Rhododendron spp.), hydrangea
(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses
(Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.),
petunias (Petunia hybrida), carnation (Dianthus caryophyllus),
poinsettia (Euphorbia pulcherima), and chrysanthemum. Conifers,
which may be employed in practicing the present invention, include,
for example, pines such as loblolly pine (Pinus taeda), slash pine
(Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine
(Pinus contorta), and Monterey pine (Pinus radiata), Douglas-fir
(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka
spruce (Picea glauca); redwood (Sequoia sempervirens); true firs
such as silver fir (Abies amabilis) and balsam fir (Abies
balsamea); and cedars such as Western red cedar (Thuja plicata) and
Alaska yellow-cedar (Chamaecyparis nootkatensis). Turf grass
include but are not limited to zoysia grasses, bentgrasses, fescue
grasses, bluegrasses, St. Augustine grasses, Bermuda grasses,
buffalo grasses, ryegrasses, and orchard grasses. Also included are
plants that serve primarily as laboratory models, e.g.,
Arabidopsis. Preferred plants for use in the present methods
include (but are not limited to) legumes, solanaceous species
(e.g., tomatoes), leafy vegetables such as lettuce and cabbage,
turf grasses, and crop plants (e.g., tobacco, wheat, sorghum,
barley, rye, rice, corn, soybean, cotton, cassava, and the like),
and laboratory plants (e.g., Arabidopsis). While any plant may be
used to carry out this aspect provided herein, tobacco plants are
particularly preferred.
[0402] Further, embodiments provided herein concern the production
of norflurazone-resistant or tolerant plants, which can be sprayed
with the herbicide in the field. In this manner, weeds and
non-transformed plants will die after contact with the herbicide
but plants containing the construct harboring the norflurazone
resistance gene will survive. In one embodiment, for example, a
norflurazone-containing herbicide is applied to the plant
comprising the DNA constructs provided herein, and the plants are
evaluated for tolerance to the herbicide. Any formulation of
norflurazone can be used for testing plants comprising the DNA
constructs provided herein. The testing parameters for an
evaluation of the norflurazone tolerance of the plant will vary
depending on a number of factors. Factors would include, but are
not limited to the type of norflurazone formulation, the
concentration and amount of norflurazone used in the formulation,
the type of plant, the plant developmental stage during the time of
the application, environmental conditions, the application method,
and the number of times a particular formulation is applied. For
example, plants can be tested in a greenhouse environment using a
spray application method. The testing range using norflurazone can
include, but is not limited to 0.5 oz/acre to 500 oz/acre. That is,
the amount of herbicide that can be applied to transgenic plants
containing a norflurazone-resistance gene in a field can be less
than, equal to, or more than 0.5, 0.6, 0.7, 0.8, 0.9. 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, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 125, 150, 175, 200, 250, 300, 350, 400, or 500 oz/acre. In
some embodiments, the norflurazone application rate is 2.24 kg to
4.48 kg ai/hectare (2 to 4 lbs ai/acre) or 2.8 to 5.6 kg
granules/hectare (2.5 to 5 lb/acre) or 234 L/hectare (25 gal/acre)
in solution. Higher amounts are preferred for finer textured soils
or when longer residual activity is desired.
[0403] The preferred commercially effective range can be from 25
oz/acre to 100 oz/acre of norflurazone, depending on the crop and
stage of plant development. A crop can be sprayed with at least one
application of a norflurazone. For testing in cotton an application
of 32 oz/acre at the 3-leaf stage may be followed by additional
applications at later stages in development. For wheat, corn,
soybean, and tobacco an application of 32 oz/acre of norflurazone
at the 3-5 leaf stage can be used. The test parameters can be
optimized for each crop in order to find the particular plant
comprising the constructs provided herein that confers the desired
commercially effective norflurazone tolerance level. The section
below describes typical curing methods which may be used to prepare
the tobacco once it is harvested.
[0404] a) Heterologous Expression
[0405] Tobacco has well-established transformation procedures and
well-characterized regulatory elements for the control of transgene
expression. Tobacco also has a high biomass yields and rapid
scalability, which makes it a very suitable platform for commercial
molecular farming. Since tobacco is a non-food and non-feed crop it
also carries a reduced risk that the transgenic material or
recombinant proteins would contaminate animal feed and would enter
the human food chain. Because conventional or wild-type tobacco has
a high content of nicotine and other toxic alkaloids, however,
investigators have not explored the ability to use tobacco as a
bioreactor. Further, the high cost of nicotine removal has steered
investigators away from this technology.
[0406] The present disclosure, however, provides several types of
genetically modified tobacco that can be used as a platform into
which genes encoding commercially valuable compounds can be
introduced. That is, by using genetically modified tobaccos having
reduced levels of nicotine, sterols, and/or TSNAs, as bioreactors,
it is contemplated that many commercially valuable industrial oils,
pharmaceuticals, dietary supplements can be obtained with fewer
processing steps (e.g., the removal of nicotine is no longer
required). Accordingly, some embodiments concern tobaccos that are
genetically modified to have a reduced level of nicotine, sterols,
and/or TSNAs, further comprising a heterologous gene that produces
a medicinal compound, industrial oil, or dietary supplement, which
can be harvested and/or isolated or purified from said tobacco.
Compounds generated in this manner can be used for a variety of
applications such as the preparation of immunogens, vaccines,
cooking oils, pharmaceuticals and dietary supplements. Techniques
for the production of medicinal compounds in low-nicotine tobacco,
such as a protein, for industrial or pharmaceutical application has
been described in the art. It is contemplated, that these
techniques can be readily used with the tobaccos and techniques
that are described herein.
[0407] As an exemplary, non-liming example, the N-terminal fragment
of SARS-CoV S protein (S1) can be expressed in low-nicotine tobacco
plants, as is known in the art and exemplified in Pogrebnyak et
al., Proc. Natl. Acad. Sci. USA (2005) 102:9062-9067. Incorporation
of the S1 fragment into plant genomes as well as its transcription
can be confirmed by PCR and RT-PCR analyses. High levels of
expression of recombinant S1 protein can be observed in several
transgenic lines by Western blot analysis using specific
antibodies. Mammals parenterally primed with tobacco-derived S1
protein can have sera containing SARS-CoV-specific IgG as detected
by Western blot and ELISA analysis.
[0408] The original gene encoding the human SARS-CoV spike
glycoprotein (strain TOR2, National Center for Biotechnology
Information no. NC 004718) is known in the art. DNA encoding a
79-kDa S protein fragment, corresponding to amino acids 14-714, can
be amplified by two consecutive rounds of PCR to generate XbaI and
SacI sites at the 5' and 3' ends, respectively, by using the
following primers: SP-F1-CCT TGC GCT TCT CAG CCA CGC AAA CTC AAG
AGG ATC GCA TCA CCA TCA CCA TCA CAG TGA CCT TGA CCG GTG CAC (SEQ ID
NO 51), XbaI-F2-ATA ATC TAG ATG ATC ATG GCT TCC TCC AAG TTA CTC TCC
CTA GCC CTC TTC CTT GCG CTT CTC AGC CAC G (SEQ ID NO 52), and
SacI-HDELR-ATT CGA GCT CTT AAA GTT CAT CAT GAG CCA TAG AAA CAG GCA
TTA CT (SEQ ID NO 53). The expression cassette of SARS-CoV 51
protein can contain the plant-derived 23-aa
{MIMASSKLLSLALFLALLSHANS (SEQ. ID. No. 54}, signal peptide (SP),
and a histidine tag {RGSHHHHHH (SEQ. ID. NO. 55} at the N-terminal
portion of the resulting 79-kDa polypeptide. After addition of the
plant-specific endoplasmic reticulum retention signal {HDEL (SEQ.
ID. NO. 56}, the cassette can be subcloned into the XbaI/SacI site
of the plant binary vector pE1801, which is known as a super
promoter, followed by a tomato etch virus translation enhancer. The
vector also can contain the npt II gene for kanamycin selection of
transgenic plants. Plasmid pE1801-79SHDEL can be electroporated
into Agrobacterium tumefaciens strain LBA4404 and used for plant
transformations.
[0409] A genetically modified reduced nicotine and reduced TSNA
tobacco, made as described herein, can be used as the platform. The
low-nicotine/TSNA tobacco can be transformed by
Agrobacterium-mediated transformation as is known in the art.
Independent kanamycin-resistant (KmR) tobacco lines can be used for
molecular analyses. The presence of the spike gene in transgenic
plants can be confirmed by PCR using genomic DNA. KmR transgenic
plants with PCR-confirmed presence of the S transgene can be
further analyzed for gene-specific mRNA expression by quantitative
RT-PCR as is known in the art. Western blot analysis of transgenic
lines with polyclonal SARS-specific antibodies Sm and Sn can
confirm the presence of SARSCoV S-specific 79-kDa protein and its
derivatives. KmR T1 tobacco lines (cv. LAMD-609) can be grown
hydroponically to obtain large amounts of root tissue for
immunological experiments. Western blot analysis of T1 lines can
reveal high levels of S protein expression comparable with the
original T0 transgenic lines.
[0410] Immunological assessment of the plant-expressed S protein
can be performed in 6- to 8-week-old female BALB/c mice. For
parenteral immunization, mice can be injected three times at 2-week
intervals with an equivalent of 50 mg of dry tobacco root material
per mouse. Powdered plant material can be reconstituted with saline
(1/1 by weight) just before immunization. First and second
immunizations can be given s.c. with complete and incomplete
Freund's adjuvant, respectively; the third dose can be administered
i.p. in saline. Sera can be collected retroorbitally from each
mouse before and 10 days after each immunization. Four weeks after
the last immunization, mice can receive an i.p. booster dose of 1
.mu.g of commercially obtained S peptide (Cell Sciences, Canton,
Mass.) without adjuvant. After 10 days, mice can be killed and
exanguinated by heart puncture, and sera can be assayed by ELISA
and Western blot analysis. Solid-phase ELISA can be carried out as
known in the art MaxiSorp 96-well plates (Nalge Nunc) coated
overnight at 4.degree. C. with the same S peptides obtained from
Cell Sciences at a concentration of 1 .mu.g/ml in PBS.
Antigen-specific antibodies can be detected by using the following
antibodies: rabbit anti-mouse IgG (total) and anti-mouse IgG1 (both
from BD Biosciences Pharmingen), anti-mouse IgG2a, IgG2b, IgG3,
IgM, and IgA (all from Organon Teknika), and anti-mouse IgE
(eBioscience, San Diego). A serum dilution with an OD450 of 0.15
units above background can be considered the ELISA titer.
III. Tobacco Products
[0411] Although the modified tobaccos described herein are
preferably used to create tobacco products for human consumption
(e.g., cigarettes, chew, snuff, plug, etc.) it should be realized
that the tobacco described herein can be used for other
applications such as animal feed, pharmaceutical production, and,
in particular, the gernation of proteins (e.g., antiviral or
anti-oncogenic peptides or antibodies or fragments thereof).
Preferably, however, the tobacco and methods provided herein can be
applied to any 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. The term
"tobacco product" includes, but is not limited to, smoking
materials (e.g., cigarettes, cigars, pipe tobacco), snuff, chewing
tobacco, gum, and lozenges. In numerous embodiments, the methods
provided herein are applied to tobacco used to create the tobacco
product.
[0412] A. Reduced Risk Tobacco Products
[0413] Provided herein are reduced risk tobacco products. A reduced
risk tobacco product provided herein can be a traditionally
configured tobacco product containing a reduced risk tobacco, such
as a modified tobacco as provided herein. A reduced risk tobacco
product provided herein also can contain conventional tobacco and
be configured to reduce the risk of using the tobacco product. An
example of such a reduced risk tobacco product is a cigarette
containing a filter designed to reduce the risk associated with
cigarette smoke. A reduced risk tobacco product also can contain a
reduced risk tobacco and be configured to reduce the risk of using
the tobacco product. An example of such a reduced risk tobacco
product is a cigarette containing a reduced risk tobacco and a
filter designed to reduce the risk associated with cigarette smoke.
In some such embodiments, the reduced risk tobacco and reduced risk
configuration act synergistically to reduce the overall risk of
using the tobacco product.
[0414] Typical configurations of a tobacco product that reduces the
risk of the tobacco product will include a filter that reduces the
risk of exposure to tobacco smoke. A filter can be configured to be
used with any smoking tobacco product, including cigars, pipes and
cigarettes, as is known in the art. In one embodiment, the reduced
risk tobacco product is a cigarette containing a filter that
reduces the risk of exposure to tobacco smoke. Any of a variety of
known filters that reduce the risk of exposure to tobacco smoke can
be used in the reduced risk tobacco products provided herein,
including, but not limited to, commercially available filters
provided in cigarette products, and other filters known in the art,
such as filters containing antioxidants, copper, carbon or
activated charcoal, and/or paper-containing filters. One exemplary
filter can be a filter containing an antioxidant or a radical
scavenger. Filters containing antioxidants or radical scavengers
can be prepared according to known methods, as exemplified in U.S.
Pat. Nos. 6,832,612 and 6,415,798, herein expressly incorporated by
reference in their entireties. Another exemplary filter is a filter
that can reduce tobacco smoke-induced modulation of cell
homeostasis, as can be assessed by determining, for example,
modulation of the transcriptome or proteome, cell viability, or
integrity of genetic material. Such filters can include a compound
that sequesters or intercepts harmful components that generate DNA
breaks, or enhance DNA breakage, thereby yielding a filter that
removes harmful smoke components. For example, a filter can contain
flat aromatic compounds that can scavenge potential carcinogens
(e.g., components of tar), where exemplary flat aromatic compounds
include caffeine and pontoxyfyllen. In some of the methods provided
herein the filter comprises an interceptor of the carcinogen that
has aromatic chemical structure: the carcinogen associates then
with interceptor forming a complex that is retained in the
filter.
[0415] The methods provided herein can be used for evaluating
modifications to tobacco product configurations. For example, the
methods provided herein can be used as assays for evaluating the
effectiveness of a cigarette filler or filter. These methods also
can be used to test any effect that can be resultant from a
particular tobacco product configuration in combination with a
modified tobacco. For example, the methods provided herein can be
used to test a selected filter in combination with a modified
tobacco. Accordingly, the methods provided herein provide a basis
for evaluating and developing a reduced risk tobacco product. The
methods provided herein futher provide a basis for evaluating any
further reduction in risk that can result from specific
combinations of modified tobaccos and filters.
[0416] As provided herein, methods can be used for testing
modulation of cell homeostasis by, for example, monitoring a
molecular marker of modulation of cell homeostasis, when the cells
are exposed to a tobacco composition from a modified tobacco
configured in a tobacco product with a filter, or configured in a
plurality of tobacco products with a plurality of different
filters. Similarly, methods can be used for testing modulation of
cell homeostasis when the cells are exposed to a tobacco
composition from a plurality of modified tobaccos configured in a
tobacco product with a filter, or configured in a plurality of
tobacco products with a plurality of different filters.
Accordingly, a variety of combinations of modified tobaccos and
tobacco configurations can be tested for their properties of
modulating cell homeostasis. The examples provided herein
demonstrate that the risk-reducing properties of a tobacco and the
risk-reducing properties of a filter can be interrelated such that
the risk-reducing properties of a particular filter can vary
depending on the type of tobacco used. The methods provided herein
can be used to evaluate the degree to which a particular filter
reduces the risk of one or more tobacco products, and also can be
used to evaluate the ability of one or more filters to reduce the
risk of a particular tobacco product. The methods provided herein
also can be used to evaluate the ability of one or more filters in
combination with one or more tobaccos to have additive
risk-reducing properties, thereby forming an even further
reduced-risk tobacco product.
[0417] B. Methods for Evaluating Tobacco and/or Tobacco Product
[0418] Methods for Determining the Risk Potential of Tobacco and
Tobacco Products
[0419] Provided herein are several methods for identifying the
propensity of a tobacco or tobacco product to contribute to a
tobacco related disease. Generally, these approaches are practiced
by providing a tobacco, obtaining smoke or a smoke condensate from
the tobacco, contacting a cell with the smoke or smoke condensate,
and identifying one or more attributes of the contacted cell.
Tobacco products contain a number of compounds that induce various
types of changes to a cell, including cell damage, change in gene
expression including mRNA and/or protein expression, mutations,
chromosomal aberrations, aberrant sister chromatid exchanges and
micronuclei. Attributes of contacted cells indicative of such
tobacco-induced cell changes can be identified in the methods
provided herein, which address changes in cell homeostasis, as
indicated by changes in gene expression, genetic mutations or
aberrations, and modulation of cell viability and/or apoptosis. The
methods provided herein can be used to determine affect of a
tobacco or a tobacco product on a cell by determination of the
presence, absence, or change in a molecular marker. For example, a
molecular marker can be monitored, which is indicative of an affect
on mRNA, protein, DNA damage, cell viability or apoptosis can be
determined according to the methods provided herein or other
methods generally known in the art, where monitoring of the
molecular marker can be used to determine the affect of a tobacco
or tobacco product on cell homeostasis. Exemplary affects of a
tobacco or tobacco product on a cell include, but are not limited
to, induction of a double-strand DNA break, inhibition of
apoptosis, inhibition of cell proliferation, and modulation of gene
expression, including modulation of the transcriptome and/or
modulation of the proteome. Accordingly, the methods provided
herein can be used to establish a profile for a particular tobacco
by employing assay methods, including assays that identify tobacco
products that modulate cell homeostasis from tobacco products that
do not. For example, assays for induction of damage of cellular
genetic material or assays for modulation of gene expression can be
used to differentiate reduced risk tobacco products from
conventional tobacco products. For example, the methods provided
herein can be used to characterize a tobacco by assay methods
including an assay for the induction of a double-strand DNA break,
inhibition of apoptosis, inhibition of cell proliferation,
modulation of transcription, or modulation of translation.
[0420] Several other assays have classically been used to analyze
tobacco for the risk of adverse health effects. Traditionally, the
first manner of testing consists of analysis of cigarette smoke for
various components that can relate to health effects associated
with smoking. A second manner of testing includes testing cigarette
smoke tar on living cells. One of these tests detects changes in
the genetic material of bacteria. Another test uses mouse cells
grown in Petri dishes to detect potential cancer-causing activity.
A third manner of testing seeks to determine if people smoke the
tested tobacco cigarettes differently than the comparable brand or
type currently on the market. If the way the cigarettes are smoked
is different, then the other manners of testing can be repeated
with the smoking machines set to reflect the change in smoking
behavior. A fourth manner of testing examines the response of
animals to cigarette smoke or tar. One such type of test looks for
inflammation in the lungs of mice in response to cigarette smoke. A
second test looks for tumor formation in the upper respiratory
tract of hamsters exposed to smoke. A third test looks for the
cancer producing ability of cigarette smoke tar by applying the tar
to the skin of mice. Each manner of testing can include comparing
tobacco cigarettes and both the effects of mainstream and
sidestream smoke can be tested.
[0421] During smoking, both mainstream smoke (inhaled by the
smoker) and sidestream smoke (mainly from the burning end of the
cigarette) are generated. While mainstream and sidestream smoke are
qualitatively similar the quantity of specific components differs
between the two. Additionally, modifications to the cigarette can
independently affect the composition of sidestream and mainstream
smoke. It is concluded, therefore, that testing of tobacco or
cigarettes can be assayed for both mainstream and sidestream
smoke.
[0422] Epidemiology is not a practical approach for addressing the
issue of the health effects of changes in a cigarette composition.
Because people can smoke cigarettes differently (ex. longer or
faster puffs) it can be important to consider whether these changes
affect smoke chemistry and therefore toxicity. For example, a new,
cigarette type can result in a smoker taking longer puffs, which
can then change the smoke chemistry and toxicity.
[0423] Testing, however, can examine the effects on toxicity of a
single design change in a cigarette or can examine the effects of a
set of design changes compared to an unchanged control. Testing
protocols can follow either a screening or a tradeoff approach. In
the screening approach new designs can be subjected to a series of
tests each with criteria for passing or failing. Designs that fail
are eliminated from further testing, while those passing are
subjected to additional scrutiny. In the tradeoff approach the
relative changes in each test would be assessed in light of other
information about the particular design.
[0424] The FTC method describes: how cigarettes are to be prepared
for smoking, the type of smoking machine to use, the way the
smoking machine should be operated, the method for collecting smoke
products, and ways to measure moisture content, nicotine, carbon
monoxide and tar. Typically in the methods provided herein, the FTC
protocol for studies of cigarette smoke chemistry and toxicity are
used.
[0425] Toxicity of cigarette smoke is directly related to the
composition of the smoke and the composition of smoke can be
changed if the way the cigarettes are smoked is changed.
[0426] There are a variety of chemical analyses that can be done to
aid in the determination of the change in toxicity of a tobacco.
These relate to the chemical composition of tobacco smoke. The
following lists the chemical composition analysis and the health
effect associated with the component or property measured: Total
Particulate Matter (TPM; carcinogen), pH (effect on nicotine
toxicity), Redox Potential (influence toxicity of whole smoke),
Carbon Monoxide (reduces ability of blood to carry oxygen),
Nitrogen Oxides (NOx; increases nitrosamine formation, inhibits
enzyme function), Hydrogen Cyanide (inhibits lung clearance, lowers
ability of body to use oxygen), Hydrocarbons (benzene, butadiene;
suspected or known carcinogens), Aldehydes (ex. formaldehyde,
acrolein; inhibit lung clearance, animal carcinogens), Volatile
nitrosamines (strong animal carcinogens), Tobacco-specific
nitrosamines (strong animal carcinogens), Nicotine (associated with
cardiovascular disease), Phenols (enhance carcinogen action)
Catechol (major carcinogen), and Polynuclear Aromatic Hydrocarbons
(PNAs; major tumor initiators).
[0427] There are also a variety of known cell toxicity tests that
can be performed in a relatively short time scale: bacterial
mutagenicity test, animal cell test to detect potential
carcinogens, and lung inflammation test in animals. One test, the
Ames test, uses certain types of Salmonella bacteria to
quantitatively assess the ability of a material to cause mutations,
such as mutations involved in the process of carcinogenesis. In
this test a solution of collected smoke particulates is mixed with
the bacteria. Bacteria with the ability to grow in the absence of a
particular nutrient are scored as mutants.
[0428] The potential cancer-causing ability of chemicals can also
be evaluated using a cell transformation assay. In this assay,
solutions of smoke particulates are given to animal cells grown in
Petri dishes in the laboratory. After several weeks the cells are
examined under the microscope. At this time the cells are scored
for abnormal growth patterns. The number of clusters of abnormally
growing cells is then compared among cigarette types.
[0429] In animal studies, inflammation of the lungs can be
assessed. The changes measured in this test can be related to the
development of chronic obstructive pulmonary disease. In these
tests mice can be exposed to whole smoke two times per day, for any
number of days according to the experimental design. At the end of
the exposure period the animals would be killed and their lungs
washed out to collect inflammatory cells. The numbers and kinds of
the cells would be measured.
[0430] Two long-term animal tests for cancer causing ability of
tobacco can be performed. In the first, test cigarette tar is
applied to the back skin of mice. Skin tumors are then scored over
the life of the animals. The use of this test is based on two
observations: (1) in studies of tumor formation by smoke in
hamsters whole smoke is active but smoke free of particulates is
not and (2) a large number of known carcinogens are contained in
the particulate portion of cigarette smoke.
[0431] The second test examines the tumor forming ability, of whole
smoke in hamsters. A positive response can be observed in the
larynx of hamsters exposed over their lifetime to whole cigarette
smoke. In this test the animals are exposed twice daily to the
diluted smoke of one cigarette every day for their entire lives.
Tumor formation is the endpoint measured in this assay. Because the
test is so labor intensive it is recommended only as a last step in
a series of tests.
[0432] These known methods for assaying tobacco toxicity have
limitations in terms of time length and/or expense relative to the
assay methods provided herein. Accordingly, there is a long felt
need for more rapid and less costly methods of analysis of tobacco
products of different compositions. Despite the inefficiencies of
the approaches above, it is contemplated herein that these methods
for assaying tobacco toxicity can be used alone or in conjunction
with the methods provided herein so as to provide additional
information regarding the properties of the tobacco being
characterized.
[0433] In the methods provided herein, one or more cells can be
contacted with a tobacco composition such as tobacco smoke (TS), a
tobacco smoke condensate (TSC), or total particulate matter (TPM),
where exemplary TS and TSC are cigarette smoke (CS) and cigarette
smoke condensate (CSC). Preparation of the tobacco composition used
in the methods provided herein can be performed in accordance with
the teachings herein and the knowledge and skill in the art. For
example, TS can be collected using a smoking machine such as an
INBIFO-Condor smoking machine, and TSC can be collected using cold
traps, and TPM can be collected using a filter, as is known in the
art. For example, CSC for testing can be prepared by passing smoke
through a series of cold traps containing glass beads upon which
CSC condenses; the CSC can then be collected by washing the beads
with acetone as described in Mathewson, H. D. Beitrage zur
Tabforschung. 3(6):430-7. September 1966. In addition, cells can be
contacted with smoke provided in diluted form, where diluted smoke
can be produced in a dilution chamber, as known in the art. For
example, a smoking setup can contain a dilution chamber where the
concentration of the smoke being applied to the cells can be varied
by dilution with air in order to produce different dosages and
intensities of smoke. The dilution chamber can be located between
the burning cigarette and the cell exposure chamber. In addition,
cigarette particulate matter for testing can be prepared by passing
smoke through a glass fiber filter which is subsequently washed
with solvent to collect the sample as described in Coresta
Recommended Method No. 23 (August 1991). Although the description
herein provides several methods in the context of characterizing
tobacco and tobacco products that undergo pyrolysis (e.g.,
cigarettes, pipe tobacco, and cigars), similar approaches can be
applied to the evaluate snuff, chew, and other tobacco products
that do not undergo pyrolysis. Accordingly, the methods provided
herein are not limited to smoke or smoke condensate, but can be
applied to any tobacco composition known in the art. The
preparation and analysis of compositions from such non-pyrolysis
tobacco products is straightforward given the teachings provided
herein or otherwise known in the art. Methods for contacting cells
with compositions from such non-pyrolysis tobacco products also is
straightforward given the teachings provided herein or otherwise
known in the art.
[0434] The tobacco derived composition (i) can originate in a
tobacco product, which can be either pure tobacco or a tobacco
formulation (such as a cigarette, cigar, pipe or chewing tobacco)
having multiple compositional elements, for example but not limited
to structural elements, flavor chemicals and/or other additives,
and (ii) can have multiple components (e.g., smoke or a smoke
condensate, also referred to collectively as "smoke products") or
can be a single known or unidentified component (e.g., a single
chemical compound). The composition can be "derived" from tobacco
or a tobacco formulation (i) by simple physical separation; (ii) as
a product of combustion or heating, (iii) by solvent extraction,
(iv) by chemical reaction(s) or (v) by enzyme activity (e.g., smoke
concentrate treated with a microsomal cellular fraction or purified
cytochrome P450).
[0435] In some methods provided herein, cells are contacted with
TS, such as CS. The contacting of the cells with the CS, CSC, TS,
TSC or TPM can be accomplished using any method known to one of
skill in the art, including but not limited to, placing said cells
into a smoking machine or smoke chamber (e.g., CULTEX.RTM.) for a
period of time to allow the cells to be contacted with smoke,
and/or providing a CSC or TSC to the media for a designated period
of time (e.g., in 0.5% dimethylsulfoxide or other formulation). The
contacting can be for any amount of time, however, preferably the
cells are contacted for an amount of time that does not result in
nonviability of more than 50% of the cells. In some embodiments,
the amount of time can be varied and the results are compared. In a
further embodiment, the cells are treated for an amount of time in
which the gene expression is modulated, but the majority of cells
are still viable. That is, in some embodiments, the cells are
treated to a point in which the cells are at least, equal to, or
more than 1% viable, including but not limited to 1%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99%, and 100% viable.
[0436] In a another embodiment, the amount of time for contacting a
cell with the CS, CSC, TS, TSC or TPM is any amount selected from
the group consisting of about at least, equal to, or more than 1
seconds to about 24 hours, including but not limited to at least,
equal to, or more than 1 second, 15 seconds, 30 seconds, 45
seconds, 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20
minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45
minutes, 60 minutes, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5
hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours,
7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10
hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13
hours, 13.5 hours, 14 hours, 14.5 hours, 15 hours, 15.5 hours, 16
hours, 16.5 hours, 17 hours, 17.5 hours, 18 hours, 18.5 hours, 19
hours, 19.5 hours, 20 hours, 20.5 hours, 21 hours, 22 hours 23
hours and 24 hours. In a further embodiment, the cells are
contacted for less than and including about 20 minutes. In yet
another embodiment, the cells are contacted for about 2 to about 20
minutes.
[0437] The amount of smoke with which the cells are contacted can
be any of a variety of amounts according to the desired level of
exposure. For example, smoke exposure can be performed in
accordance with FTC parameters: 2.0 second puff duration, 35 mL
puff every 60 seconds. Puff duration, volume and frequency can be
increased or decreased to achieve different levels of smoke
exposure, as desired. Similarly, smoke condensate or other tobacco
compositions can be contacted with cells at a variety of different
concentrations and for a variety of different durations, as
desired. For example, smoke condensate at 20 mg/mL can be contacted
with cells for any of the above-provided amounts of time, as
desired.
[0438] Tobacco smoke or smoke products can be treated prior to
contacting the cells with the smoke or smoke product. For example,
the smoke or smoke concentrate can be contacted with a filter, such
as a filter provided herein, for example by obtaining smoke or
smoke condensate from a cigarette after passing through a filter
attached to the tobacco product, such as a cigarette.
[0439] The cells suitable for use in the methods provided herein
include human as well as non-human cells, but are preferably human
pulmonary cells (e.g., lung or bronchial cell), although cells of
other systems impacted by smoking, including but not limited to
cells of the upper aerodigestive tract (e.g., oral cavity including
cheek, pharynx, larynx, and esophagus), bladder, stomach, kidney,
pancreas, and blood (e.g., lymphocytes, monocytes, neutrophils,
esoinophils or basophils, or neoplastic blood cells such as myeloid
leukemia cells); cells of the cardiovascular system (including
endothelial cells, smooth muscle cells (e.g. from vessel walls,
myocardial cells, etc.) and cells of the female reproductive system
(e.g. cells of the uterus, cervix, fallopian tubes, ovary, and
placenta), can also be used. The cells can be normal or can be
neoplastic, metaplastic, dysplastic or malignant. The cells can be
collected from a living organism (e.g., a pulmonary lavage
specimen, tissue section such as a lung or bronchial section, oral
mucosa sample, cheek swab, or sputum sample), can be primary cell
cultures, or can be established cell cultures. In some embodiments,
the cells can be obtained from a living organism, including a
human, after the organism is contacted with a tobacco composition,
for example, after a human consumes a cigarette. Cells collected
from a living organism can be collected using any of a variety of
known methods known in the art, according to the cell type to be
collected (e.g., a cheek scrape or lung lavage). In specific,
non-limiting embodiments, the cells can be NHBE cells, or can be
human epithelian pulmonary type II cells, such as A549 cells, or
can be cells obtained from a human primary culture.
[0440] Many embodiments described herein employ NHBE cells that are
maintained in culture, and other embodiments employ human lung
carcinoma cells (A549 cells). Although NHBE and A549 cells are
preferred for the methods described herein, it should be understood
that many other cells that are typically contacted with tobacco or
TS during the process of smoking (e.g., lung cells, bronchial
cells, cells of the oral mucosa, pharynx, larynx, and tongue) can
also be used. Additionally, many immortal cell lines can be used
with the methods described herein. Preferred cells for use with the
embodied approaches include, but are not limited to, human
bronchial cells (e.g., BEP2D or 16HBE140 cells), human bronchial
epithelial cells (e.g., HBEC cells, 1198, or 1170-I cells), NHBE
cells, BEAS cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell
lung cancer (NSCLC) cells or human alveolar cells (e.g., H460,
H1792, SK-MES-1, Calu, H292, H157, H1944, H596, H522, A549, and
H226), tongue cells (e.g., CAL 27), and mouth cells (e.g.,
Ueda-1)). Many of such cultures are available commercially or
through a public repository (e.g., ATCC). Further, several
techniques exist that allow for one to generate primary cultures of
said cells and these primary cultures can be used with the methods
described herein.
[0441] Conventional approaches in tissue culture can be used to
establish and maintain said cells in preparation for the methods
described herein. That is, the cells may be grown in culture by any
method known to one of skill in the art and with the appropriate
media and conditions. The cells grown in culture may require feeder
layers, for example. The cells may be grown to confluence or may be
grown to less than confluence before, during, or after treatment.
In some embodiments the cells are grown to between about 10% and
about 90% confluence, including but not limited to, at least, equal
to, or more than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% confluence before
contact with CS, CSC, TS, or TSC.
[0442] In some embodiments, the cells contacted and assayed in
accordance with the methods provided herein are manipulated to
control and/or modify the percentage of cells that are in one or
more phases of the cell cycle. For example, the cells can be
manipulated such that at least 50% of the cells of the population
of cells are in the S phase. The cells used herein can be
manipulated to control the population of cells in one or more of
G0, G1, S, G2, or M phases of the cell cycle. For example, cells
can be manipulated such that at least 50%, 60%, 70%, 75%, 80%, 85%,
90%, 95%, or 100%, of the population of cells are in G0, G1, S, G2,
or M phase. In another example, cells can be manipulated such that
greater than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of
the population of cells are in G0, G1, S, G2, or M phase. In
another example, cells can be manipulated such that 50%, 60%, 70%,
75%, 80%, 85%, 90%, 95%, or 100%, of the population of cells are in
G0, G1, S, G2, or M phase. The section below describes several
preferred methods for characterizing tobacco and tobacco products
in greater detail.
[0443] Exemplary Assays
[0444] The methods provided herein for characterizing tobacco or a
tobacco product can be used in a variety of applications,
including, but not limited to, the comparison of two or more
tobaccos or two or more tobacco products, identifying a modulation
of cell homeostasis, identifying an induction of damage of cellular
genetic material, or identifying a modulation of gene expression
including mRNA and/or protein expression. The methods provided
herein for characterizing tobacco also can be used for identifying
a tobacco product that has a reduced potential to contribute to a
tobacco-related disease, and making a tobacco product that has a
reduced potential to contribute to a tobacco-related disease. In
addition to methods provided herein for characterizing tobacco or a
tobacco product, additional methods known in the art for
characterizing tobacco or a tobacco product can be used in the
place of, or in conjunction with, the methods provided herein.
[0445] The methods of identifying a tobacco, identifying a compound
in tobacco, identifying a tobacco product, and making a tobacco
product provided herein, can in addition be utilized in methods of
identifying two or more tobaccos, identifying a compound in two or
more tobaccos, identifying a tobacco product by comparing two or
more tobacco products, and making a tobacco product by comparing
two or more tobacco products. In some embodiments, the two or more
tobaccos or tobacco products can be compared for their effect on
cell homeostasis, gene expression including mRNA and/or protein
expression, or damage to genetic material of cells. In some
embodiments, at least one tobacco or tobacco product can be a
reduced risk tobacco or tobacco product, respectively. In some
embodiments, at least one tobacco can be a modified tobacco, such
as a chemically modified tobacco or a genetically modified tobacco.
In some embodiments, at least one tobacco can be a reduced risk
tobacco product, such as a tobacco product configured to contain a
filter that reduces the risk of tobacco exposure such as a filter
provided herein. In one example, two or more tobaccos can be
compared to identify a compound in tobacco that modulates gene
expression including mRNA and/or protein expression. In one
example, two or more tobacco products can be compared to identify a
compound in tobacco that modulates gene expression including mRNA
and/or protein expression.
[0446] In some embodiments, a second tobacco product (e.g., a
cigarette) is compared to a first tobacco product (e.g., a
cigarette) using the methods above so as to identify which of the
two tobacco products is less likely to contribute to a
tobacco-related disease. For example, a first population of
isolated human cells of the mouth, tongue, oral cavity, or lungs
(e.g., NHBE cells), is contacted with a CS from a first tobacco
product (e.g., a "reduced risk full flavor" cigarette) in an amount
and for a time sufficient to modulate cell homeostasis, such as
inducing damage of cellular genetic material or modulating gene
expression including mRNA and/or protein expression. A second
population of isolated human cells of the mouth, tongue, oral
cavity, or lungs (e.g., NHBE cells), preferably the same type of
cell as used in the analysis of the first tobacco product, is also
contacted with a CS from a second tobacco product (e.g., a
cigarette) in an amount and for the same amount of time as used
with the first product or for a time sufficient to modulate cell
homeostasis by, for example, inducing damage of cellular genetic
material or modulating gene expression including mRNA and/or
protein expression.
[0447] The data obtained from the analysis of the first tobacco
product can be compared to the data obtained from the analysis of
the second tobacco product so as to identify, for example, an
increased risk tobacco or a compound in tobacco. The data also can
be used to identify a reduced risk tobacco. The data further can be
used to identify a tobacco product configuration, such as a filter,
with increased risk or with reduced risk. Thus, by analyzing the
differences between the tobacco products, one can identify a
tobacco product that has less potential to contribute to a tobacco
related disease or to identify, for example, a first tobacco
product that has a reduced risk to contribute to a tobacco-related
disease, as compared to a second tobacco product or vice versa. By
one technique, a tobacco product that is less likely to contribute
to a tobacco-related disease is identified because it causes less
modulation of cell homeostasis. By another technique, a tobacco
product that is less likely to contribute to a tobacco-related
disease is identified because it causes less modulation of cell
homeostasis under the same level of damage induced to cellular
genetic material. In another technique, a tobacco product that is
less likely to contribute to a tobacco-related disease is
identified because it induces less damage to cellular genetic
material. In another technique, a tobacco product that is less
likely to contribute to a tobacco-related disease is identified
because it induces fewer or smaller in degree changes in gene
expression such as changes in the transcriptome or changes in the
proteome.
[0448] The methods provided herein can be used not only to identify
a tobacco product that has a reduced potential to contribute to a
tobacco-related disease, as compared to a second tobacco product,
but also to develop tobacco products that have a reduced potential
to contribute to a tobacco-related disease, as compared to a second
tobacco product. For example, by screening modified tobacco (e.g.,
chemically or genetically modified tobacco) or a tobacco product
with a modified configuration in accordance with the methods
disclosed herein, one can rapidly determine whether the modified
tobacco or modified tobacco product has an increased or decreased
potential to contribute to a tobacco-related disease, as compared
to the tobacco or tobacco product that is not modified.
[0449] More embodiments concern methods to identify components of a
tobacco product that contribute to a tobacco-related disease, the
selective removal or inhibition of production of these components,
and the determination that the removal of the component(s) results
in a reduced risk tobacco product. Such a determination that the
removal of the component(s) results in a reduced risk tobacco
product can be indicated by, for example, a molecular marker that
is associated with a tobacco-related disease. Exemplary molecular
markers of a tobacco-related disease include, but are not limited
to a molecular marker indicative of apoptosis, a molecular marker
indicative of double-stranded DNA breaks, an overexpressed or
underexpressed mRNA, an overexpressed or underexpressed
polypeptide. In one example methods are provided to identify
components of a tobacco product that contribute to a
tobacco-related disease, the selective removal or inhibition of
production of these components, and the determination that the
removal of the component(s) modulates expression of a gene that is
associated with a tobacco-related disease in a manner that reduces
the potential for the tobacco product to contribute to a tobacco
related disease. It is contemplated that particular components of
tobacco products are the factors that modulate responses in human
cells that contribute to tobacco-related disease. It is further
contemplated that modification of the tobacco product will,
concomitantly, result in a modulation of the response in human
cells contacted with the smoke from said modified tobacco product,
which modulates the likelihood to contribute to a tobacco-related
disease relative to an unmodified tobacco product. For example,
modification of genes that contribute to the production of these
toxic components in tobacco (e.g., genetic engineering or chemical
treatment) will, concomitantly, result in a modulation of the
response in human cells contacted with the smoke from said modified
tobacco, which modulates the likelihood to contribute to a
tobacco-related disease relative to tobacco prior to modification
of the component-producing gene. Accordingly, by selectively
removing the components from a tobacco product (e.g., by modifying
a tobacco or a tobacco product configuration) that induce the
events that contribute to tobacco-related disease in a human, one
can develop tobacco products that are less likely to contribute to
a tobacco-related disease.
[0450] Methods for Identifying a Tobacco or Tobacco Product that
Modulates Cell Homeostasis
[0451] Provided herein are methods for identifying a tobacco that
modulates cell homeostasis by providing a tobacco, obtaining a
tobacco composition from the tobacco, contacting a cell with the
tobacco composition, and identifying the presence or absence of a
modulation of cell homeostasis after contact with the tobacco
composition. In some embodiments, the methods provided herein can
be used to identify a tobacco that affects cell homeostasis as can
be monitored by, for example, determining that the tobacco induces
double strand DNA breaks, modulates apoptosis, modulates cell
proliferation, or induces expression of a gene that is silent
during homeostasis or repress a gene that is active during
homeostasis. In some embodiments, the tobacco composition can be
smoke or smoke condensate. By modulation of homeostasis of a cell
is meant the change in the state of a cell upon contact of the cell
by a tobacco or tobacco composition (e.g., tobacco smoke or tobacco
smoke condensate), where the state of the cell can be the cell
cycle, the apoptotic state, the expression levels of one or more
genes as represented by mRNA levels and/or protein levels or
post-translational modifications to proteins. Typically modulation
of cell homeostasis can be monitored by measurement of one or more
molecular markers of the state of a cell. It is contemplated herein
that any tobacco-induced change in cell homeostasis can serve as an
indicator that said tobacco or tobacco composition may contribute
to a tobacco-related disease. Because it is known that tobacco or
tobacco compositions can have a plurality of adverse affects on
cells, it is contemplated that typically a less pronounced
modulation of cell homeostasis after the cell is contacted with a
tobacco or tobacco composition (e.g., tobacco smoke or tobacco
smoke condensate), the greater propensity that the tobacco or
tobacco composition will present a reduced level of risk for
developing a tobacco related disease relative to a conventional or
reference tobacco. Accordingly, it is contemplated herein that a
tobacco or tobacco composition characterized as reduced risk
tobacco or tobacco composition is one that, upon contact with a
cell, does not modulate cell homeostasis or modulates cell
homeostasis to a lesser degree than the cell homeostasis modulation
induced by a conventional or reference tobacco.
[0452] Also provided herein are methods of identifying a compound
in tobacco that modulates cell homeostasis by providing a first
tobacco, obtaining a tobacco composition from the first tobacco,
contacting a first population of cells with the tobacco composition
from the first tobacco, identifying the degree of modulation of
cell homeostasis in the first population of cells after contact
with the tobacco composition from the first tobacco, providing a
second tobacco that has been modified to reduce a compound in the
second tobacco, obtaining a tobacco composition from the second
tobacco, contacting a second population of cells with the tobacco
composition from the second tobacco, and identifying the degree of
modulation of cell homeostasis after contact with the tobacco
composition from the second tobacco, where an identification of a
reduction in the degree of modulation of cell homeostasis after
contact with the tobacco composition from the second tobacco
identifies the compound as one that modulates cell homeostasis. In
some embodiments, the methods provided herein can be used to
identify a compound in tobacco that modulates apoptosis. In some
embodiments, the methods provided herein can be used to identify a
compound in tobacco that modulates cell proliferation. In some
embodiments, the tobacco composition can be smoke or smoke
condensate.
[0453] Also provided herein are methods of identifying a tobacco
product that has a reduced potential to contribute to a
tobacco-related disease by providing a first tobacco product,
obtaining a tobacco composition from the first tobacco product,
contacting a first population of cells with the tobacco composition
from the first tobacco product, identifying the degree of
modulation of cell homeostasis in the first population of cells
after contact with the tobacco composition from the first tobacco
product, providing a second tobacco product, obtaining a tobacco
composition from the second tobacco product, contacting a second
population of cells with the tobacco composition from the second
tobacco product, and identifying the degree of modulation of cell
homeostasis after contact with the tobacco composition from the
second tobacco product, where an identification of a reduction in
the degree of modulation of cell homeostasis after contact with the
tobacco composition from the second tobacco product as compared to
the degree of modulation of cell homeostasis after contact with the
tobacco composition from the first tobacco product identifies the
second tobacco product as one that has a reduced potential to
contribute to a tobacco-related disease. In some embodiments, the
second tobacco product has been modified to reduce a compound in
the second tobacco. In some embodiments, the second tobacco product
can be genetically modified to reduce the expression of at least
one gene that regulates production of the compound. In some
embodiments of the methods provided herein, the degree of
modulation of cell homeostasis can be determined by identifying the
degree of modulation of apoptosis. In some embodiments of the
methods provided herein, the degree of modulation of cell
homeostasis can be determined by identifying the degree of
modulation of cell proliferation. In some embodiments, the tobacco
composition can be smoke or smoke condensate.
[0454] Also provided herein are methods of making a tobacco product
that has a reduced potential to contribute to a tobacco-related
disease by providing a first tobacco, obtaining a tobacco
composition from the first tobacco, contacting a first population
of cells with the tobacco composition from the first tobacco,
identifying the degree of modulation of cell homeostasis in the
first population of cells after contact with the tobacco
composition from the first tobacco, providing a second tobacco,
obtaining a tobacco composition from the second tobacco, contacting
a second population of cells with the tobacco composition from the
second tobacco, identifying the degree of modulation of cell
homeostasis after contact with the tobacco composition from the
second tobacco product, where an identification of a reduction in
the degree of modulation of cell homeostasis after contact with the
tobacco composition from the second tobacco as compared to the
degree of modulation of cell homeostasis after contact with the
tobacco composition from the first tobacco identifies the second
tobacco as one that has a reduced potential to contribute to a
tobacco-related disease, and incorporating the second tobacco,
which has a reduced potential to contribute to a tobacco-related
disease, into a tobacco product. In some embodiments, the second
tobacco has been modified to reduce a compound in the second
tobacco. In some embodiments, the second tobacco can be genetically
modified to reduce the expression of at least one gene that
regulates production of the compound. In some embodiments of the
methods provided herein, the degree of modulation of cell
homeostasis can be determined by identifying the degree of
modulation of apoptosis. In some embodiments of the methods
provided herein, the degree of modulation of cell homeostasis can
be determined by identifying the degree of modulation of cell
proliferation. In some embodiments, the tobacco composition can be
smoke or smoke condensate.
[0455] The methods provided herein can be used to determine the
effect of a tobacco product or a compound from a tobacco product,
on cell homeostasis. Cells of an organism contacted with a tobacco
composition, e.g., mammalian epithelial cells, can undergo
apoptosis and can proliferate at particular levels under "normal"
conditions, where "normal" as used in this context refers to
conditions in which cells are not contacted with tobacco or a
tobacco composition and are not otherwise placed under atypical
(e.g., stressful) environmental conditions. Environmental
conditions, for example, contacting the cells with a tobacco
composition, can modulate apoptosis of the contacted cells and also
can modulate the proliferation of the contacted cells. Such
modulation can result in processes that can directly lead to
cellular events in tobacco-related disease (e.g., apoptosis can be
decreased, which can lead to neoplastic cell growth) or can
indirectly lead to cellular events in tobacco-related disease
(e.g., apoptosis can be increased, which can trigger a cell growth
response in an organism, which can lead to neoplastic cell growth).
The methods provided herein can be used to examine the affect of a
tobacco product or a compound from a tobacco product, on cell
homeostasis by, for example, determining the affect of a tobacco or
tobacco compound on apoptosis in a cell or a cell population, or,
for example, determining the affect of a tobacco or tobacco
compound on cell proliferation of a cell or a cell population. In
some embodiments, a first tobacco that causes a lesser degree of
modulation of cell homeostasis relative to a second tobacco can be
characterized as a reduced risk tobacco. In some embodiments, a
first tobacco that causes a lesser degree of inhibition of
apoptosis relative to a second tobacco can be characterized as a
reduced risk tobacco. In some embodiments, a first tobacco that
causes a lesser degree of inhibition of cell proliferation relative
to a second tobacco can be characterized as a reduced risk tobacco.
Any of a variety of known methods for determining modulation of
cell homeostasis by, for example, modulating apoptosis, modulating
cell proliferation, modulating gene expression (e.g., mRNA or
protein levels) as exemplified herein, can be used in the methods
provided herein.
[0456] Also provided herein are methods for determining cell
response to cell damage. Cells can be exposed to environmental
input, such as a tobacco composition, that causes cell damage. The
response of these cells to the environmental input-mediated damage
can be indicative of the likelihood of the environmental input
leading to an environmental input-related disease. In one
embodiment, cells can be contacted with a tobacco composition, and
the response of the cells to the contact by the tobacco composition
can indicate the likelihood of the tobacco composition leading to a
tobacco-related disease.
[0457] As provided herein, cells contacted by different
environmental inputs, for example, different tobacco compositions,
can respond differently to cell damage caused by the environmental
input, where some cell responses are more indicative of leading to
a disease state compared to other cell responses. Thus,
contemplated herein, two or more tobacco compositions can be
compared and characterized according to the cell responses in
reaction to damage induced by exposure to the tobacco compositions.
In such methods, exposure conditions can be manipulated such that
the amount of damage to the cells is equivalent for each different
tobacco composition, resulting in a determination of different
characteristic cell responses to the same amount of cell
damage.
[0458] Accordingly, methods are provided herein for comparing two
or more tobacco compositions by contacting a first tobacco
composition with a first population of cells, and contacting a
second composition with a second population of cells, where the two
different contacting steps are performed in such a manner that the
first and second population of cells undergo equivalent amount of
cell damage, and then determining the degree of modulation of cell
homeostasis in the first and second populations of cells, where the
tobacco composition that is characterized by the lowest degree of
cell modulation can be identified as a tobacco with reduced
likelihood of causing a tobacco-related disease. In such methods,
damage to the cells caused by the tobacco compositions can be
measured by, for example, measuring the degree of damage to the
genetic material of the cells, in accordance with the methods
provided herein or otherwise known in the art. Also in such
methods, the degree of modulation of cell homeostasis can be
determined by the degree of modulation of apoptosis or cell
proliferation relative to cells not contacted by a tobacco
composition or relative to cells contacted by a tobacco composition
from a tobacco, such as a reduced risk tobacco with a known degree
of modulation of cell homeostasis. The following section describes
several methods for differentiating tobaccos and tobacco products
that induce genetic damage from those that do not.
[0459] Analysis of Changes to Cell Homeostasis: Identifying a
Tobacco that Induces Genetic Damage
[0460] Provided herein are methods of identifying a tobacco that
induces genetic damage by providing a tobacco, obtaining a tobacco
composition from the tobacco, contacting a cell with the tobacco
composition, and identifying the presence or absence of damage of
cellular genetic material in the cell after contact with the
tobacco composition. In some embodiments, the methods provided
herein can be used to identify a tobacco that induces a
double-strand DNA break. In some embodiments, the tobacco
composition can be smoke or smoke condensate.
[0461] Also provided herein are methods of identifying a compound
in tobacco that induces damage of cellular genetic material by
providing a first tobacco, obtaining a tobacco composition from the
first tobacco, contacting a first population of cells with the
tobacco composition from the first tobacco, identifying the amount
of damage of cellular genetic material in the first population of
cells after contact with the tobacco composition from the first
tobacco, providing a second tobacco that has been modified to
reduce a compound in the second tobacco, obtaining a tobacco
composition from the second tobacco, contacting a second population
of cells with the tobacco composition from the second tobacco, and
identifying the amount of damage of cellular genetic material after
contact with the tobacco composition from the second tobacco, where
an identification of a reduction in the amount of damage of
cellular genetic material after contact with the tobacco
composition from the second tobacco identifies the compound as one
that induces the damage of cellular genetic material. In some
embodiments, the methods provided herein can be used to identify a
compound in tobacco that induces a double-strand DNA break. In some
embodiments, the tobacco composition can be smoke or smoke
condensate.
[0462] Also provided herein are methods of identifying a tobacco
product that has a reduced potential to contribute to a
tobacco-related disease by providing a first tobacco product,
obtaining a tobacco composition from the first tobacco product,
contacting a first population of cells with the tobacco composition
from the first tobacco product, identifying the amount of damage of
cellular genetic material in the first population of cells after
contact with the tobacco composition from the first tobacco
product, providing a second tobacco product, obtaining a tobacco
composition from the second tobacco product, contacting a second
population of cells with the tobacco composition from the second
tobacco product, and identifying the amount of damage of cellular
genetic material after contact with the tobacco composition from
the second tobacco product, where an identification of a reduction
in the amount of damage of cellular genetic material after contact
with the tobacco composition from the second tobacco product as
compared to the amount of damage of cellular genetic material after
contact with the tobacco composition from the first tobacco product
identifies the second tobacco product as one that has a reduced
potential to contribute to a tobacco-related disease. In some
embodiments, the second tobacco product has been modified to reduce
a compound in the second tobacco. In some embodiments, the second
tobacco product can be genetically modified to reduce the
expression of at least one gene that regulates production of the
compound. In some embodiments of the methods provided herein, the
amount of damage of cellular genetic material can be determined by
identifying the induction of double-strand DNA breaks. In some
embodiments, the tobacco composition can be smoke or smoke
condensate.
[0463] Also provided herein are methods of making a tobacco product
that has a reduced potential to contribute to a tobacco-related
disease by providing a first tobacco, obtaining a tobacco
composition from the first tobacco, contacting a first population
of cells with the tobacco composition from the first tobacco,
identifying the amount of damage of cellular genetic material in
the first population of cells after contact with the tobacco
composition from the first tobacco, providing a second tobacco,
obtaining a tobacco composition from the second tobacco, contacting
a second population of cells with the tobacco composition from the
second tobacco, identifying the amount of damage of cellular
genetic material after contact with the tobacco composition from
the second tobacco product, where an identification of a reduction
in the amount of damage of cellular genetic material after contact
with the tobacco composition from the second tobacco as compared to
the amount of damage of cellular genetic material after contact
with the tobacco composition from the first tobacco identifies the
second tobacco as one that has a reduced potential to contribute to
a tobacco-related disease, and incorporating the second tobacco,
which has a reduced potential to contribute to a tobacco-related
disease, into a tobacco product. In some embodiments, the second
tobacco has been modified to reduce a compound in the second
tobacco. In some embodiments, the second tobacco can be genetically
modified to reduce the expression of at least one gene that
regulates production of the compound. In some embodiments of the
methods provided herein, the amount of damage of cellular genetic
material can be determined by identifying the induction of
double-strand DNA breaks. In some embodiments, the tobacco
composition can be smoke or smoke condensate.
[0464] Also provided herein are methods, compositions and kits for
evaluating the ability of a tobacco-derived substance to produce
DSBs in chromosomal DNA. The presence of DSBs is detected using an
appropriate marker, which, in preferred embodiments provided
herein, is phosphorylated histone H2AX (also referred to herein as
".DELTA.H2AX"). The presence of DSBs also can be detected by
detecting (i) activation of one or more of the protein kinases that
are responsible for H2AX phosphorylation (e.g., ATM, ATR and/or
DNA-PK); (ii) appearance of nuclear foci that are induced by
histone H2AX phosphorylation; or (iii) activation of one or more
protein components of nuclear foci induced by H2AX phosphorylation
that are associated with DNA repair. The term "activation" in
regard to proteins activated by DSBs refers to a chemical
modification such as phosphorylation, acetylation, ubiquitinylation
or poly(ADP)ribosylation, and/or a change in protein conformation,
occurring in response to formation of DSBs. Activated proteins can
be detected, for example, immunocytochemically.
[0465] Some of the assays provided concern methods of detecting,
quantifying, identifying and/or evaluating (e.g., for harmfulness)
a tobacco-derived substance in the course of research or in the
environment via its promotion of DSBs in the chromosomal DNA of a
test cell. A correlation with harmful potential is drawn based upon
the known relationship between DSBs and genetic mutations
(including cancer-causing and teratogenic mutations) as well as
cell damage and death.
[0466] Accordingly, one set of preferred embodiments provided
herein are methods of detecting a harmful tobacco-derived substance
comprising the steps of (a) exposing a test cell (or test cell
population) to a tobacco test composition; (b) measuring the degree
of H2AX phosphorylation in the test cell or cell population; and
(c) comparing the degree of H2AX phosphorylation determined in the
test cell or cell population to the degree of H2AX phosphorylation
in a control cell or control cell population; wherein a higher
degree of H2AX phosphorylation in the test cell compared to the
control cell indicates the presence of a harmful tobacco derived
substance in the tobacco test composition. The presence of DSBs
also can be detected by detecting (i) activation of one or more of
the protein kinases that are responsible for H2AX phosphorylation
(e.g., ATM, ATR and/or DNA-PK); (ii) appearance of nuclear foci
that are induced by histone H2AX phosphorylation; or (iii)
activation of one or more protein components of nuclear foci
induced by H2AX phosphorylation that are associated with DNA
repair.
[0467] Another set of non-limiting embodiments, provided herein
include methods for identifying one or more harmful components of
TS comprising the steps of: (a) exposing a first test cell
population to a first smoke product generated from a first tobacco
composition; (b) exposing a second test cell population to a second
smoke product generated from a second tobacco composition, wherein
the first and second smoke products are prepared using essentially
equivalent protocols; (c) measuring the degree of H2AX
phosphorylation in the first and second test cell populations; and
(d) comparing the degree of H2AX phosphorylation in the first and
second test cell populations; (e) identifying the tobacco
composition associated with a greater degree of H2AX
phosphorylation in steps (a)-(d); and (f) comparing the components
of the first and second tobacco composition to identify one or more
component present in the tobacco composition of step (e) but absent
in the other tobacco composition. Methods for detecting activation
of protein kinases such as ATM, ATR and/or DNA-PK as well as
formation of nuclear foci and protein components of the nuclear
foci can be performed according to the same steps. According to
such embodiments, the first tobacco composition can differ from the
second tobacco composition in its ingredients and/or in the way it
was processed. The information obtained by this method can be used
to develop a tobacco product that lacks or has lower levels of the
identified harmful component(s), which can render the product
lower-risk. Alternatively, the information can be used in an
environmental context: for example, air purifiers can be modified
to extract the harmful component from smoke-contaminated air.
[0468] Another set of non-limiting embodiments provided herein
concern methods for identifying one or more harmful components of
TS comprising the steps of: (a) exposing a first test cell
population to a first smoke product generated from a tobacco
composition; (b) exposing a second test cell population to a second
smoke product generated from the tobacco composition, wherein the
first and second smoke products are prepared differently; (c)
measuring the degree of H2AX phosphorylation in the first and
second test cell populations; (d) comparing the degree of H2AX
phosphorylation in the first and second test cell populations; and
(e) identifying the method of smoke product preparation associated
with a greater degree of H2AX phosphorylation in steps (a)-(d);
wherein the method of smoke product preparation identified in step
(e) has greater harmful potential. Methods for detecting activation
of protein kinases such as ATM, ATR and/or DNA-PK as well as
formation of nuclear foci and protein components of the nuclear
foci can be performed according to the same steps. In such
embodiments, the methods of smoke product preparation can differ in
the rate of combustion of the tobacco composition (including
whether the tobacco composition is burned or heated), or can differ
in the filtering of the smoke product (e.g., unfiltered, filtered
with a traditional filter, or filtered with a filter containing an
antioxidant), or can differ by other known methods of altering
tobacco smoke products. The components of the different smoke
products can be compared to identify one or more harmful
components. As above, the identification of a harmful component can
facilitate the development of lower-risk tobacco products and/or
environmental safeguards.
[0469] Also provided herein are methods for comparing the harmful
potentials of a first and a second tobacco composition comprising
the steps of: (a) exposing a first test cell population to a first
smoke product generated from the first tobacco composition; (b)
exposing a second test cell population to a second smoke product
generated from the second tobacco composition, wherein the first
and second smoke products are prepared using essentially equivalent
protocols; (c) measuring the degree of H2AX phosphorylation in the
first and second test cell populations; and (d) comparing the
degree of H2AX phosphorylation in the first and second test cell
populations; wherein the tobacco composition which generated the
smoke product that produced a higher degree of H2AX phosphorylation
has greater harmful potential. Methods for detecting activation of
protein kinases such as ATM, ATR and/or DNA-PK as well as formation
of nuclear foci and protein components of the nuclear foci can be
performed according to the same steps.
[0470] Accordingly, the methods provided herein include one or more
steps of determining whether damage of cellular genetic material
has occurred. Typically, such methods include assays for damage to
the genomic DNA of the cell. Any of a variety of methods known in
the art for assaying damage of cellular genetic material, such as
genomic DNA, can be used in the methods provided herein. Exemplary
known assays include assays for double-strand DNA breaks, assays
for single-strand DNA breaks, and assays for modulated properties
of DNA resultant from damage, such as assays for micronuclei and
assays for chromosome exchange. Assays for DNA breaks are known in
the art, as exemplified in U.S. Pat. Pub. No. 20040132004 and U.S.
Pat. No. 6,309,838, all of which are hereby expressly incorporated
by reference in their entireties.
[0471] In one example, the methods provided herein can include
detection of double-strand DNA breaks by detection of
phosphorylation of histone H2AX. Mammalian cells respond to agents
that introduce DNA double-stranded breaks with the immediate and
substantial phosphorylation of histone H2AX. While not wishing to
be bound to the following theory, which is only offered to explain
one possible mechanism, H2AX is thought to be involved in the
recognition of regions of chromatin containing a DNA
double-stranded break. Formation of the phosphorylated H2AX
protein, termed gamma-H2AX, can be detected as an indicator of DNA
double-stranded breaks. Known antibodies or antigenically-reactive
fragments thereof that specifically bind to a C-terminal
phosphorylated serine in an H2AX histone protein can be used for
the detection of gamma-H2AX, and, thus can be used to indicate the
presence of double stranded breaks in a cell. Thus, in the methods
provided herein, the presence or absence of DNA damage can be
detected by detecting the presence or absence of phosphorylation of
histone H2AX. For example, the presence or absence of
phosphorylation of histone H2AX can be identified with an antibody
or fragment thereof, which binds to phosphorylated H2AX but not
unphosphorylated H2AX. Antibodies and fragments thereof, and
related methods for selectively detecting gamma-H2AX, are known in
the art, as exemplified in U.S. Pat. Nos. 6,362,317 and 6,884,873,
all of which hereby expressly incorporated by reference in their
entireties.
[0472] In some embodiments provided herein, the methods include
assaying a cell for double-strand DNA breaks (DSBs). DSBs are
generated by a variety of genotoxic agents, and are among the most
critical lesions that lead either to apoptosis, mutations or the
loss of significant sections of chromosomal material. Detection of
DSBs upon cell exposure to a potential carcinogen, therefore,
provides the means to assess the potential hazard of the exposure
in terms of cancer induction. In one embodiment, a sensitive assay
of DSBs detection based on analysis of histone H2AX phosphorylation
can be used. Histone H2AX, a variant of a family of at least eight
protein species of the nucleosome core histone H2A, becomes
phosphorylated in live cells upon induction of DNA double strand
breaks (DSBs). The phosphorylation of H2AX on Ser 139 at sites
flanking the DSBs is carried out by ATM-, ATR-, and/or
DNA-dependent protein kinases (DNA-PKs). The phosphorylated form of
H2AX is denoted .DELTA.H2AX.
[0473] The availability of antibodies to .DELTA.H2AX allow for
immunocytochemical detection of DSBs. After induction of DSBs, the
appearance of .DELTA.H2AX in chromatin manifests in the form of
discrete foci, each focus considered to represent a single DSB.
Checkpoint and DNA repair proteins such as Rad50, Rad51 and Brcal
co-localize with .gamma.H2AX. The intensity of .DELTA.H2AX
immunofluorescence (IF) measured by cytometry was reported to
strongly correlate with the dose of ionizing radiation and thus
with the number of the induced DSBs. However, because untreated
cells, particularly cells replicating DNA, express .DELTA.H2AX, to
obtain a stoichiometric relationship between DSBs and the intensity
of .gamma.H2AX IF, it is necessary to compensate for the extent of
this "programmed" H2AX phosphorylation. Following compensation, the
.DELTA.H2AX IF measured by cytometry offers a sensitive and
convenient means to detect and measure DSBs in individual cells
following radiation. In fact, .DELTA.H2AX IF can be a surrogate for
cell killing in viability assays of radiated cells.
[0474] .DELTA.H2AX antibody ("Ab") in conjunction with
multiparameter flow- and laser scanning cytometry can be used in
assays of DSBs, to detect and measure their induction in
individual, live cancer cells exposed to antitumor drugs in vitro.
The intensity of .gamma.H2AX IF correlates well with the drug
concentration and duration of cell exposure to the drug, indicating
a relationship between the incidence of DSBs induced by these drugs
and yH2AX IF intensity. Multiparameter analysis of .DELTA.H2AX IF
and cellular DNA content made it possible to relate the abundance
of DSBs (extent of DNA damage) to the position of the cell in the
cycle.
[0475] The ability of the tobacco-derived substance to promote the
formation of DSBs is measured using an appropriate DSB marker,
which is preferably .DELTA.H2AX (phosphorylated histone H2AX), but
which can be another associated molecule, such as, but not limited
to, Rad50, Rad51 and Brcal, and other proteins that are
characteristic of nuclear foci formation. Formation of DSBs also
can be detected by detecting activate protein kinases associated
with DSBs such as ATM, ATR or DNA-PK. The presence of such markers
can be determined using a marker-specific antibody (or derivative
or fragment thereof), preferably an antibody (or fragment or
derivative thereof) specific for .DELTA.H2AX, or an antibody (or
fragment or derivative thereof) specific for Rad50, Rad51 or Brcal,
or ATM, ATR or DNA-PK. The presence of such markers can be
determined using a marker-specific antibody (or derivative or
fragment thereof), preferably an antibody (or fragment or
derivative thereof) specific for a polypeptide encoded by a gene
provided in Tables 1 and 2. The genes provided in Table 4 encode
polypeptides that are involved in homologous recombination
processes in the cell, and these genes can be activated in response
to cellular damage of genetic material. Accordingly, detection of
one or more products of the genes of Table 4 can be indicative of
cellular damage of genetic material, for example, double-strand DNA
breaks. The genes provided in Table 5 encode polypeptides that are
involved in non-homologous nucleic acid end-joining processes in
the cell, and these genes can be activated in response to cellular
damage of genetic material. Accordingly, detection of one or more
products of the genes of Table 5 can be indicative of cellular
damage of genetic material, for example, double-strand DNA breaks.
Provided herein is an exemplary use of antibody directed to
.DELTA.H2AX; analogous methods can be applied using antibodies
directed to Rad50, Rad51, Brcal, ATM, ATR or DNA-PK, or the
products of the genes listed in Tables 1 and 2. In preferred
non-limiting embodiments provided herein, antibody binding can be
detected by immunofluorescence-based techniques. Various antibodies
for Rad50, Rad51, Brcal, ATM, ATR, DNA-PK, and the products of the
genes listed in Tables 1 and 2 are known in the art and can be
readily obtained for use in accordance with the methods provided
herein; for example, Anti-phospho-ATM (Ser1981), is available from
Upstate USA as clone 10H11.E12. Such techniques can optionally be
used in conjunction with automated cytometry, such as, for example,
flow and/or laser scanning cytometry.
TABLE-US-00010 TABLE 4 Homologous recom- bination Top of Page RAD51
Homologous pairing 15q15.1 NM_002875 RAD51L1 Rad51 homolog 14q24.1
NM_002877 (RAD51B) RAD51C Rad51 homolog 17q23.2 NM_002876 RAD51L3
Rad51 homolog 17q12 NM_002878 (RAD51D) DMC1 Rad51 homolog, meiosis
22q13.1 NM_007068 XRCC2 DNA break and crosslink 7q36.1 NM_005431
XRCC3 repair 14q32.33 NM_005432 XRCC2, XRCC3 RAD52 Accessory
factors for 12p13.33 NM_002879 RAD54L recombination 1p34.1
NM_003579 RAD54B RAD52, RAD54L, 8q22.1 NM_012415 RAD54B BRCA1
Accessory factor for 17q21.31 NM_007295 transcription and
recombination. E3 Ubiquitin ligase BRCA2 Cooperation with RAD51,
13q13.1 NM_000059 essential function SHFM1 BRCA2 associated 7q21.3
NM_006304 (DSS1) RAD50 ATPase in complex with 5q23.3 NM_005732
MRE11A, NBS1 MRE11A 3' exonuclease 11q21 NM_005590 NBS1 Mutated in
Nijmegen 8q21.3 NM_002485 breakage syndrome MUS81 A
structure-specific DNA 11q13.1 NM_025128 EME1 nuclease 17q21.33
NM_152463 (MMS4L) MUS81, MMS4
TABLE-US-00011 TABLE 5 Non-homologous end-joining G22P1 (Ku70)
22q13.2 NM_001469 XRCC5 (Ku80) 2q35 NM_021141 PRKDC 8q11.21
NM_006904 LIG4 13q33.3 NM_002312 XRCC4 5q14.2 NM_003401 DCLRE1C
(Artemis) 10p13 NM_022487
[0476] The term "immunofluorescence-based techniques" or
"immunocytochemical-based techniques" encompasses various forms of
such assays, as are known in the art. For example, and not by way
of limitation, an immunofluorescence-based technique can use an
unlabelled primary antibody and a fluorescently labeled secondary
antibody (as illustrated, for example, in Example 1); or can use a
primary antibody that carries a fluorescent tag to detect the
phosphorylated H2AX molecule directly; or the primary antibody can
carry a biotin molecule while the secondary antibody can carry both
an avidin molecule (which binds specifically to biotin) and a
fluorescence molecule. In the biotin/avidin approach, the binding
of the secondary antibody is based on binding of biotin by avidin
rather than the binding of an antibody of one species directed
against a protein of another species. Other variations of such
techniques that would be known to the skilled artisan as
"immunfluorescence-based techniques" or "immunocytochemical-based
techniques" can be used according to the invention. Likewise,
detection can be made using analogous methods that utilize a
modality other than fluorescence, such as chromogenic or
colorimetric assays, radiologic assays, and so forth.
[0477] Techniques such as immunocytochemical-based techniques can
be used in conjunction with methods for counting cells, sorting
cells, or other method for further characterizing cells. Exemplary
methods include, but are not limited to, flow cytometry, laser
scanning cytometry, fluorescence image analysis, chromogenic
product imaging, fluorescence microscopy or transmission
microscopy.
[0478] The "degree of phosphorylation of H2AX" as used herein
refers to the relative, rather than absolute, amount of
.DELTA.H2AX. This is because .DELTA.H2AX is produced during normal
progression of the cell cycle. As discussed in Example 1, allowance
can be made for normally occurring phosphorylation of H2AX. For
example, the data can preferably be subjected to two normalization
processes. First, allowance can be made for the normally occurring
"programmed" phosphorylation of H2AX. Second, correction can be
made for the fact that histone content is exactly doubled over the
course of a cell cycle, doubling the size of the target (histone).
In a specific non-limiting embodiment, a data value from cells with
twice the DNA content (e.g., G2 and mitotic cells) with twice the
histone target can be divided by 2 while a data value from cells in
S phase having an intermediate in histone content can be divided by
1.5. In this manner, the amount of .DELTA.H2AX detected beyond what
occurs in an untreated control cell or cell population is
normalized to a unit of histone so that one can refer to the
"degree of histone H2AX phosphorylation" on a per unit of histone
basis.
[0479] In another example, the methods provided herein can include
detection of DNA breaks and other forms of genomic damage by Comet
assay. Comet assay can be used to detect damaged DNA pulled from
the nucleus of cells exposed to an electric field. Comet assay is a
fluorescent microscopic method to examine DNA damage and repair at
individual cell level. For example, cells can be embedded in
agarose on a microscope slide and lysed with detergent and high
salt to form nucleoids containing supercoiled loops of DNA linked
to the nuclear matrix, and electrophoresis at high pH can result in
structures resembling comets, observed by fluorescence microscopy.
The intensity of the comet tail relative to the head reflects the
number of DNA breaks. This assay can be used for detecting various
forms of DNA damage (e.g., single- and double-strand breaks,
oxidative DNA base damage, and DNA-DNA/DNA-protein/DNA-Drug
cross-linking) and DNA repair in many eukaryotic cell types. Comet
assay not only provides an estimate of how much damage is present
in cells, but what form it takes. Although it is primarily a method
for measuring DNA breaks, modifications of the methods, for
example, by introducing lesion-specific endonucleases, allows
detection of, for example, pyrimidine dimers, oxidized bases, and
alkylation damage. Thus, in the methods provided herein, the
presence or absence of DNA damage can be identified by, for
example, the presence or absence of comet tails when cells are
analyzed using the Comet assay. Various methods for performing
Comet assays are known in the art, as exemplified in Collins,
(2004) Mol. Biotechnology 26:249-261, Tice, et al. (2000) Environ.
Mol. Mutagen. 35:206-221 and Gichner et al. (2004) Mutation Res.
559:49-57, all of which are hereby expressly incorporated by
reference in their entireties.
[0480] In another example, the methods provided herein can include
detection of double-strand DNA breaks by TUNEL assay. TUNEL assay
can be used to measure double-strand breaks by incorporation of
labeled nucleotides at the site of double-strand breaks using
terminal transferase. The labeled nucleotides can then be detected
with antibodies. TUNEL assay is frequently used to detect
apoptosis-induced DNA fragmentation through a quantitative
fluorescence assay. In one exemplary protocol, terminal
deoxynucleotidyl transferase (TdT) catalyzes the incorporation of
bromo-deoxyuridine (BrdU) residues into the fragmenting nuclear DNA
at the 3'-hydroxyl ends by nicked end labeling. A TRITC-conjugated
anti-BrdU antibody can then label the 3'-hydroxyl ends for
detection. The TUNEL assay distinguishes two populations of cells:
non-apoptotic cells (TUNEL-negative) and apoptotic cells
(TUNEL-positive). Thus, in the methods provided herein, the
presence or absence of DNA damage can identified by, for example,
detecting the presence or absence of labeled nucleotides at the
site of double-strand breaks, incorporated by, for example,
terminal transferase. A variety of methods of performing TUNEL
assays is known in the art, as exemplified in Doolin et al., J.
Burn Care Rehabil. 20: 374-376, 1999; Kalyuzhny (2002) Methods Mol.
Biol. 203:219-34; Lawry, Methods Mol. Med. (2004) 88:183-90; U.S.
Pat. No. 6,506,609 and U.S. Pat. Pub. No. 20030017462, all of which
are hereby expressly incorporated by reference in their
entireties.
[0481] In another example, the methods provided herein can include
detection of double-strand DNA breaks by sister chromatid exchange
assay. Sister chromatid exchange assays detect late damage when
genetic material is exchanged between sister chromatids. Sister
chromatid exchange refers to a reciprocal interchange of the two
chromatid arms within a single chromosome. This exchange can be
visualized during the metaphase portion of the cell cycle and can
be mediated by the enzymatic incision, translocation and ligation
of at least two DNA helices. Thus, in the methods provided herein,
the presence or absence of DNA damage can identified by, for
example, detecting the presence or absence of interchange of
chromatid arms within a single chromosome by, for example, sister
chromatid exchange assay. A variety of methods for performing
sister chromatid exchange assays are known in the art, as
exemplified in 40 C.F.R. .sctn.79.65, 40 C.F.R. .sctn.798.5915,
Renqing et al., (2000) Toxicology Letters 115:23-32, Deen et al.
and Cancer Res. (1986) 46:1599-602, all of which are hereby
expressly incorporated by reference in their entireties.
[0482] In another example, the methods provided herein can include
detection of double-strand DNA breaks by micronuclei assays.
Micronuclei assays can be used to detect late damage occurring
after cells attempt to divide so that non-centromeric DNA forms as
micronuclei in daughter cells. The test is based on the observation
that a secondary nucleus (micronucleus) is formed around a
chromosomal fragment, outside the main nucleus of a dividing cell.
A micronucleus may also be produced due to a lagging whole
chromosome formed as a result of a chromosome loss at anaphase.
Thus, in the methods provided herein, the presence or absence of
micronuclei can be identified. Micronuclei can be detected by
microscopic methods, flow cytometric methods and automated image
recognition methods, as known in the art and exemplified in Offer
et al., FASEB J. (2005) 19:485-7; Smolewski et al., Cytometry
(2001) 45:19-26; Driessens et al., Ann N Y Acad Sci. (2003)
1010:775-9; and U.S. Pat. Pub. No. 20050002552, all of which are
hereby expressly incorporated by reference in their entireties.
[0483] In another example, the methods provided herein can include
detection of chromosomal translocations. Chromosomal translocations
can occur as a result of DNA damage. Methods for detecting
chromosomal translocations can include fluorescence in situ
hybridization methods (FISH), in which probe hybridization patterns
in cells containing chromosomal translocation are altered relative
to wild type. Thus, in the methods provided herein, the presence or
absence of DNA damage can identified by, for example, detecting the
presence or absence of chromosomal translocations by, for example,
FISH. Methods for detecting chromosomal translocations are known in
the art, as exemplified by U.S. Pat. Nos. 5,997,869, 6,576,421, and
6,416,948, and U.S. Pat. Pub. Nos. 20040235039 and 20020192692, all
of which are hereby expressly incorporated by reference in their
entireties.
[0484] The example below provides one non-limiting specific example
of the DSB detection methods and materials. Variations of the assay
method used in terms of materials, assay times, instrumentation and
protocols would be apparent to the skilled artisan for detecting
and/or quantifying DSBs, for example via .DELTA.H2AX.
Example 1
Preparation of Cigarette Smoke Condensates
[0485] Smoke was generated from a commercially available nationally
sold brand of American cigarettes (non-menthol, full-flavor type
with averaged FTC measured values of 14.5 mg tar/1.04 mg nicotine)
using an INBIFO-Condor smoking machine under Federal Trade
Commission (FTC) smoking parameters (2.0 second puff duration 35
milliliter puff every 60 seconds). The cigarettes had been
equilibrated at 23.9.degree. C..+-.1.1.degree. C. and 60%.+-.2%
relative humidity for a minimum of 24 hours and a maximum of 14
days. CSC was collected from the smoke via a series of three cold
traps (-10.degree. C., -40.degree. C., and -70.degree. C.) onto
impingers filled with glass beads. The smoke condensate was
dissolved in acetone, which was then removed by rotary evaporation
at 35.degree. C. The resulting smoke condensate was weighed and
dissolved in dimethylsulfoxide (DMSO) to make a stock solution at a
concentration of 20 mg/mL, which was stored at -20.degree. C. prior
to use.
[0486] NHBE Cell Culture and Smoke Condensate Treatment
[0487] NHBE cells were purchased from Cambrex Corporation, East
Rutherford, N.J. The cells were cultured in complete Bronchial
Epithelial Cell Growth Medium (BEGM), prepared by supplementing
Bronchial Epithelial Basal Medium with retinoic acid, human
epidermal growth factor, epinephrine, transferrin,
triiodothyronine, insulin, hydrocortisone, bovine pituitary extract
and gentamicin by addition of SingleQuots, TM (both medium and the
supplements were purchased from Cambrex Corporation, East
Rutherford, N.J.). Dual-chambered slides (Nunc Lab-Tek II, Fisher
Scientific, Pittsburgh, Pa.) were seeded with 1 ml of 8.times.104
cells/ml cell suspension per chamber. All incubations were at
37.degree. C. in a humidified atmosphere of 5% CO2 in air. Cells
were grown to 50% confluency, at which time they were treated with
medium containing smoke condensate. Appropriate dilutions of the 20
mg/ml smoke condensate in DMSO stock solution were used to prepare
culture medium containing 10, 25, or 50 .mu.g/mL smoke condensate.
The final DMSO concentration was 0.5%. Cells were treated by
carefully aspirating the culture medium from each chamber and
replacing it with 1 ml per chamber of smoke condensate-containing
medium at 37.degree. C. For control slides, the medium was replaced
with 1 mL of either fresh medium (mock-treated control) or medium
containing 0.5% DMSO (vehicle control). Slides were immediately
returned to the incubator for up to 24 hours. At the end of the
treatment, medium from each chamber was carefully aspirated and 1
ml of 1% fresh paraformaldehyde in 1.times. Dulbecco's PBS was
added to each chamber and the cells fixed by gently rocking the
slides at room temperature for 15 minutes. Following aspiration of
the fixative, the chamber slides were disassembled and the slides
submerged in 50 ml conical tubes filled with 70% ethanol. The fixed
slides were stored at 4.degree. C. prior to analysis.
[0488] A549 Cell Culture and Smoke Treatment
[0489] A549 cells were purchased from American Type Culture
Collection (ATCC #CCL-185, Manassas, Va.). The cells were cultured
in Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5
g/L sodium bicarbonate (ATCC, Manassas, Va.) and supplemented with
10% fetal bovine serum (ATCC, Manassas, Va.). Dual-chambered slides
(Nunc Lab-Tek II) were seeded with 1 ml of 105 cells/ml cell
suspension per chamber 48 hours before exposure. All incubations
were at 37.degree. C. in a humidified atmosphere of 5% CO2 in air.
Cells were grown to 70% confluency, at which time they were treated
with smoke. The cell culture medium was replaced with 37.degree. C.
Dulbecco's PBS (D-PBS) containing calcium and magnesium (Sigma, St.
Louis, Mo.) for the smoke exposure. Slide chamber covers were
removed and the slides were placed in a smoke exposure chamber
(20.6 cm.times.6.7 cm.times.6.3 cm--L.times.W.times.H). Smoke was
generated from IM16 (Industry Monitor #16, Philip-Morris, Richmond
Va.) cigarettes under FTC smoking conditions using a KC 5 Port
Smoker (KC Automation, Richmond, Va.). The smoke was diluted by
drawing it through a 250 mL round-bottom flask prior to its
reaching the exposure chamber. The time and distance that the smoke
traveled from the end of the cigarette to the exposure chamber was
minimized by using the shortest lengths of tubing possible between
the parts of the apparatus. Cigarettes were smoked to within 3 mm
of the filter tip. Cells were exposed to smoke for up to 40
minutes. Mock-exposed (control) cells were treated under identical
conditions as the exposed cells except for the absence of a
cigarette in the smoking port. They were mock-exposed for 10
minutes. Following treatment or mock treatment, the D-PBS was
aspirated and replaced with 1 ml per chamber of fresh culture
medium at 37.degree. C. The slides were placed in the 37.degree.
C., 5% CO2 incubator and incubated for 15 minutes. Following
incubation, the medium was aspirated and the cells fixed as
described above for the NHBE experiment.
[0490] Immunocytochemical Detection of Phosphorylated Histone H2AX
and Caspase-3 Activation
[0491] Cells were treated with smoke (i.e., A549) or smoke
condensate (i.e., NHBE) and fixed as described above, then rinsed
twice in PBS and immersed in 0.2% Triton X-100 (Sigma) in a
solution of 1% (w/v) bovine serum albumin (BSA; Sigma) in PBS for
30 min to suppress non-specific antibody binding. The cells were
then incubated in 100 .mu.l volume of 1% BSA containing 1:200
dilution of anti-phosphorylated histone H2AX (.gamma.-H2AX) rabbit
polyclonal Ab (Trevigen, Gaithersburg, Md.). After overnight
incubation at 4.degree. C., the slides were washed twice with PBS
and then incubated in 100 .mu.l of 1:200 dilution of Alexa Fluor
488 goat anti-rabbit IgG (H+L) (Molecular Probes, Eugene, Oreg.)
for 45 min at room temperature in the dark. Parallel samples were
incubated with 1:100 diluted anti-cleaved (activated) caspase-3
rabbit polyclonal Ab (Cell Signaling Technology, Beverly, Mass.)
overnight at 4.degree. C., washed twice with PBS and incubated with
1:30 diluted FITC-conjugated F(ab')2 fragment of swine anti-rabbit
immunoglobulin (DAKO, Carpinteria, Calif.) for 30 min in room
temperature in the dark. The cells were then counterstained with 1
.mu.g/ml 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes,
Eugene, Oreg.) in PBS for 5 min. Each experiment was performed with
an IgG control in which cells were labeled only with secondary
antibody, Alexa Fluor 488 goat anti-rabbit IgG (H+L) or
FITC-conjugated F(ab')2 fragment of goat anti-mouse
immunoglobulins, without primary antibody incubation to estimate
the extent of nonspecific binding of the secondary antibody to the
cells.
[0492] Measurement of Cell Fluorescence by Laser Scanning
Cytometry
[0493] Cellular green (phosphorylated histone H2AX and cleaved
caspase 3), and blue (DNA-bound DAPI) fluorescence emission was
measured using a Laser Scanning Cytometer (LSC; CompuCyte,
Cambridge, Mass.), utilizing standard filter settings; fluorescence
was excited with 488-nm argon ion and violet diode lasers,
respectively. The intensities of maximal pixel and integrated
fluorescence were measured and recorded for each cell. At least
3,000 cells were measured per sample.
[0494] Statistical Analysis
[0495] To compare the changes in immunofluorescence intensity, the
mean fluorescence intensity (integral values of individual cells)
was calculated for cells in each phase of the cycle by gating G1, S
and G2/M cells based on differences in DNA content. The means of
the fluorescence value for G1, S and G2/M populations of cells in
the IgG control groups were then subtracted from the respective
means of the smoke condensate or smoke-treated cells. All
experiments were run under identical instrument settings. Data is
presented as mean .DELTA.H2AX fluorescence of each cell cycle
compartment or where not indicated, of the entire population (G1, S
and G2M). Each experiment was run in duplicate or triplicate. All
experiments were repeated at least three times.
[0496] Filter Comparison
[0497] Tests for phosphorylated histone H2AX also were applied to
tests of several filters attached to different tobaccos. Filters
and tobacco were obtained from: (1) the industry standard reference
tobacco IM16 (Philip Morris.RTM. USA); (2) reduced risk cigarette
Omni.RTM. (Vector Tobacco Ltd.); and (3) reduced risk cigarette
Quest 3.RTM. (Vector Tobacco Ltd.). A549 cells were exposed to mock
treatment (control) and cigarette smoke substantially as provided
in the above smoke treatment description.
[0498] Each of IM16, Omni.RTM. and Quest 3.RTM. were tested, and
the .DELTA.H2AX (smoke-mock) time course for each is presented in
FIG. 44A. FIG. 44A demonstrates that each of IM16, Omni.RTM. and
Quest 3.RTM. have clearly different influences on .DELTA.H2AX
levels, where the yH2AX levels parallel the expected level of risk
attributed to the tobacco product (IM16 is highest expected risk
and has the highest .DELTA.H2AX levels, while Quest 3.RTM. is
lowest expected risk and has the lowest .DELTA.H2AX levels).
[0499] Next, the influence of IM16, Omni.RTM. and Quest 3.RTM.
filters were compared by configuring a cigarette with IM16 tobacco,
and each of the IM16, Omni.RTM. and Quest 3.RTM. filters. FIG. 44B
demonstrates that the cigarette configured with the IM16 filter
resulted in the highest .DELTA.H2AX levels, while the cigarette
configured with the Quest 3.RTM. filter resulted in the lowest
.DELTA.H2AX levels. Thus, when the same tobacco (IM16) is used, the
.DELTA.H2AX levels reflect the influence of the filter on the
number of double stranded DNA breaks caused by tobacco smoke. In
the instant example, when IM16 tobacco is used, the .DELTA.H2AX
levels parallel the expected level of risk attributed to the
tobacco filter (IM16 is highest risk filter and has the highest
.DELTA.H2AX levels, while Quest 3.RTM. is lowest risk filter and
has the lowest .gamma.H2AX levels).
[0500] Next, the filters were tested using Omni.RTM. and Quest
3.RTM. tobaccos. FIG. 44C demonstrates that when Omni.RTM. tobacco
is used, a cigarette containing the IM16 filter results in
comparable .DELTA.H2AX levels as compared to a cigarette containing
the Omni.RTM. filter. Thus, FIG. 44C demonstrates that the
risk-reducing properties of the tobacco and the risk-reducing
properties of the filter can be interrelated such that the
risk-reducing properties of a particular filter can vary depending
on the type of tobacco used. FIG. 44D demonstrates that when Quest
3.RTM. tobacco is used, a cigarette containing the IM16 filter
results in higher .gamma.H2AX levels as compared to a cigarette
containing the Quest 3.RTM. filter.
[0501] Exposure of A549 cells to TS induces H2AX phosphorylation,
which can be detected immunocytochemically (FIG. 12). Though the
intensity of green .DELTA.H2AX IF varies from cell to cell, its
distribution is nuclear and punctate. Mock-treated cells have
minimal, but still detectable levels of .DELTA.H2AX IF.
[0502] FIG. 13 illustrates the raw data in the form of scattergrams
of the A549 cells untreated (0 time) and exposed to TS for 30 min.
A scattergram representing cells immunostained with an irrelevant
isotype control IgG is also included in the figure. The intensity
of fluorescence of the mock-exposed cells is distinctly higher than
that of the isotype control. This is a reflection of the
"programmed" phosphorylation of H2AX, known to occur during normal
progression through the cell cycle. Exposure of A549 cells to
smoke, in this instance, markedly increased cellular .DELTA.H2AX
IF. The increase, however, was proportional for the cells in each
phase of the cell cycle.
[0503] As noted above, the mean "programmed" H2AX IF was subtracted
from the mean .DELTA.H2AX IF of the cells exposed to either smoke
or smoke condensate, separately for cells in each phase of the cell
cycle, for each data-point shown in the FIGS. 14 and 15. In
addition, since the amount of histone doubles as cells proceed from
G.sub.1 to G.sub.2 phase, .DELTA.H2AX IF was normalized to
DNA/histone content by dividing the mean .DELTA.H2AX IF of the S
and G.sub.2M phase cells by 1.5 and 2, respectively. The normalized
data, therefore, does not represent the total amount of
phosphorylated H2AX per cells but rather the degree of H2AX
phosphorylation, independent of the increase in total H2AX IF that
occurs during progression through S.
[0504] During the initial 10 min exposure of A549 cells to smoke,
no change in .gamma.H2AX IF was apparent (FIG. 14). However,
between 10 and 20 min exposure to smoke, .DELTA.H2AX IF increased
by 71%, 67.5% and 45.7% for G.sub.1, S and G.sub.2M phase cells,
respectively. An additional 10 min of exposure to smoke (30 min in
total) resulted in an additional increase in .DELTA.H2AX IF
compared to mock-exposed cells: 151.2%, 132.2% and 109.3% for
G.sub.1, S or G.sub.2M phase cells.
[0505] The plots shown in FIG. 15 display the increase in the level
of H2AX phosphorylation as a function of length of exposure of NHBE
cells to 10, 25 or 50 .mu.g/ml concentrations of smoke condensate.
At each concentration, the maximal rate of increase in H2AX IF was
seen during the initial 4 h of cell treatment. However, whereas at
10 and 25 .mu.g/ml of smoke condensate the peak of H2AX
phosphorylation occurred at 4 h, followed by a plateau up to 24 h,
at a smoke condensate concentration of 50 .mu.g/ml, H2AX
phosphorylation increased during the entire 24 h time course of the
experiment. No cell cycle phase specificity was apparent in H2AX
phosphorylation when cells were exposed to 10 .mu.g/ml smoke
condensate (FIG. 16). The same was true for these cells exposed to
25 or 50 .mu.g/ml.
[0506] Activation of caspase-3 was measured in samples parallel to
those that were subjected to analysis of H2AX phosphorylation, by
detecting the presence of activated caspase-3 immunocytochemically.
Exposure of A549 cells to smoke for up to 40 min followed by their
fixation at 15 minutes had no effect on caspase-3 activation: less
than 0.5% of the cells demonstrated the presence of activated
caspase-3 in either mock-exposed or smoke treated cultures (Table
6). Caspase-3 activation could be shown, however, if A549 cells
exposed to smoke for 20 min were allowed to grow in culture for an
extended period of time (24 h) at which point virtually half the
cells were positive for activated caspase-3 (Table 6).
TABLE-US-00012 TABLE 6 Effect of Smoke on Caspase-3 Activation
Exposure to smoke Time in culture % Caspase-3 positive (min)
following exposure (h) cells (%)* 0 0.25 0.1 10 0.25 0.4 20 0.25
0.1 30 0.25 0.4 40 0.25 0.1 0 24 0.2 20 24 49.9 *Caspase-3 positive
cells were detected immunocytochemically, as described
elsewhere.
[0507] The present results demonstrate that exposure of A549 cells
to TS or NHBE cells to TSC induces phosphorylation of H2AX. The
extent of H2AX phosphorylation is concentration-dependent. It also
correlated with the duration of exposure. In the case of NHBE
cells, while at lower smoke condensate concentrations (10 and 25
.mu.g/ml), a plateau is achieved after 4 h, at 50 .mu.g/ml
concentration, progressive phosphorylation continues for up to 24
h. H2AX phosphorylation in the A549 cells exposed to smoke also
progresses with time of exposure, although it appears to plateau
after 30 min. Phosphorylation of H2AX is a specific marker of
induction of DSBs; the present data indicate that TS and TSC both
induce such breaks in A549 cells and NHBE cells in a dose and time
dependent manner.
[0508] It should be noted that H2AX is intensely phosphorylated in
response to DNA fragmentation that occurs upon induction of
apoptosis. Caspase activation, however, is required to trigger
apoptosis-related DNA fragmentation. In fact, inhibition of
caspase-3 activity (e.g. by z-VAD-FMK) can prevent the
apoptosis-associated H2AX phosphorylation. In the present study, no
caspase-3 activation was detected in the cells exposed for up to 40
min to smoke (Table 6). Thus, apoptosis-associated phosphorylation
of H2AX did not contribute to the .DELTA.H2AX IF measured in A549
cells exposed to smoke for up to 40 min, when the cells were
collected within 15 min of exposure.
[0509] The present assay provides quantitative results.
Specifically, the number of H2AX phosphorylation foci is considered
to correspond to the number of DSBs. Assuming that the individual
foci have comparable intensity of IF, the integrated value H2AX IF,
as presently measured, would be expected to correspond to the
number of foci, hence, to the number of DSBs. Furthermore, the mean
.DELTA.H2AX IF of the mock-exposed cells was subtracted from each
mean of cells exposed to smoke or smoke condensate, to ensure that
the measurement was not affected by the level of "programmed" H2AX
phosphorylation in these cells (see FIG. 13). Though not applicable
in the present instance in which the time between exposure to smoke
or smoke condensate and harvesting of the cells was relatively
short (55 min or less), a phosphatase inhibitor such as calyculin A
or okadaic acid can be included in the culture to prevent possible
dephosphorylation of H2AX molecules. The data presented in the
plots, therefore, represent the smoke-induced differential
.DELTA.H2AX IF. Furthermore, since the H2AX content increases as
cells traverse through S phase, the mean values .DELTA.H2AX IF for
S and G.sub.2/M cells were compensated for the H2AX increase. The
intensity of .DELTA.H2AX IF so compensated, thus, reflects the
degree of H2AX phosphorylation in the cell, i.e. is unrelated to
H2AX content.
[0510] There is little evidence that CS and specific smoke
constituents can cause single strand breaks (SSBs) in the normal
human genome, but no evidence for the induction of DSBs. DSBs are
among the most deleterious types of DNA damage in mammalian cells.
A cell that incurs DSBs is at major risk for developing genomic
instability, which can result in an array of specific defects such
as chromosome fragmentation, translocation, rearrangement and loss.
More importantly, each of these chromosomal abnormalities can play
a pivotal role in the etiology or progression of a wide range of
human cancers. Consequently, in order to ensure the faithful repair
of DSBs and maintain genomic integrity, the cell has evolved
sensitive DNA damage-activated checkpoint control pathways that are
coupled to an interconnected web of efficient repair mechanisms,
the most prominent of which are homologous recombination and
non-homologous end joining. Individuals who either have
debilitating alterations or deletions of the genes involved in
detecting and repairing DSBs tend to manifest the dual syndromes of
chromosome instability and higher incidence of various cancers.
Clearly, therefore, the induction of DSBs by an exogenous agent
like TS can be a potentially hazardous genetic event in the
long-term smoker. In particular, if overall repair efficiencies of
DSBs are not as efficient as for other types of DNA damage, e.g.,
single strand breaks (SSBs), and/or if an individual smoker has
specific polymorphisms in the relevant genes that reduce their
effectiveness, then cells chronically exposed to TS can manifest
the genetically dangerous combination of increased levels of DSBs
and compromised repair capacities. Furthermore, in addition to DSB
level and repair capacity, the genomic positioning of DSBs can be
another factor that determines how successfully a cell responds to
this type of damage. For example, the probability that a DSB break
is inaccurately rejoined is relatively low when DSBs are spatially
separated but increases considerably when multiple breaks
coincide.
[0511] The successful repair of DSBs appears also to depend on cell
cycle position. The data, however, show no obvious cell cycle
specificity in terms of accumulation of DSBs. Thus, if
proliferating cells exposed to TS experience similar levels of DSBs
during each phase of the cell cycle but dissimilar repair rates,
they can be particularly susceptible to accumulating deleterious
DNA defects during that specific phase. It is relevant to point out
that although the rates of DSB induction and repair in noncycling
cells, which are one of the initial primary target cells in lungs
exposed to CS, can be different than in cycling cells, the lungs of
persistent smokers undergo a significant increase in the number of
proliferating cells due to smoke-induced damage. Moreover, cells
actively dividing at the time of carcinogen exposure are at
particular risk for transformation-related events.
[0512] The methods of identifying a tobacco, identifying a compound
in tobacco, identifying a tobacco product, and making a tobacco
product provided herein, can additionally be used to compare two or
more tobaccos so as to identify a toxic compound, evaluate the
potential risk posed by the tobacco products, or to develop reduced
risk tobacco products. In some embodiments, the two or more
tobaccos are compared for their ability to induce damage to the
genetic material of cells. In some embodiments, at least one
tobacco is a reduced risk tobacco or identified as a reduced risk
tobacco. In some embodiments, at least one tobacco is a modified
tobacco, such as a chemically modified tobacco or a genetically
modified tobacco.
[0513] Example 2 below provides one non-limiting specific example
of methods for comparing tobaccos in accordance with the methods
provided herein. Variations of the assay method used in terms of
assay methodologies (e.g., assay for apoptosis or for cell
proliferation) would be apparent to the skilled artisan for
comparing tobaccos.
Example 2
[0514] A549 cells were exposed to whole smoke from IM16 cigarettes
for various lengths of time, washed and allowed to grow for an
additional hour before being harvested for analysis. DNA damage was
identified as an increase in phosphorylation of histone H2AX
denoted as .DELTA.H2AX.
[0515] In order to compare DNA damage as a function of the cell's
position in the cell cycle, .DELTA.H2AX values were normalized to
DNA content since histone content doubles as cells proceed from G1
to G2 phase. Thus, in order to determine any change in histone H2AX
phosphorylation independent of changes in histone/DNA content or
DNA ploidy, the values for S and G2M phase populations, gated
according to DNA content, were multiplied by 0.75 and 0.5,
respectively. In instances where "normalized" values of .DELTA.H2AX
are presented, these values were obtained by subtracting the mean
values of each cell cycle population (or the total population) from
the mean of the mock-treated population whose .DELTA.H2AX values
represent "scheduled" .DELTA.H2AX expression. In all instances, the
values presented represent the mean .DELTA.H2AX fluorescence of the
population; typically 3-5.times.103 cells were analyzed for each
condition.
[0516] As illustrated in FIG. 17, there was little or no change in
.DELTA.H2AX when exposure of A549 cells to whole smoke was limited
to 5 min. However, as the time of exposure exceeded 5 min there was
a more or less linear increase in .DELTA.H2AX. Initially, S phase
cells appeared most sensitive to DNA damage expressing
approximately 37% higher levels of .DELTA.H2AX than G1 phase cells
following 10 min of exposure to smoke. When the length of exposure
was increased to 20 min, G1 phase cells invariably expressed 10-20%
higher levels of .DELTA.H2AX-associated fluorescence.
[0517] In another set of experiments it was determined that the
extent of DNA damage varied with the length of time of recovery
following exposure to whole smoke. Previous studies have shown that
following exposure to whole smoke for times in excess of 20 min
leads to a significant increase in apoptotic cells in the
population depending upon when the assay is performed. Apoptotic
cells contained significantly increased levels .gamma.H2AX compared
to what one sees when assessing the primary breaks due to DNA
damaging agents. Based on the absence of activation of caspase 3,
there is little or no induction of apoptosis in A549 cells within
the first 3 h following 20 min exposure to whole smoke from
IM16.
[0518] Within 15 min of exposure to whole smoke, A549 cells already
displayed a dramatic increase in .DELTA.H2AX relative to mock
exposed cells (FIG. 18A). Increasing the recovery time following
exposure led to continued increase in DNA damage. As noted above,
G1 cells appear to be the most sensitive to smoke especially when
the cells are harvested 30 min or longer after exposure to whole
smoke. The relative increase in .DELTA.H2AX following 60 min of
recovery is illustrated in FIG. 18B where it can be observed that
virtually all smoke-exposed cells (right) express levels of
.DELTA.H2AX in excess of the expression observed in the
mock-treated cells (left).
[0519] The response of NHBE cells to whole smoke from IM16
cigarettes was more or less identical to that observed for A549
cells (FIG. 18C). The one difference between the two cell lines was
that S phase cells in NHBE cultures always expressed higher
"scheduled" amounts of .DELTA.H2AX. Nevertheless, as with A549
cells, G1 cells are the most sensitive to smoke-induced DNA damage
in these cultures. FIG. 18D demonstrates both the increased basal
level of .DELTA.H2AX in mock-treated cultures (left) and the
extensive increase in .DELTA.H2AX expression observed 60 min
following a 20 min exposure of cells to whole smoke (right).
[0520] In the next series of experiments, the DNA damage caused by
whole smoke from different sources was compared. Using an exposure
time of 20 min, damage due to whole smoke from two other cigarettes
could be compared to that caused by IM16 following various recovery
times. The curves of .DELTA.H2AX following exposure of A549 cells
to IM16 (FIG. 19, top right) were comparable to that displayed in
FIG. 18A. Exposure of the same cells to whole smoke from Quest
3.RTM. on the other hand resulted in an initial increase in
.DELTA.H2AX at 30 min that returned to near background levels when
assayed after longer recovery times (FIG. 19, bottom left). Whole
smoke from Omni.RTM. cigarettes caused damage intermediate between
that of Quest 3.RTM. and IM16 (FIG. 19, bottom right). The DNA
damage caused by Omni.RTM. increased until 60 min after which it
more or less plateaued. Smoke from Quest 3.RTM. cigarettes affects
S phase cells to a greater extent than any other phase while G1
cells are invariably most sensitive to smoke from IM16 and
Omni.RTM.. Importantly, these data demonstrate that tobacco
products containing modified tobacco (i.e., Omni.RTM. and Quest
3.RTM.) induced less DNA damage than a reference tobacco product
(i.e., IM16). Accordingly, the modified tobacco products Omni.RTM.,
and Quest 3.RTM. have a reduced potential to contribute to a
tobacco related disease (i.e., Omni.RTM., and Quest 3.RTM. are
reduced risk tobacco products) according to the double strand break
assay.
[0521] In the next series of experiments, it was determined that
DNA damage caused by whole smoke can be mitigated by the presence
of NAC. Using a standardized set of conditions (20 min of exposure
followed by a 1 h recovery), DNA damage caused by whole smoke from
IM16 cigarettes was assayed in both A549 and NHBE cells. NAC at a
concentration of 25 mM was either absent or present during exposure
and absent or present during the 1 h recovery time. In this
instance, the background or "scheduled" .DELTA.H2AX expression
observed in Mock-treated cells was subtracted from each
measurement. The remaining fluorescence should be indicative of the
level of DNA DSBs under each set of conditions.
[0522] In A549 cells (FIG. 20, top), IM16 caused a dramatic
increase in H2AX phosphorylation in the absence of NAC (PBS, PBS).
Applying NAC to the media following exposure to smoke did nothing
to mitigate the DNA damage caused by whole smoke. However, if NAC
was present during exposure to smoke, DNA damage was suppressed by
greater than 80% for the entire population; the suppression was
greatest for G1 cells (91%), intermediate for G2M (88%) and least
for S (82%) phase cells. The presence of NAC both during exposure
to smoke and during the 1 h recovery period provided slightly more
protection increasing suppression of .DELTA.H2AX to 90% for the
entire population.
[0523] As with A549 cells, when NHBE cells were exposed to whole
smoke from IM16 cigarettes, the cells in G1 phase were the most
sensitive. However, since the S phase cells express somewhat higher
levels of "scheduled" .DELTA.H2AX and are not as sensitive as G1
cells to smoke (FIG. 18C), the value for S phase cell DNA damage
was considerably less than for cells in G1 or G2M phase (FIG. 20,
bottom). Addition of NAC only during recovery had little effect on
the level of DNA damage induced by whole smoke. NAC present during
exposure diminished the damage observed in G1 cells by nearly 69%;
the decrease was about 65% for G2M cells but S phase cells were
afforded no protection. NAC present both during exposure and
recovery provided a small degree of additional protection.
[0524] Next, the effect of NAC on DNA damage caused by whole smoke
from various sources was evaluated. A549 cells were exposed to
smoke from IM16, Omni.RTM. and Quest 3.RTM. cigarettes in the
presence and absence of NAC during exposure. As illustrated in FIG.
21, NAC dramatically reduced the effects of smoke from IM16 cells.
Omni.RTM. produced less damage than IM16 but NAC reduced the damage
to near background levels. Quest 3.RTM. smoke caused the least
amount of damage which could also be reduced to background levels
by the presence of 25 mM NAC during exposure. In all instances, the
level of damage following exposure to smoke in the presence of NAC
was approximately the same, just slightly more than the background
or scheduled level of .DELTA.H2AX expression. As above, the data
from this assay demonstrates that tobacco products containing
modified tobacco (i.e., Omni.RTM. and Quest 3C)) induced less DNA
damage than a reference tobacco product (i.e., IM16). Again, the
double strand break assay has shown that the modified tobacco
products Omni.RTM., and Quest 3.RTM. have a reduced potential to
contribute to a tobacco related disease (i.e., Omni.RTM., and Quest
3.RTM. are reduced risk tobacco products).
[0525] In more experiments, the cell cycle specific inhibition of
whole smoke-induced DNA damage by NAC was analyzed. A549 cells were
exposed to whole smoke in the presence and absence of various
concentrations of NAC. Exposure was always for 20 min and recovery
was 1 h. In each instance, the background or "scheduled" expression
of .gamma.H2AX was subtracted from the value obtained for each
population in each cell cycle phase. Since G1 phase cells were the
most sensitive and had the highest value, all other measurements
were normalized to that of G1 phase cells exposed to IM16 smoke in
the absence of NAC (plotted as 0.1 mM NAC on the log plot).
[0526] As can be seen in FIG. 22, damage by whole smoke from IM16
to S phase A549 cells was unaffected by the presence of NAC up to a
concentration of 5 mM. In contrast, damage caused to both G1 and
G2M cells began to decrease when as little as 1 mM NAC was present
during exposure. The damage caused to S phase cells decreased
sharply as the NAC concentration was increased to 10 mM and, by 25
mM, there was little difference in residual .DELTA.H2AX expression
between cells in any phase of the cycle.
[0527] The concentration of NAC that reduced DNA damage by 50% for
each cell cycle phase can be determined from the graph in FIG. 22.
For G1, S and G2M phase cells the values were approximately 4.5,
2.6 and 7.5 mM NAC.
[0528] In more experiments, it was determined that the vapor phase
of smoke induces damage that is abrogated by the presence of NAC.
FIG. 23 (top) illustrates the ability of the vapor phase of smoke
from various tobacco sources to cause DNA damage to A549 cells in
comparison to whole smoke from IM16 cigarettes. Thus, the vapor
phase from IM16 cigarettes using standard conditions of exposure
and recovery caused only about 26% of the DNA damage (.gamma.H2AX)
as whole smoke from the same source. In the same comparison, the
vapor phase from Quest 1.RTM. and Quest 3.RTM. caused only 8.1% and
5.6% of the damage caused by whole smoke from IM16. As a direct
comparison, the vapor phase of smoke from Quest 1.RTM. and
Quest.RTM. caused 68.8% and 78.5%, respectively, less damage than
the vapor phase of smoke from IM16.
[0529] The presence of 25 mM NAC during exposure of A549 cells to
whole smoke form IM16 cigarettes reduced .DELTA.H2AX by nearly 90%
(89.1%) compared to cells exposed to whole smoke in the absence of
NAC. NAC present during cell exposure to the vapor phase of smoke
from IM16, Quest 1.RTM. and Quest 3.RTM., reduced .DELTA.H2AX by
93.2%, 98.9% and 100%, respectively compared to the damage caused
by the vapor phase of smoke in the absence of NAC.
[0530] The same experiment performed on NHBE cells resulted in more
or less comparable results (FIG. 23, bottom). Whole smoke from IM16
cells produced less damage in NHBE cells under standard conditions
compared to A549 cells (note the greater background observed in
NHBE cells). The vapor phase from IM16 CS caused only about 30%
(29.7%) of the damage caused by whole smoke whereas the vapor phase
of smoke from Quest 1.RTM. caused 97% less damage than whole smoke
from IM16 cigarettes. The vapor phase of smoke from Quest 3.RTM.
produced no increase in .DELTA.H2AX over background in NHBE
cells.
[0531] The presence of NAC during exposure of NHBE cells to whole
smoke from IM16 cigarettes reduced .DELTA.H2AX by about 78%
(77.9%). The presence of NAC during exposure of cells the vapor
phase of IM16, Quest 1.RTM. or Quest 3.RTM. abolished virtually all
DNA damage relative to mock-treated cells; i.e., .DELTA.H2AX was
reduced to background levels or below.
[0532] The cell cycle phase specific results are comparable to that
for the whole populations (FIG. 24). The vapor phase of smoke from
IM16 caused comparable amounts of damage in each cell cycle phase
in A549 cells though the reduction of damage in G1 phase by NAC was
somewhat higher than it was for S and G2M phase; 98.5% versus 89.0%
and 92.2%, respectively. The vapor phase from both Quest 1.RTM. and
Quest 3.RTM. caused more damage to S phase cells though in each
instance, the presence of NAC reduced damage to background levels
for each cell cycle phase.
[0533] NHBE cells as noted earlier have higher .DELTA.H2AX levels
in S phase of mock-treated cells as can be seen in FIG. 24. The
largest increase in damage caused by the vapor phase of smoke from
IM16 occurred in G1 phase cells (54.4% and 66.9% greater than for
cells in S or G2M, respectively). The presence of NAC reduced the
damage caused by the vapor phase of smoke from IM16 to background
levels or below. The vapor phase of smoke from Quest 1.RTM. and
Quest 3.RTM. cigarettes had only a small effect on DNA damage in
cells in G1 or S but not G2M phase. All damage caused by the vapor
phase of smoke from Quest.RTM. cigarettes in NHBE cells was
inhibited in the presence of NAC. Importantly, this data provide
more evidence that the tobacco products containing modified tobacco
(i.e., Quest 1.RTM. and Quest 3.RTM.) induced significantly less
DNA damage (i.e., double strand DNA breaks) than that of a
reference tobacco product (i.e., IM16). Accordingly, the modified
tobacco products Quest 1.RTM., and Quest 3.RTM. have a reduced
potential to contribute to a tobacco related disease (i.e., Quest
1.RTM. and Quest 3.RTM. are reduced risk tobacco products,
according to the double strand DNA break assay.
[0534] FIGS. 30, 32 and 33 show additional comparisons of reactions
of A549 cells to smoke from various cigarettes, where the affect
can vary for different cigarettes, and can vary according to the
cell cycle of the cells, and can vary according to the presence of
antioxidant.
[0535] Further performed was a test of double-strand DNA breaks in
the cells of a human subject exposed to tobacco smoke. The level of
.DELTA.H2AX expression in the buccal mucosa of a smoker was
compared to the level of .DELTA.H2AX expression in the buccal
mucosa of a nonsmoker. A cheek swab was collected from a subject
(smoker) within 5 min completion of smoking a Marlboro Light.RTM.
cigarette, and a second check swab was collected from a subject
that did not smoke a cigarette (non-smoker). Levels of .DELTA.H2AX
were then measured for both cell samples. As seen in FIG. 31 the X
axis depicts .DELTA.H2AX associated fluorescence (.gamma.H2AX), and
the Y axis depicts the number of cells having the corresponding
.gamma.H2AX fluorescence level. There were 358 cells with a very
low value of .DELTA.H2AX in the non-smoker sample, whereas the
smoker sample had cells with .DELTA.H2AX values spread over a wide
range. Each histogram represents 3.times.103 cells. The buccal
cells from the smoker showed a low number of cells having little or
no .DELTA.H2AX fluorescence signal, and showed a large number of
cells with higher .DELTA.H2AX fluorescence levels. In contrast,
almost all cells of the non-smoker had little or no .DELTA.H2AX
fluorescence. Thus, human buccal cells exposed to tobacco smoke
have an increased level of double strand DNA breaks relative to
human buccal cells not exposed to tobacco smoke. These results
parallel the in vitro results observed for A549 cells and for NHBE
cells. Thus, the in vitro approaches described herein are
predictive of in vivo responses.
[0536] Accordingly, the methods that were applied to A549 cells and
NHBE cells for comparing different tobacco products, analyzing
cells at different stages in cell cycle, and determining protection
provided by the presence of an antioxidant, will be performed on
human samples of buccal cells and it is expected, as shown in the
in vitro experiments, that modified tobaccos, in particular
genetically modified tobaccos that have a reduced amount of one or
more compounds that contribute to a tobacco related disease (e.g.,
genetically modified tobacco having a reduced nicotine, TSNA,
and/or sterol content) will induce fewer or a reduced amount of
double strand DNA breaks in humans that are contacted with smoke
from said modified tobaccos than will be observed in humans that
are contacted with smoke from conventional tobacco products,
reference tobacco products, or non-transgenic (wild-type tobacco of
the same variety as the parental strain prior to genetic
modification). The following section describes several methods to
evaluate the ability of a tobacco or a tobacco product to modulate
apoptosis in greater detail.
[0537] Analysis of Changes in Cell Homeostasis: Changes in the
Fidelity of the DNA, Double Strand Breaks
[0538] By one approach, for example, CS is generated using a
smoking machine from a first tobacco modified product, e.g., a
product containing tobacco that has been genetically modified to
have a reduced amount of a compound. A first population of NHBE
cells is contacted with said CS obtained from the modified tobacco
product, and the cells contacted with CS are assayed for
double-strand DNA breaks. A second population of NHBE cells is then
contacted with CS generated from an unmodified tobacco product,
wherein the unmodified tobacco product retains the component that
was removed or inhibited in the modified tobacco product. An
unmodified tobacco product can be, for example a product containing
the parental variety of tobacco, where the parental variety of
tobacco is the unmodified tobacco variety used to generate the
modified tobacco variety. The second population of cells contacted
with CS is then assayed for double-strand DNA breaks. A comparison
of the data obtained from the analysis of the first and second
tobacco products will reveal that the difference in double-strand
DNA breaks caused by the modified tobacco product relative to the
unmodified tobacco product. By this approach, one can effectively
identify the contribution of individual components of a tobacco
product to double-strand DNA breaks, or other assay conditions
provided herein. These methods can thereby be used to identify the
contribution of individual components of a tobacco product to a
tobacco-related disease. This approach can be used to develop
tobacco products that are less likely to contribute to a
tobacco-related disease and reduced risk tobacco products
identified by these methods are embodiments provided herein.
Further, tobacco products prepared by these approaches can be
prepared according to good manufacturing processes (GMP) (e.g.,
suitable for or accepted by a governmental regulatory body, such as
the Federal Drug Administration (FDA), and containers that house
said tobacco products can comprise a label or other indicia, with
or without structure-function indicia, which reflects approval of
said tobacco product from said regulatory body.
[0539] Thus, the methods provided herein can be used to
characterize a first and a second tobacco product by providing the
first and second tobacco products, obtaining a first and second
tobacco composition from the first and second tobacco products,
respectively, contacting a first cell with the first tobacco
composition and contacting the second cell with the second tobacco
composition, and identifying one or more attributes of the
contacted cells. Different tobacco products can contain different
levels of carcinogens that can induce various types of cell damage
including mutations, chromosomal aberrations, aberrant sister
chromatid exchanges and micronuclei. Comparison of attributes of
cells contacted with different tobacco compositions can be
performed in the methods provided herein, and such attributes
include, but are not limited to, differential levels of mRNA,
differential levels of protein, induction of damage of cellular
genetic material or modulation of cell homeostasis. Accordingly,
the methods provided herein can be used to compare two or more
tobacco products by assay methods including assay for differential
levels of mRNA, differential levels of protein, induction of damage
of cellular genetic material or modulation of cell homeostasis.
Exemplary assay methods include microarray assays, ELISA assays,
Western blot assays, assays of a double-strand DNA break,
inhibition of apoptosis, or inhibition of cell proliferation.
[0540] In some embodiments, the first and second smoke products are
prepared using essentially equivalent protocols. The phrase,
"wherein the first and second smoke products are prepared using
essentially equivalent protocols," as used herein, means that the
two smoke products can be validly compared. For example, both
products can be smoke or both products can be smoke
concentrates.
[0541] The methods provided herein include methods of identifying a
compound in tobacco that induces damage of cellular genetic
material or modulates cell homeostasis by providing a first
tobacco, obtaining smoke or a smoke condensate from the first
tobacco, contacting a first population of cells with the smoke or
smoke condensate from the first tobacco, identifying induction of
damage of cellular genetic material or modulation of cell
homeostasis in the first population of cells after contact with the
smoke or smoke condensate from the first tobacco, providing a
second tobacco that has been modified to reduce a compound in the
second tobacco, obtaining smoke or a smoke condensate from the
second tobacco, contacting a second population of cells with the
smoke or smoke condensate from the second tobacco, and identifying
an induction of damage of cellular genetic material or modulation
of cell homeostasis in the second population of cells after contact
with the smoke or smoke condensate from the second tobacco, where
an identification of a reduction in the induction of damage of
cellular genetic material or modulation of cell homeostasis in the
second population of cells after contact with the smoke or smoke
condensate from the second tobacco identifies the compound as one
that induces damage of cellular genetic material or modulates cell
homeostasis. Compounds identified in accordance with the methods
provided herein can be, for example, compounds that induce the
double strand DNA breaks, inhibit apoptosis, or inhibit cell
proliferation. In some embodiments, the second tobacco can be
genetically modified to reduce the expression of at least one gene
that regulates production of the compound.
[0542] The compound in tobacco that induces damage of cellular
genetic material or modulates cell homeostasis identified by the
methods provided herein can be a tobacco-derived substance
associated with double-strand DNA breaks (DSBs). The
tobacco-derived substance associated with DSBs can be detected in
the context of comparing the harmful potential of two different
tobacco or smoke products (as provided herein elsewhere) or can be
detected in an environmental context, such as TS in a business
office, train car, or restaurant. The ability to detect the tobacco
derived substance can depend on not only its presence, but also its
concentration in the "tobacco test composition" (which can be
smoke, a smoke concentrate, or, for example, an air sample
containing or potentially containing TS). To that end, useful
parameters for assessing the degree of harmfulness can include, for
example, not only the degree of phosphorylation of H2AX (or
accumulation of another DSB marker), but also the initial rate of
DSB accumulation, the period of time required to reach a plateau
and the degree of phosphorylated DSB at the plateau level where a
rapid rise in the degree of H2AX phosphorylation, a protracted
period of time to reach a plateau, and a high plateau level can be
correlated with increased harmful potential (for example, see FIGS.
14 and 15 and accompanying text). Note that where assay conditions
are relatively prolonged (for example, longer than 55 minutes) it
can be desirable to include, in the assay, a phosphatase inhibitor
such as calyculin A or okadaic acid to inhibit and/or prevent
possible dephosphorylation of H2AX molecules.
[0543] Also provided herein are methods of identifying a tobacco
product that has a reduced potential to contribute to a
tobacco-related disease by providing a first tobacco product,
obtaining smoke or a smoke condensate from the first tobacco
product, contacting a first population of cells with the smoke or
smoke condensate from the first tobacco product, identifying the
presence or absence of an induction of damage of cellular genetic
material or modulation of cell homeostasis in the first population
of cells after contact with the smoke or smoke condensate from the
first tobacco product, providing a second tobacco product,
obtaining smoke or a smoke condensate from the second tobacco
product, contacting a second population of cells with the smoke or
smoke condensate from the second tobacco product, and identifying
the presence or absence of an induction of damage of cellular
genetic material or modulation of cell homeostasis in the second
population of cells after contact with the smoke or smoke
condensate from the second tobacco product, where an identification
of a reduction in the amount or the absence of an induction of
damage of cellular genetic material or modulation of cell
homeostasis in the second population of cells after contact with
the smoke or smoke condensate from the second tobacco product, as
compared to the amount or presence of an induction of damage of
cellular genetic material or modulation of cell homeostasis
identified in the first population of cells identifies the second
tobacco product as one that has a reduced potential to contribute
to a tobacco-related disease. Tobacco products identified as having
a reduced potential to contribute to a tobacco-related disease in
accordance with the methods provided herein can be, for example,
tobacco products that are characterized by a reduced induction of
double strand DNA breaks, a lower level of inhibition of apoptosis,
or a lower level of inhibition of cell proliferation.
[0544] Also provided herein are methods of making a tobacco product
that has a reduced potential to contribute to a tobacco-related
disease by providing a first tobacco, obtaining smoke or a smoke
condensate from the first tobacco, contacting a first population of
cells with the smoke or smoke condensate from the first tobacco,
identifying the presence or absence or amount of induction of
damage of cellular genetic material or modulation of cell
homeostasis in the first population of cells after contact with the
smoke or smoke condensate from the first tobacco, providing a
second tobacco that is genetically modified to reduce the
expression of at least one gene that regulates production of a
compound in the second tobacco, obtaining smoke or a smoke
condensate from the second tobacco, contacting a second population
of cells with the smoke or smoke condensate from the second
tobacco, identifying the presence or absence or amount of induction
of damage of cellular genetic material or modulation of cell
homeostasis in the second population of cells after contact with
the smoke or smoke condensate from the second tobacco, where an
identification of a reduction in the presence or amount of
induction of damage of cellular genetic material or modulation of
cell homeostasis in the second population of cells after contact
with the smoke or smoke condensate from the second tobacco, as
compared to the presence or amount of induction of damage of
cellular genetic material or modulation of cell homeostasis
identified in the first cell population identifies the second
tobacco as one that has a reduced potential to contribute to a
tobacco-related disease, and incorporation of the second tobacco,
which has a reduced potential to contribute to a tobacco-related
disease, into a tobacco product. Tobacco products identified as
having a reduced potential to contribute to a tobacco-related
disease in accordance with the methods provided herein, which are
incorporated into a tobacco product, can be, for example, tobacco
products that are characterized by a lower induction of double
strand DNA breaks, lower level of inhibition of apoptosis, lower
level of inhibition of cell proliferation, or reduced level of
modulation of cell homeostaisis (e.g., a reduced amount of
perturbation of gene expression; such as reduced amount of
expression of genes involved in oncogenesis or a reduced inhibition
of genes involed in oxidative repair as comparied to a conventional
tobacco product). The section that follows describes several
methods for identifying a tobacco or tobacco products that modulate
cell homeostasis.
[0545] Analysis of Changes to Cell Homeostasis: Modulation of
Apoptosis
[0546] In some embodiments, modulation of cell homeostasis can be
identified by determining a modulation of apoptosis. Thus, provided
herein are methods of identifying a tobacco that modulates
apoptosis by providing a tobacco, obtaining a tobacco composition
from the tobacco, contacting a cell with the tobacco composition,
and identifying a modulation of apoptosis in the cell after contact
with the tobacco composition. Also provided herein are methods of
identifying a compound in tobacco that modulates apoptosis, methods
of identifying a tobacco product that has a reduced potential to
contribute to a tobacco-related disease, and methods of making a
tobacco product that has a reduced potential to contribute to a
tobacco-related disease, in accordance with the methods of
identifying a tobacco or tobacco compound that modulates cell
homeostasis provided herein elsewhere. Also provided herein are
methods of identifying a compound in tobacco that modulates
apoptosis, methods of identifying a tobacco product that has a
reduced potential to contribute to a tobacco-related disease, and
methods of making a tobacco product that has a reduced potential to
contribute to a tobacco-related disease, in conjunction with the
methods of identifying a tobacco or tobacco compound that modulates
cell proliferation provided herein.
[0547] Also provided herein are methods of comparing two or more
tobacco products. In some embodiments, a tobacco or tobacco
compound that induces a lower degree of apoptosis can be
characterized as a tobacco that has a potential to contribute to a
tobacco-related disease. In some embodiments, a first tobacco that
induces a lower degree of apoptosis than a second tobacco can be
characterized as a tobacco that has an increased potential to
contribute to a tobacco-related disease. In some embodiments, a
first tobacco that induces a higher degree of apoptosis than a
second tobacco can be characterized as a tobacco that has a reduced
potential to contribute to a tobacco-related disease. In some
embodiments, a tobacco or tobacco compound that induces a higher
degree of apoptosis can be characterized as a tobacco that has a
potential to contribute to a tobacco-related disease. In some
embodiments, a first tobacco that induces a higher degree of
apoptosis than a second tobacco can be characterized as a tobacco
that has an increased potential to contribute to a tobacco-related
disease. In some embodiments, a first tobacco that induces a lesser
degree of apoptosis than a second tobacco can be characterized as a
tobacco that has a reduced potential to contribute to a
tobacco-related disease. In some embodiments, the methods of
identifying a tobacco that modulates apoptosis can be used to
identify modified tobacco that modulates apoptosis as provided
herein or otherwise known in the art.
[0548] Also provided herein are methods of comparing two or more
tobacco products. In some embodiments, a tobacco or tobacco
compound that inhibits apoptosis can be characterized as a tobacco
that has a potential to contribute to a tobacco-related disease. In
some embodiments, upon inducing the same degree of DNA damage
(DSBs) a first tobacco that induces lesser degree of apoptosis than
a second tobacco can be characterized as a tobacco that has an
increased potential to contribute to a tobacco-related disease. In
some embodiments, upon inducing the same degree of DNA damage
(DSBs) a first tobacco that induces lesser degree of apoptosis than
a second tobacco can be characterized as a tobacco that has a
reduced potential to contribute to a tobacco-related disease. In
some embodiments, a tobacco or tobacco compound that increases
apoptosis can be characterized as a tobacco that has a potential to
contribute to a tobacco-related disease. In some embodiments, a
first tobacco that increases apoptosis to a greater degree than a
second tobacco can be characterized as a tobacco that has an
increased potential to contribute to a tobacco-related disease. In
some embodiments, a first tobacco that increases apoptosis to a
lesser degree than a second tobacco can be characterized as a
tobacco that has a reduced potential to contribute to a
tobacco-related disease. In some embodiments, the methods of
identifying a tobacco that modulates apoptosis can be used to
identify modified tobacco that modulates apoptosis as provided
herein or otherwise known in the art.
[0549] As used herein, a tobacco or tobacco compound that induces a
lower or higher degree of apoptosis refers to a tobacco or tobacco
compound that causes a cell or cell population to decrease or
increase, respectively, apoptosis in that cell or cell population
relative to a cell or cell population that is not contacted by the
tobacco or tobacco compound. Any of a variety of methods can be
used to determine apoptosis in a cell or cell population, including
those provided herein, and other methods known in the art.
[0550] While not intending to be limited by the following
explanation, a decreased degree of apoptosis in cells may result in
cells with damaged DNA that can survive and be tumorigenic rather
than die and be eliminated. In other cellular functions, extensive
apoptosis may induce compensatory stem cell proliferation and
result in tumorigenesis. Accordingly, as contemplated herein an
increase or decrease in apoptosis can lead to a tobacco-related
disease.
[0551] Also provided herein are methods of comparing two or more
tobacco products when the two or more tobacco products induce the
same level of damage to cells. In some embodiments, a tobacco or
tobacco compound that inhibits apoptosis can be characterized as a
tobacco that has a potential to contribute to a tobacco-related
disease. In some embodiments, upon inducing the same degree of DNA
damage (DSBs) a first tobacco that induces lesser degree of
apoptosis than a second tobacco can be characterized as a tobacco
that has an increased potential to contribute to a tobacco-related
disease. In some embodiments, upon inducing the same degree of DNA
damage, a first tobacco that induces lesser degree of apoptosis
than a second tobacco can be characterized as a tobacco that has a
reduced potential to contribute to a tobacco-related disease. In
some embodiments, upon inducing the same degree of DNA damage, a
tobacco or tobacco compound that increases apoptosis can be
characterized as a tobacco that has a potential to contribute to a
tobacco-related disease. In some embodiments, upon inducing the
same degree of DNA damage, a first tobacco that increases apoptosis
to a greater degree than a second tobacco can be characterized as a
tobacco that has an increased potential to contribute to a
tobacco-related disease. In some embodiments, upon inducing the
same degree of DNA damage, a first tobacco that increases apoptosis
to a lesser degree than a second tobacco can be characterized as a
tobacco that has a reduced potential to contribute to a
tobacco-related disease. In some embodiments, the methods of
identifying a tobacco that modulates apoptosis can be used to
identify modified tobacco that modulates apoptosis as provided
herein or otherwise known in the art.
[0552] The methods provided herein can include one or more steps of
determining modulation of apoptosis. Typically, such methods
include assays for modulation of apoptosis in a population of
cells. Any of a variety of methods known in the art for assaying
apoptosis can be used in the methods provided herein. Exemplary
known assays include assays for activation of apoptosis-related
proteins, assays for double-strand DNA breaks, and assays for
membrane permeability.
[0553] In one exemplary method, modulation of apoptosis can be
identified by determining caspase activation. Caspases are
proteases involved in apoptosis. Activation of caspases can lead to
apoptosis in the cell. Accordingly, measurement of activated
caspases can be used to identify apoptosis in cells. Typically,
caspases are activated by a cleavage reaction. Thus, activated
caspase can be determined by detecting activated cleaved caspases.
For example, caspase activation can be identified using an antibody
or fragment thereof, which binds to activated caspase but not
inactive caspase. There are a number of caspases that can be
screened in accordance with the methods provided herein, including
but not limited to, caspase 1, 3 and 9. In another example,
activation of caspase by its catalytic activity can be determined.
For example, caspase-3 has substrate selectivity for the amino acid
sequence Asp-Glu-Val-Asp (DEVD) (SEQ. ID. NO. 1). A fluorogenic
indicator such as Ac-DEVD-AMC can be used for fluorometric assay of
caspase-3 activity. A variety of caspase activation assays are
known in the art, as exemplified in Gown et al., J. Histochem.
Cytochem. (2002) 50:449-54; Iordanov et al., Apoptosis (2005)
10:153-66; and Kahlenberg et al., J. Leukoc. Biol. (2004)
76:676-84, all of which are hereby expressly incorporated by
reference in their entireties.
[0554] In another exemplary method, modulation of apoptosis can be
identified by determining cleavage of the protein poly(ADP-ribose)
polymerase (PARP). Enzymatic cleavage of the PARP occurs uniquely
during apoptosis. Activation of caspases results in cleavage of
PARP, which produces inactive PARP fragments. One inactive PARP
fragment binds DNA and inhibits DNA repair. Thus, cleavage of PARP
can be determined using an antibody specific to cleaved PARP
fragments. Cleavage of PARP also can be determined by measuring
decrease in PARP activity. PARP catalyzes the NAD-dependent
addition of poly(ADP-ribose) to nuclear proteins such as histone.
Thus, in one exemplary assay, incorporation of biotinylated
poly(ADP-ribose) onto histone proteins can be measured as an
indicator of PARP activity. Methods for determining PARP cleavage
are known in the art, as exemplified in Mullen, Methods Mol. Med.
(2004) 88:171-81; Yu et al., Science (2002) 297:259-63; and Saldani
et al. Eur. J. Histochem. (2001) 45:389-92, all of which are hereby
expressly incorporated by reference in their entireties.
[0555] In another exemplary method, modulation of apoptosis can be
identified by determining annexin V binding. Annexin V binds to
phosphotidylserine on the cell membrane, a phenomenon that occurs
only in cells undergoing apoptosis. In one exemplary assay,
fluorescently labeled annexin V can be added to cells, and presence
of the fluorescent marker on the cells is indicative of annexin
binding. In another example, antibodies specific for annexin V can
be used to detect the presence of annexin V on the cell membrane.
This technique is often combined with the use of fluorescent dyes
that are normally not able to penetrate the cell membrane unless it
is damaged these include dyes such as propidium iodide and acridine
orange. Methods for determining annexin V binding are known in the
art, as exemplified in U.S. Pat. No. 5,767,247, Vermes et al., J.
Immunol. Methods (1995) 184:39-51; Wilkins et al., Cytometry (2002)
48:14-9; and Peng et al., Chin. Med. Sci. J. (2002) 17:17-21, all
of which are hereby expressly incorporated by reference in their
entireties.
[0556] In another exemplary method, modulation of apoptosis can be
identified by determining chromatin condensation. Chromatin
condensation is a well-established indicator of apoptosis.
Chromatin condensation can be detected by a variety of methods, for
example, detection by decreased forward angle light scatter or
decreased right angle light scatter, and detection by presence of a
specific dye such as Hoechst 33342. Methods for determining
chromatin condensation are known in the art, as exemplified in
Tounekti et al., Exp. Cell Res. (1995) 217:506-16 and Dobrucki et
al., Micron (2001) 32:645-52, all of which are hereby expressly
incorporated by reference in their entireties.
[0557] In another exemplary method, modulation of apoptosis can be
identified by determining an increase sensitivity of chromatin in
cells to acid or heat-induced denaturation. Sensitivity of
chromatin in cells can be a marker of apoptosis. Chromatin
sensitivity to acid or heat-induced denaturation can be detected by
a variety of methods known in the art, such as detecting the
altered binding of the metachromatic dye acridine orange. Methods
for assaying chromatin sensitivity to denaturation are known in the
art, as exemplified in Frankfurt et al., (1996) Exp. Cell Res.
226:387-397, Frankfurt et al., (2001) J. Histochem. Cytochem.
49:369-378, Frankfurt et al., (2001) J. Immunol. Methods. 253:
133-144, Groos et al., (2003) Anat. Rec. 272A:503-513, Zamzani et
al., (1999) Nature 401:127-128, and Allera et al., (1997) J. Biol.
Chem. 272:10817-10822, all of which are hereby expressly
incorporated by reference in their entireties.
[0558] In another exemplary method, modulation of apoptosis can be
identified by determining fractional DNA content. Under appropriate
conditions, small molecular weight DNA fragments occurring as the
result of the apoptotic process can be removed from cells,
resulting in cells with decreased DNA content. Assays can be used
to detect cells with decreased (fractional) DNA content by using,
for example, DNA dyes in flow cytometry according to known methods.
Methods for assaying fractional DNA content are known in the art,
as exemplified in Mazur et al., Hum. Exp. Toxicol. (2002) 21:335-41
and Gorczyca, Endocrine-Related Cancer (1999) 6:17-19, all of which
are hereby expressly incorporated by reference in their
entireties.
[0559] In another exemplary method, modulation of apoptosis can be
identified by determining TUNEL assay, as discussed herein
elsewhere. TUNEL assay can detect DNA strand breaks occurring
following activation of an apoptosis-specific nuclease.
Incorporation of labeled nucleotides at the site of the
double-strand breaks can be detected by, for example, binding of
antibodies or other molecules (biotin-avidin) carrying a
fluorescent tag.
[0560] An exemplary assay for cell apoptosis determination is
provided in Example 1 for caspase-3 activation measurement.
Briefly, cells were treated with smoke (i.e., A549) or smoke
condensate (i.e., NHBE) and fixed as described above, then rinsed
twice in PBS and immersed in 0.2% Triton X-100 (Sigma) in a
solution of 1% (w/v) bovine serum albumin (BSA; Sigma) in PBS for
30 min to suppress non specific antibody binding. The cells were
then incubated in 100 .mu.l volume of 1% BSA containing 1:100
dilution of anti-cleaved (activated) caspase-3 rabbit polyclonal Ab
(Cell Signaling Technology, Beverly, Mass.) overnight at 4.degree.
C., washed twice with PBS and incubated with 1:30 diluted
FITC-conjugated F(ab')2 fragment of swine anti-rabbit
immunoglobulin (DAKO, Carpinteria, Calif.) for 30 min in room
temperature in the dark. The cells were then counterstained with 1
.mu.g/ml 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes,
Eugene, Oreg.) in PBS for 5 min. Each experiment was performed with
an IgG control in which cells were labeled only with secondary
antibody, FITC-conjugated F(ab')2 fragment of goat anti-mouse
immunoglobulins, without primary antibody incubation to estimate
the extent of nonspecific binding of the secondary antibody to the
cells. The following section describes several assays that can be
used to evaluate the ability of a tobacco or a tobacco product to
modulate cell proliferation.
[0561] Analysis of Changes to Cell Homeostasis: Modulation of Cell
Proliferation
[0562] In some embodiments, modulation of cell homeostasis can be
identified by determining modulation of cell proliferation. Thus,
provided herein are methods of identifying a tobacco that modulates
cell proliferation by providing a tobacco, obtaining a tobacco
composition from the tobacco, contacting a cell with the tobacco
composition, and identifying a modulation of cell proliferation in
the cell after contact with the tobacco composition. Also provided
herein are methods of identifying a compound in tobacco that
modulates cell proliferation, methods of identifying a tobacco
product that has a reduced potential to contribute to a
tobacco-related disease, and methods of making a tobacco product
that has a reduced potential to contribute to a tobacco-related
disease, in accordance with the methods of identifying a tobacco or
tobacco compound that modulates cell homeostasis provided herein
elsewhere. Also provided herein are methods of identifying a
compound in tobacco that modulates cell proliferation, methods of
identifying a tobacco product that has a reduced potential to
contribute to a tobacco-related disease, and methods of making a
tobacco product that has a reduced potential to contribute to a
tobacco-related disease, in conjunction with the methods of
identifying a tobacco or tobacco compound that modulates cell
proliferation provided herein.
[0563] Also provided herein are methods of comparing two or more
tobacco products. In some embodiments, a tobacco or tobacco
compound that inhibits cell proliferation can be characterized as a
tobacco that has a potential to contribute to a tobacco-related
disease. In some embodiments, a first tobacco that inhibits cell
proliferation to a greater degree than a second tobacco can be
characterized as a tobacco that has an increased potential to
contribute to a tobacco-related disease. In some embodiments, a
first tobacco that inhibits cell proliferation to a lesser degree
than a second tobacco can be characterized as a tobacco that has a
reduced potential to contribute to a tobacco-related disease. In
some embodiments, a tobacco or tobacco compound that increases cell
proliferation can be characterized as a tobacco that has a
potential to contribute to a tobacco-related disease. In some
embodiments, a first tobacco that increases cell proliferation to a
greater degree than a second tobacco can be characterized as a
tobacco that has an increased potential to contribute to a
tobacco-related disease. In some embodiments, a first tobacco that
increases cell proliferation to a lesser degree than a second
tobacco can be characterized as a tobacco that has a reduced
potential to contribute to a tobacco-related disease. In some
embodiments, the methods of identifying a tobacco that modulates
cell proliferation can be used to identify modified tobacco that
modulates cell proliferation as provided herein or otherwise known
in the art.
[0564] As used herein, a tobacco or tobacco compound that inhibits
or increases cell proliferation refers to a tobacco or tobacco
compound that causes a cell or cell population to proliferate at a
decreased or increased rate, respectively, relative to a cell or
cell population that is not contacted by the tobacco or tobacco
compound. Any of a variety of methods can be used to determine cell
proliferation in a cell or cell population, including those
provided herein, and other methods known in the art.
[0565] Any of a variety of assays can be used that monitor
alterations to the viability and growth potential of cells in vitro
when challenged by exposure to a vast array of insults (e.g.,
ionizing radiation, ultraviolet radiation, drugs, toxins,
carcinogens, CS, CSC, TPM, viruses, chemicals, free radicals,
pollution, and the like). Assays that can be used in the methods
provided herein can include assays that monitor proliferative rates
(cell proliferation assays) and assays that monitor survivability
and proliferation with time (e.g., clonogenic survival assay).
[0566] In one example, clonogenic survival can be monitored. The
clonogenic survival assay can be used to study the ability of
specific agents to impact the proliferation of cells. This assay is
frequently employed in cancer research laboratories to determine
the effect, if any, of a range of substances (e.g., drugs,
radiation, chemicals, organic mixtures, etc), on the proliferation
of tumor cells. The term "clonogenic" refers to the fact that these
cells are clones of one another. Any of a variety of cell types can
be used in such experiments. The cells used typically come from
established cell lines, which have been well-studied and whose
general characteristics are known. Typically, a clonogenic survival
assay has four major steps: (1) inoculating cells into culture
dishes and incubate the cells (e.g., 24-48 hours); (2) upon the
cells reaching the logarithmic phase of growth, the treating the
cells with a tobacco composition (e.g., contacting the cells with
freshly prepared and diluted CS for different periods of time); (3)
allowing the cells to recover for a set number of hours (e.g., up
to 24 hours), then treating the cells and allowing the cells to
grow further (e.g., trypsinizing the cells, replating the cells at
specific dilutions, and allowing the cells to grow for 5-7 days);
and (4) fixing, staining and counting the cells. Experimental
specifics such as time of incubation and growth, number of cells to
use for plating, and the like, can be readily determined by one
skilled in the art according to the type of cell used. Typically,
the number of surviving colonies of 25-50 cells is representative
of the percentage of cells that survived the treatment. A graphical
representation of survival versus exposure time to a tobacco
composition can then be generated. The surviving fraction can be
determined by dividing the number of colonies in the dish by the
number of the colonies in the control (non-treated) dish.
[0567] In addition to clonogenic assays, any of a variety of cell
proliferation assays can be used to monitor an increase or decrease
in proliferative capacity and which can be used in context with
exposure to a tobacco composition such as CS, CSC and/or TPMs.
[0568] In one example of cell proliferation assays, intake and
conversion of a dye can be an indicator of cell proliferation. One
example of such an assay is a resazurin-based assay. Resazurin is a
redox dye which is not fluorescent, but upon reduction by
metabolically active cells, is converted into a highly fluorescent
product (resorufin). Living cells can readily reduce this non-toxic
reagent and the resulting increase in fluorescence intensity is
monitored using a fluorescence spectrophotometer or plate reader.
Exemplary commercially available assays include alamarBlue.TM.
reagent from BioSource International, Camarillo Calif.
[0569] Another example of dye intake and conversion-based cell
proliferation assay is a tetrazolium salt-based assay. The
tetrazolium salt assay is a colorimetric assay is based on the
conversion of a tetrazolium salt (MTT, WST, or other) to formazan,
a purple dye. This cellular reduction reaction involves the
pyridine nucleotide cofactors NADH/NADPH and is only catalyzed by
living cells. The formazan product has a low aqueous solubility and
is present as purple crystals. Dissolving the resulting formazan
with a solubilization buffer permits the convenient quantification
of product formation. The intensity of the product color is
directly proportional to the number of living cells in the culture.
Exemplary commercially available assays include Quick Cell
Proliferation Assay Kit from BioVision Inc., Mountain View,
Calif.
[0570] In another example of cell proliferation assays, cells can
be monitored for plasma membrane damage. Plasma membrane
damage-based assays can be used to monitor cell death or
cytotoxicity. Typical assays quantitate molecules released from
damaged cells such as adenylate kinase and lactate dehydrogenase.
Exemplary commercially available assays include LDH-Cytotoxicity
Assay Kit from BioVision Inc., Mountain View, Calif.
[0571] In another example of cell proliferation assays, cells can
be monitored for dye exclusion/dye uptake assays. Dye
exclusion/uptake assays distinguish live from dead cells based on
dyes which specifically stain either live or dead cells. Exemplary
commercially available assays include trypan blue dye exclusion,
Live-Dye.TM. (a cell-permeable green fluorescent dye that stains
live cells) from BioVision Inc., Mountain View, Calif.
[0572] In another example of cell proliferation assays, cells can
be monitored for ATP and ADP levels. ATP and ADP level-based assays
utilize the phenomenon that increased levels of ATP and decreased
levels of ADP have been recognized in proliferating cells.
Exemplary commercially available assays include ApoSENSOR.TM. Cell
Viability Assay Kit from MBL International, Woburn Mass.
[0573] In another example of cell proliferation assays, cells can
be monitored for protein or DNA levels in the cells. Cell
proliferation is associated with increased protein and DNA
synthesis. DNA quantitation-based assays can use, for example,
[3H]-thymidine incorporation, the fluorescence of a DNA-dye complex
from lysed cells, or other known markers of DNA synthesis.
Similarly, protein synthesis can be monitored for incorporation of
labeled amino acids into the proteins. Exemplary commercially
available assays include Quantos.TM. Cell Proliferation Assay Kit
from Stratagene, La Jolla, Calif.
[0574] Example 3 below provides one non-limiting specific example
of the clonogenic survival assay methods provided herein.
Variations of the assay method used in terms of materials, assay
times, instrumentation and protocols would be apparent to the
skilled artisan.
Example 3
[0575] A clonogenic survival assay was used to study the ability of
tobaccos and tobacco products to impact the proliferation of cells.
The experiment involves four major steps: (1) inoculate cells into
culture dishes and incubate for 24-48 hours; (2) upon reaching the
logarithmic phase of growth, the treatment is applied; the
treatment in this case is freshly prepared and diluted CS for
increasing periods of time; (3) the cells are then allowed to
recover for a set number of hours (up to 24), then the cells are
trypsinized, replated at specific dilutions, and allowed to
continue growing for 5-7 days; the number of cells used depends
largely on the plating efficiency of the cell line and must be
determined empirically prior to the experiment; and (4) at the
conclusion of the experiment, the cells are fixed, stained, and
counted. The primary measure is to count surviving colonies of
25-50 cells which is presented as the percentage of cells which
survived the treatment. A graphical representation of survival
versus exposure time to CS is then generated. The surviving
fraction is determined by dividing the number of colonies in the
dish by the number of the colonies in the control (non-treated)
dish.
[0576] A549 cells were exposed to smoke as described above.
Following smoke exposure the medium is aspirated and the cells
rinsed refed with 37.degree. C. BEGM and placed in a 37.degree. C.,
5% CO2 humidified incubator for two to three hours. The cells are
harvested by trypsinization with trypsin-EDTA (0.25% trypsin-0.38
mg/ml EDTA, Invitrogen). Cells are centrifuged at 260.times.g for 8
min. Cell pellets are resuspended in 1 ml of Ham's F-12K medium,
10% FBS (complete medium) per pellet and counted. Cells are
serially diluted so that the mock treated have .about.65 cells per
well and smoke treated have .about.300 cells per well when seeded
onto 96-well flat bottom tissue culture plates; one plate per
condition. The plates are incubated for five days in a 37.degree.
C., 5% CO2 humidified incubator. The colonies of cells are fixed
with 5% formaldehyde/PBS and colored with 0.8% crystal violet
solution for visualization. The colonies are counted with the aid
of a macroscopic dissecting microscope. The cloning efficiency
results are expressed in relation to the mock exposed cells. Unless
otherwise indicated, each bar in the graphs represents three
replicate data points per experiment.
[0577] A549 cells were exposed to whole smoke from IM16 or
Marlboro.RTM. cigarettes for various lengths of time after which
clonogenic assays were performed. FIG. 25 is a summary of multiple
experiments. The numbers in parentheses indicate the number of
experiments represented by each bar. The industry monitor reference
cigarette IM16 shows an effect on viability essentially identical
to that of the Marlboro.RTM. cigarettes. In both cases there was a
linear decrease in cell viability with increasing smoke
exposure.
[0578] In one set of experiments, A549 cells were exposed to smoke
from various cigarettes for 20 min and clonogenic assays were
performed. IM16, Omni.RTM., Marlboro.RTM., Quest 1.RTM., or Quest
3.RTM. brand cigarettes were compared. Each graph of FIG. 26
represents a separate experiment. The assay distinguishes between
the cigarettes, with Quest 3.RTM. treatment having the least impact
on cell viability and IM16 having the greatest. An overall ranking
of the cigarettes in terms of impact on viability can be seen:
Quest 3.RTM.<Quest 1.RTM. and
Omni.RTM.<Marlboro.RTM.<IM16. Thus, the tobacco products
containing modified tobacco (i.e., Omni.RTM., Quest 1.RTM., and
Quest 3.RTM. had the an impact on cell viability that was
significantly less than a reference tobacco product (i.e., IM16)
and a conventional, commercially available, traditional tobacco
product (i.e., Marlboro.RTM.). Accordingly, the modified tobacco
products Omni.RTM., Quest 1.RTM., and Quest 3.RTM. have a reduced
potential to contribute to a tobacco related disease (i.e.,
Omni.RTM., Quest 1.RTM., and Quest 3.RTM. are reduced risk tobacco
products) according to the clonogenic assay.
[0579] In a next set of experiments, the mitigation of the effect
of whole smoke on cell viability by the presence of NAC was
evaluated. A549 cells were exposed to 20 min IM16 smoke in the
presence of various concentrations of the free radical scavenger
N-acetylcysteine (NAC) and the clonogenic assay performed. NAC
protected the viability of the cells in a dose-dependent manner.
FIG. 27 shows the increasing degree of proliferation resulting from
increasing concentrations of NAC.
[0580] In another series of experiments, the effect of NAC on the
viability of cells contacted with whole smoke from different
cigarettes was evaluated. A549 cells were exposed to smoke from
various cigarettes for 20 min in the presence or absence of 25 mM
NAC and the clonogenic assay performed. IM16, Omni.RTM., and Quest
3.RTM. cigarettes were compared. NAC completely protected the cells
exposed to Quest 3.RTM. smoke, and partially protected cells
exposed to Omni.RTM. or IM16 (FIG. 28). Again, these data show that
tobacco products containing modified tobacco (i.e., Omni.RTM. and
Quest 3.RTM.) had the an impact on cell viability that was
significantly less than a reference tobacco product (i.e., IM16).
Accordingly, the modified tobacco products Omni.RTM. and Quest
3.RTM. have a reduced potential to contribute to a tobacco related
disease (i.e., Omni.RTM. and Quest 3.RTM. are reduced risk tobacco
products).
[0581] In yet another series of experiments, the effect of NAC on
cell death caused by the VAPOR phase of smoke from different
cigarettes was evaluated. A549 cells were exposed to the vapor
phase of smoke for 20 min by inserting a Cambridge filter pad
immediately after the cigarette in the smoking apparatus so as to
filter out the particulate matter ("tar") and leave only the vapor
phase. Three different cigarettes were used: IM16, Quest 1.RTM. and
Quest 3.RTM.. Cells were exposed in the presence or absence of 25
mM NAC. The clonogenic assay was subsequently performed.
[0582] The vapor phase of all cigarettes showed less effect on cell
viability than the corresponding whole smoke exposure, with Quest
3.RTM. exhibiting almost no effect (FIG. 29). The effect of various
cigarette modifications on vapor phase toxicity can thus be
selectively monitored. In all vapor phase exposures, the presence
of the free radical scavenger NAC protected the cells against
viability loss. These experiments provide more evidence that the
tobacco products containing modified tobacco (i.e., Quest 1.RTM.,
and Quest 3.RTM. had an impact on cell viability that was
significantly less than a reference tobacco product (i.e., IM16)
and, thus, Quest 1.RTM., and Quest 3.RTM. have a reduced potential
to contribute to a tobacco related disease (i.e., Quest 1.RTM. and
Quest 3.RTM. are reduced risk tobacco products).
[0583] Filter Comparison
[0584] Clongenic assays also were applied to tests of several
filters attached to different tobaccos. Filters and tobacco were
obtained from: (1) the industry standard reference tobacco IM16
(Philip Morris.RTM. USA); (2) reduced risk cigarette Omni.RTM.
(Vector Tobacco Ltd.); (3) reduced risk cigarette Quest 1.RTM.
(Vector Tobacco Ltd.), and (4) reduced risk cigarette Quest 3.RTM.
(Vector Tobacco Ltd.). A549 cells were exposed to mock treatment
(control) and cigarette smoke substantially as provided in the
above smoke treatment description.
[0585] Numerous combinations of tobacco and filters from IM16,
Omni.RTM., Quest 1.RTM. and Quest 3.RTM. were tested, and the
cloning efficiency relative to mock is presented in FIGS. 45-47.
FIG. 45 shows clonogenic results for each of IM16, Omni.RTM., and
Quest 3.RTM. with the cigarette in tact, and the filter cut and
then reattached to the same tobacco rod. FIG. 45 further shows
clonogenic results for Omni.RTM. and Quest 3.RTM. filters attached
to IM16 tobacco rods, and IM16 filters attached to Omni.RTM. and
Quest 3.RTM. tobacco rods. The results show that while there was
some variation in cloning efficiency when filters were cut and
reattached to the same tobacco rod, Omni.RTM. and Quest 3.RTM.
filters attached to IM16 tobacco rods provided increased cloning
efficiency, while the IM16 filter attached to the Quest 3.RTM.
tobacco rod provided decreased cloning efficiency. These results
show that different filters attached to the same tobacco rod have
different influences on cloning efficiency, where the cloning
efficiencies are inversely related to the expected levels of risk
attributed to the tobacco product (IM16 is highest expected risk
and has the lowest cloning efficiencies, while Quest 3.RTM. is
lowest expected risk and has the highest cloning efficiencies).
Similar experiments were repeated: (1) where IM16, Quest 1.RTM. and
Quest 3.RTM. tobaccos and filters were exchanged and compared (FIG.
46) and (2) where cloning efficiency was tested at 7 days (FIG.
47). The results in FIGS. 46 and 47 are comparable to those of FIG.
45 and again reflect inverse relationship between the expected
levels of risk attributed to the tobacco product and cloning
efficiency. The following section describes several epidemiological
approaches to determine the potential of a tobacco or a tobacco
product to contribute to a tobacco related disease.
[0586] Analysis of Changes in Cell Homeostasis: Modulation of the
Transcriptome or Proteome
[0587] Provided herein are methods for identifying a tobacco that
modulates cell homeostasis by providing a tobacco, obtaining a
tobacco composition from the tobacco, contacting a cell with the
tobacco composition, and identifying any modulation of the cell
transcriptome or proteome after contact with the tobacco
composition. In some embodiments, the methods provided herein can
monitor induction of expression of a gene that is silent during
homeostasis or repression a gene that is active during homeostasis.
In some embodiments, the tobacco composition can be smoke or smoke
condensate.
[0588] The methods provided herein can be used to characterize a
first and a second tobacco product by providing the first and
second tobacco products, obtaining a first and second tobacco
composition from the first and second tobacco products,
respectively, contacting a first cell with the first tobacco
composition and contacting the second cell with the second tobacco
composition, and identifying one or more attributes of the
transcriptome or proteome of the contacted cells. Different tobacco
products can contain different levels of carcinogens that can
induce various types of changes to mRNA or protein levels, or
modifications of mRNA or protein molecules. Comparison of
attributes of cells contacted with different tobacco compositions
can be performed in the methods provided herein, and such
attributes include, but are not limited to, differential levels of
mRNA, differential levels of protein and changes to the
post-tranlsational protein modifications. Accordingly, the methods
provided herein can be used to compare two or more tobacco products
by assay methods including assay for differential levels of mRNA,
differential levels of protein, and changes to post-translational
protein modification. Exemplary assay methods include microarray
assays, qRT-PCR assays, Western bloat assays, and ELISA assays.
[0589] By one approach, for example, CS is generated using a
smoking machine from a first tobacco modified product, e.g., a
product containing tobacco that has been genetically modified to
have a reduced amount of a compound. A first population of NHBE
cells is contacted with said CS obtained from the modified tobacco
product, and the cells contacted with CS are assayed for mRNA or
protein levels. A second population of NHBE cells is then contacted
with CS generated from an unmodified or reference tobacco product.
The second population of cells contacted with CS is then assayed
for mRNA or protein levels. A comparison of the data obtained from
the analysis of the first and second tobacco products will reveal
that the difference in mRNA or protein levels caused by the
modified tobacco product relative to the unmodified tobacco
product. By this approach, one can effectively identify the
contribution of individual components of a tobacco product to mRNA
or protein levels, or other assay conditions provided herein or
otherwise known in the art. These methods can thereby be used to
identify the contribution of individual components of a tobacco
product to a tobacco-related disease. This approach can be used to
develop tobacco products that are less likely to contribute to a
tobacco-related disease and reduced risk tobacco products
identified by these methods are embodiments provided herein.
Further, tobacco products prepared by these approaches can be
prepared according to good manufacturing processes (GMP) (e.g.,
suitable for or accepted by a governmental regulatory body, such as
the Federal Drug Administration (FDA), and containers that house
said tobacco products can comprise a label or other indicia, with
or without structure-function indicia, which reflects approval of
said tobacco product from said regulatory body.
[0590] In a first series of experiments, the influence of cigarette
smoke condensates (CSC) from two different tobacco products
(cigarettes) on the gene expression of NHBE cells was examined. In
a second set of experiments, the influence of cigarette smoke (CS)
generated from one tobacco product (a cigarette) on the gene
expression of NHBE cells was examined. Although NHBE cells are
preferred for the methods described herein, other cells of the
mouth, oral cavity, trachea, and lungs, either normal or
immortalized cell lines (e.g., human bronchial cells (e.g., BEP2D
or 16HBE140 cells), human bronchial epithelial cells (e.g., HBEC
cells, 1198, or 1170-I cells), normal human bronchial epithelial
cells, BEAS cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell
lung cancer (NSCLC) cells or human alveolar cells (e.g., H460,
H1792, SK-MES-1, Calu, H292, H157, H1944, H596, H522, A549, and
H226) tongue cells (e.g., CAL 27), and mouth cells (e.g., Ueda-1))
can be used. Accordingly, several embodiments concern methods of
identifying one or more genes present in human cells of the mouth,
tongue, oral cavity, trachea, or lungs (e.g., NHBE cells) that are
modulated by exposure to CS, CSC, TS, TSC or TPM.
[0591] In some embodiments, the methods include providing a first
population of isolated human cells of the mouth, tongue, oral
cavity, or lungs (e.g., NHBE cells), contacting the cells with a
CS, CSC, TS, TSC or TPM from a first tobacco product (e.g., a
cigarette) in an amount and for a time sufficient to modulate
expression or modification of one or more genes or gene products,
and identifying the gene that is modulated or the modified gene
product (e.g., phosphorylated) or the level or amount of gene
expression or modification. The identification of a gene that is
modulated or modified gene product or the level or amount of gene
expression or presence or absence of a modification on a gene
product can be accomplished using any technique available that
analyzes transcription (e.g., microarray, genechip, oligonucleotide
array, an amplification technique, qRT-PCR, or hybridization),
protein production (e.g., ELISA, Western blot, or other antibody
detection techniques), or modifications of proteins (e.g.,
oxidation or phosphorylation, such as detection methods that employ
anti-phospho-tyrosine antibodies). Additionally, the appearance or
disappearance of metabolites associated with genes that are
modulated in response to exposure to CS, CSC, TS, TSC or TPM can
also be monitored (e.g., cysteine, glutathione, fragments of
proteins or lipids or fatty acids) using techniques that are
available.
[0592] In some embodiments, the pattern and/or level of gene
expression or gene product modification of a control population
(e.g., a second population of isolated human cells of the mouth,
tongue, oral cavity, or lungs (e.g., NHBE cells)), is compared to
the level of expression or gene product modification in the first
population of isolated cells. By this approach, preferably using
the same type of cells for each of the two populations, a first
population is contacted with a CS, CSC, TS, TSC or TPM and the
second population of isolated cells is not. In this manner, the
second population of isolated cells is a control population, which
will exhibit the baseline pattern or level or amount of gene
expression or gene product modification (homeostasis). Data
generated from the first or second population of isolated cells
before or after exposure to CS, CSC, TS, TSC, TPM or air (control)
can be recorded on a computer readable media and databases
containing this information can be used to identify a gene that is
modulated in response to contact with a CS, CSC, TS, TSC or TPM or
to investigate the gene expression pathways that lead to a
particular tobacco-related disease.
[0593] In some embodiments, a second tobacco product (e.g., a
cigarette) is compared to a first tobacco product (e.g., a
cigarette) using the analysis above. That is, for example, a first
population of isolated human cells of the mouth, tongue, oral
cavity, or lungs (e.g., NHBE cells), is contacted with a CS, CSC,
TS, TSC or TPM from a first tobacco product (e.g., a cigarette) in
an amount and for a time sufficient to modulate expression of one
or more genes or to modify a gene product, and identification of a
gene that is modulated or modified gene product (e.g.,
phosphorylated) or the level or amount of gene expression or
modification can be determined using any technique available that
analyzes transcription (e.g., qRT-PCR or hybridization), protein
production (e.g., ELISA, Western blot, or other antibody detection
techniques), modifications of proteins (e.g., oxidation or
phosphorylation), or the appearance or disappearance of metabolites
associated with genes that are modulated in response to exposure to
CS, CSC, TS, TSC or TPM (e.g., cysteine, glutathione, fragments of
proteins or lipids or fatty acids). A second population of isolated
human cells of the mouth, tongue, oral cavity, or lungs (e.g., NHBE
cells), preferably the same type of cell as used in the analysis of
the first tobacco product, is also contacted with a CS, CSC, TS,
TSC or TPM from a second tobacco product (e.g., a cigarette) in an
amount and for a time sufficient to modulate expression of one or
more genes or to modify a gene product. Identification of a gene
that is modulated or modified gene product (e.g., phosphorylated)
or the level or amount of gene expression or modification can also
be accomplished using any technique available that analyzes
transcription (e.g., qRT-PCR or hybridization), protein production
(e.g., ELISA, Western blot, or other antibody detection
techniques), modifications of proteins (e.g., oxidation or
phosphorylation), or the appearance or disappearance of metabolites
associated with genes that are modulated in response to exposure to
CS, CSC, TS, TSC or TPM (e.g., cysteine, glutathione, fragments of
proteins or lipids or fatty acids).
[0594] The data obtained from the analysis of the first tobacco
product can be compared to the data obtained from the analysis of
the second tobacco product so as to identify, for example, a
gene(s) that is induced in response to exposure to the first
tobacco product but not the second tobacco product or vice versa.
Additionally, the comparison will reveal that the level of
expression of one or more genes induced by both tobacco products
differs with respect to the two tobacco products or that the first
product has more, less, or no modification of a particular gene
product (e.g., phosphorylation), as compared to the second tobacco
product or vice versa. These data (e.g., the types of genes
expressed, the amount of expression, and modification) allow one to
develop a profile for each tobacco product analyzed (in this
example only two products are being compared but a plurality of
products can be compared using the same approach). These tobacco
product profiles can be recorded on a computer readable media and
databases containing this information can be created. Many of the
genes that are expressed, the amount of expression, and/or
modification can be associated with molecular events that
contribute to a tobacco related disease. By analyzing the
differences between the tobacco products analyzed, (e.g., the types
of genes expressed, the amount of expression, and modification),
one can identify a tobacco product that has less potential to
contribute to a tobacco related disease or that, for example, a
first tobacco product has a reduced risk to contribute to a
tobacco-related disease, as compared to a second tobacco product or
vice versa. Thus, reduced risk tobacco products identified by the
approaches described herein are embodiments of the invention.
[0595] More embodiments concern methods to identify components of
CS, CSC, TS, TSC or TPM that modulate the expression of a gene that
contributes to a tobacco-related disease. In one embodiment, the
pattern or level of gene expression or modification of a gene
product in cells of the mouth, oral cavity, trachea, or lung (e.g.,
NHBE cells) that are exposed to a first tobacco product that lacks
a component associated with a tobacco-related disease (e.g.,
nicotine) is compared to a second tobacco product (preferably of
the same type of tobacco as the first tobacco product) that
contains the component (e.g., nicotine) and the impact on the types
of genes expressed, the amount of expression, and modification of
gene products is analyzed (e.g., microarray analysis, Western blot,
ELISA, and/or qRT-PCR). By this approach, the genes or
modifications of a gene product, which are modulated as a result of
the presence or absence of the component (e.g., nicotine), can be
identified. Because many of these modulated genes or modifications
of gene products will be associated with molecular events that
contribute to a tobacco-related disease, one can rapidly identify
whether the presence or absence of a particular component in a
tobacco product elevate the risk of acquiring a particular
tobacco-related disease. Once a component that contributes to a
tobacco-related disease has been identified using the approaches
described herein, one can use various techniques to remove this
component from tobacco (e.g., genetic modification, chemical
treatment, or adjustments in the harvesting, curing, or processing
of the tobacco) and thereby develop reduced risk tobacco products
(e.g., cigarettes). Thus, reduced risk tobacco products identified
by these approaches are embodiments of the invention.
[0596] Many embodiments described herein employ normal human
bronchial cells (NHBE cells) that are maintained in culture.
Although NHBE cells are preferred for the methods described herein,
it should be understood that many other cells that are typically
contacted with tobacco or tobacco smoke during the process of
smoking (e.g., lung cells, bronchial cells, cells of the mouth,
pharynx, larynx, and tongue) can also be used. Additionally, many
immortal cell lines can be used with the methods described herein.
Preferred cells for use with the embodied approaches include, but
are not limited to, human bronchial cells (e.g., BEP2D or 16HBE140
cells), human bronchial epithelial cells (e.g., HBEC cells, 1198,
or 1170-I cells), normal human bronchial epithelial cells, BEAS
cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer
(NSCLC) cells or human alveolar cells (e.g., H460, H1792, SK-MES-1,
Calu, H292, H157, H1944, H596, H522, A549, and H226), tongue cells
(e.g., CAL 27), and mouth cells (e.g., Ueda-1)). Many of such
cultures are available commercially or through a public repository
(e.g., ATCC). Further, several techniques exist that allow for one
to generate primary cultures of said cells and these primary
cultures can be used with the methods described herein.
Example 4
Treatment of NHBE Cells with CSCs
[0597] The tobacco smoke condensates were prepared as follows.
Smoke was generated from two commercially available nationally sold
brands of American cigarettes (Brand A and Brand B) using an
INBIFO-Condor smoking machine under Federal Trade Commission (FTC)
smoking parameters (2.0 second puff duration, 35 milliliter puff
every 60 seconds). Both brands of cigarettes were non-menthol,
full-flavor types of American-blended cigarettes with averaged FTC
measured values of 13.2 mg tar/0.88 mg nicotine (Brand A), and 14.5
mg tar/1.04 mg nicotine (Brand B). Brand A contains tobacco that
has been chemically modified to reduce carcinogens (see U.S. Pat.
No. 6,789,548, herein expressly incorporated by reference in its
entirety), whereas Brand B contains conventional tobacco. Smoke
condensates extracted from these two cigarette brands and
designated CSC-A and CSC-B, respectively, were collected from the
smoke via a series of three cold traps (-10.degree. C., -40.degree.
C., and -70.degree. C.) onto impingers filled with glass beads. The
condensates were dissolved in acetone, which was then removed by
rotary evaporation at 35.degree. C. The resulting cigarette smoke
condensates (CSCs) were weighed and dissolved in dimethylsulfoxide
(DMSO) to make stock solutions of each condensate at a
concentration of 40 mg/mL, which were stored at -20.degree. C.
prior to use.
[0598] NHBE (Normal Human Bronchial Epithelial) cells were
purchased from Cambrex Corporation, East Rutherford, N.J. The cells
were cultured in complete Bronchial Epithelial Cell Growth Medium
(BEGM), prepared by supplementing Bronchial Epithelial Basal Medium
with retinoic acid, epidermal growth factor, epinephrine,
transferrin, T3, insulin, hydrocortisone, antimicrobial agents and
bovine pituitary extract by addition of SingleQuots,.TM. (both
purchased from Cambrex Corporation, East Rutherford, N.J.). S9
metabolic fraction from Aroclor 1254-treated rats was obtained from
BioReliance Corporation, Rockville, Md. A 5.times. concentration of
S9 microsomal fraction with cofactors was prepared immediately
before treating the cells, and contained 10% S9 microsomal
fraction, 4 mM NADP, 5 mM glucose-6-phosphate, 50 mM phosphate
buffer pH 8.0, 30 mM KCl, and 10 mM CaCl.sub.2.
[0599] Twenty-eight flasks were seeded with 14.6 ml of a
2.52.times.10.sup.4 cells/ml cell suspension and an additional 15.4
ml pre-warmed BEGM were added to each flask for a final volume of
30 mL/flask. All incubations were at 37.degree. C. in a humidified
atmosphere of 5% CO.sub.2 in air. Cells were grown to 40%
confluence, at which time the cultures were treated. Four flasks
were used as untreated control cultures. Following medium removal
in these four control flasks, the cells were re-fed with 30 ml
pre-warmed BEGM and their RNA harvested at 0 h (2 flasks) and 20 hr
(2 flasks). The remaining 24 experimental flasks were treated with
either CSC-A in the presence of 2% S9 microsomal fraction, CSC-B in
the presence of 2% S9 fraction, or 2% S9 microsomal fraction alone.
Following medium removal, each flask received 9.0 ml of fresh BEGM,
15.0 mL BEGM containing CSC or vehicle (400 .mu.g/ml of CSC-A or
CSC-B and 1% DMSO for the CSC-treated groups, 15.0 mL containing 1%
DMSO for the S9-only group), and 6 ml of 5.times.S9 fraction for a
final concentration of 2% S9 and a final media volume of 30 mL.
Incubation was carried out under the incubation conditions
described above. Duplicate flasks were used for each treatment/time
point of the experiment (i.e., 2, 4, 8, and 12 h).
[0600] The monolayer cultures of NHBE cells were treated in
logarithmic phase of growth for up to 12 hours with CSC-A or CSC-B
in the presence of 2% S9 microsomal fraction, or with 2% S9
fraction alone. Cell viability after 12 hours exposure was 84% and
73% for CSC-A and CSC-B treatments, respectively, when compared to
untreated cells. RNA was then extracted from cells at 2, 4, 8, and
12 hours post-treatment, fluorescently labeled and hybridized to
genome-scale microarrays, as described in the examples that
follow.
[0601] Treatment of NHBE Cells with CS
[0602] Two identical and independent smoke exposure experiments
using NHBE cells were performed. In both experiments, the cells
were exposed to cigarette smoke (CS) or air ("mock-exposed") for 15
min, after which the cells were re-fed with fresh media and allowed
to incubate for either 4 h or 24 h (the "washout" period). In
preparation for exposure, cells were seeded into 35 mm Petri dishes
(Fisher Scientific, Falcon #35-3001, Pittsburgh, Pa.) at a density
of 105 cells/dish. This resulted in no more than 70% confluence at
the time of smoke treatment 48 hours later.
[0603] Experiment 1 used cells from a 23-year-old nonsmoking,
non-diabetic male donor purchased from Cambrex Corporation
(Walkersville, Md.). A total of ten Petri dishes were treated: two
dishes were mock-exposed with a 4 h washout, two dishes were
CS-exposed with a 4 h washout, three dishes were mock-exposed with
a 24 h washout, and three dishes were CS-exposed with a 24 h
washout.
[0604] Experiment 2 was performed in an essentially identical
manner as Experiment 1, except for the cell donor (a 13-year-old
nonsmoking, non-diabetic male, purchased from Cambrex Corporation,
Walkersville, Md.), and the number of Petri dishes used for the
mock- and CS-exposed samples with a 24 h washout (two instead of
three). This resulted in a total of eight Petri dishes treated for
Experiment 2.
[0605] Smoke was generated from a commercially available,
nationally sold, non-menthol, full-flavor brand of American filter
cigarettes (averaged FTC measured values of 14.5 mg tar/1.04 mg
nicotine) using a KC 5 Port Smoker (KC Automation, Richmond, Va.)
smoking machine under Federal Trade Commission (FTC) smoking
parameters (35.+-.0.3 cc puff volume, one puff every 60 seconds,
2-second puff duration with none of the ventilation holes blocked,
using cigarettes which have been equilibrated at 23.9.degree.
C..+-.1.1.degree. C. and 60%.+-.2% relative humidity for a minimum
of 24 hours and a maximum of 14 days).
[0606] Immediately prior to smoke exposure the culture medium was
removed from each dish and replaced with pre-warmed Dulbecco's
Phosphate Buffered Saline (PBS) containing calcium and magnesium
(BioSource, Rockville, Md.). The Petri dishes were placed in a
smoke exposure chamber (20.6 cm.times.6.7 cm.times.6.3 cm). Each 35
cc puff was diluted to 500 cc using compressed air containing 5%
CO2 and then was drawn over the cells with the aid of a vacuum pump
in order to keep a constant flow of smoke over the cells with
minimal accumulation in the exposure chamber. Cigarettes were
smoked to a maximum of seven puffs per cigarette, within 3 mm of
the filter tip. Mock exposure conditions were identical to smoke
conditions without a cigarette placed in the smoking port.
Immediately after exposure, the PBS was removed from each dish and
replaced with fresh pre-warmed cell culture medium. The Petri
dishes were transferred to a 37.degree. C. 5% CO2 incubator and
incubated for 4 or 24 hours post-exposure.
[0607] Cells were cultured in complete Bronchial Epithelial Cell
Growth Medium, prepared by supplementing Bronchial Epithelial Basal
Medium with retinoic acid, epidermal growth factor, epinephrine,
transferrin, T3, insulin, hydrocortisone, antimicrobial agents and
bovine pituitary extract by addition of SingleQuots, TM (Cambrex
Corporation, Walkersville, Md.). All incubations were at 37.degree.
C. in a humidified atmosphere of 5% CO2 in air. All cells were used
before their fifth passage, although NHBE cells can be used up to
10 passages or more in the methods described herein.
[0608] Once the cells are contacted with a CS, CSC, TS, TSC or TPM,
an approach to analyze the genes that are modulated in response to
the exposure is employed. In some embodiments, the identification
of at least one gene that is modulated by exposure to CS, CSC, TS,
TSC or TPM is accomplished using an array technology, an
oligonucleotide array technology, a genechip technology, any type
of hybridization or blot, PCR, QRT-PCR, another amplification
technology or protein detection methodologies, such as antibody
detection methods, ELISA and Western blot. In some embodiments, the
identification is made by observing a modulation (up-regulation or
down-regulation) in the level or activity of an mRNA and/or a
protein. In some embodiments, the modulation is seen as an increase
in mRNA or protein production. In other embodiments, the modulation
is seen as a decrease in mRNA or protein production. In some
embodiments, the modulation is identified as being statistically
relevant. In some embodiments, the presence or absence of a
modification of a gene product (e.g., phosphorylation, acylation,
or cleavage of a peptide) or the presence or absence of a
metabolite (e.g., cysteine or glutathione) is analyzed. In still
more embodiments the modulation, modification, metabolite or
amounts thereof are recorded on a computer readable medium (e.g.,
disc drive, floppy, CD-ROM, DVD-ROM, zip disc, memory cache, and
the like). Accordingly, specific genes or patterns of genes and
modified gene products that appear in response to exposure to CS,
CSC, TS, TSC or TPM can be identified, recorded on a computer
readable medium and this data can be used to generate a profile for
each product tested.
[0609] In the example that follows, approaches that were used to
analyze the pattern and level of expression of genes from NHBE
cells exposed to a tobacco smoke condensate (CSC) from two
different tobacco products are described.
Example 5
Isolation of RNA from CSC-Treated Cells and Production of cDNA
[0610] After NHBE cells were exposed to the cigarette smoke
condensates (CSC-A and CSC-B), as explained in Example 4, RNA was
prepared by harvesting cells for total RNA extraction after 0
(untreated), 2, 4, 8, and 12 hours of treatment. The medium was
aspirated and the flasks were rinsed twice with pre-warmed 15 mL
Dulbecco's Phosphate Buffered Saline. After the second rinse, 5.0
mL of cold TRIzol.RTM. (Invitrogen Corp., Carlsbad, Calif.) were
added to cover the cells in each flask. Each flask was vigorously
vortexed for approximately one minute. The TRIzol.RTM. was pipetted
up and down over the surface of the flask at least five times to
suspend the cell lysate. The resulting TRIzol.RTM./cell lysate was
allowed to remain in the flask for at least 10 minutes at room
temperature after which it was transferred to microfuge tubes and
extracted with 0.2 ml chloroform per 1.0 ml TRIzol/cell lysate. The
tubes were capped and shaken vigorously to initiate the RNA
extraction, and centrifuged at >15,000.times.g for two 5-minute
spins. Following the second 5-minute centrifugation, the aqueous
layer was collected (.about.500 .mu.l) and transferred to a second
set of microfuge tubes containing an equal volume of isopropyl
alcohol. The samples were centrifuged for 30 minutes at
>15,000.times.g. Following centrifugation, most (.about.90%) of
the liquid was removed from the microfuge tube. The remaining RNA
pellet was frozen and stored at <-60.degree. C. RNA was
resuspended in diethylpyrocarbonate-treated water. RNA integrity
was assessed using capillary gel electrophoresis (Agilent
Technologies, Palo Alto, Calif.) to determine the ratio of 28s:18s
rRNA in each sample. cDNA was synthesized with a direct
incorporation of Cy3-dUTP from 2 .mu.g total RNA using Clontech
Powerscript (Clontech, Palo Alto, Calif.) reverse transcriptase.
Labeled cDNA was then purified using a Montage 96-well vacuum
system.
[0611] Microarray Printing and Processing in CSC Experiments
[0612] The microarrays used in experiments involving CSC-treated
cells were purchased from the Oklahoma Medical Research Foundation
Microarray Research Facility. Slides were produced using
commercially available libraries of 70 nucleotide long DNA
molecules whose length and sequence specificity were optimized to
reduce the cross-hybridization problems encountered with cDNA-based
microarrays (Human Genome Oligo Set Version 2.0, Qiagen, Valencia,
Calif.). The microarrays had 21,329 human genes represented. The
oligonucleotides were derived from the UniGene and RefSeq
databases. The RefSeq database is an effort by the NCBI to create a
true reference database of genomic information for all genes of
known function. For the genes present in this database, information
on gene function, chromosomal location, and reference naming are
available. All 11,000 human genes of known or suspected function
are represented on these arrays. In addition, most undefined open
reading frames were represented (approximately 10,000 additional
genes). Oligonucleotides were resuspended at 40 .mu.M
concentrations in 3.times.SSC and spotted onto Corning.RTM.
U1traGAPS.TM. amino-silane coated slides, rehydrated with water
vapor, snap dried at 90.degree. C., and then covalently fixed to
the surface of the glass using 300 mJ, 254 nm wavelength
ultraviolet radiation. Unbound free amines on the glass surface
were blocked for 15 min with moderate agitation in a 143 mM
solution of succinic anhydride dissolved in
1-methyl-2-pyrolidinone, 20 mM sodium borate, pH 8.0. Slides were
rinsed for 2 min in distilled water, immersed for 1 min in 95%
ethanol, and dried with a stream of nitrogen gas.
[0613] The cDNA generated above was added to hybridization buffer
containing Cot-1 DNA (0.5 mg/ml final concentration), yeast tRNA
(0.2 mg/ml), and poly(dA).sub.40-60 (0.4 mg/ml). Hybridization was
performed on a Ventana Discovery system for 6 hr at 42.degree. C.
(Ventana Medical Systems, Tucson, Ariz.). Microarrays were washed
to a final stringency of 0.1.times.SSC. Microarrays were scanned on
a dual-channel, dynamic auto focus, fluorescent scanner at 10 um
resolution (Agilent Technologies, Palo Alto, Calif.). Fluorescent
intensity was determined using Imagene.TM. software (BioDiscovery,
Marina del Rey, Calif.).
[0614] Genechip Analysis in CSC Experiments
[0615] CSC-induced changes in gene expression were then determined
in a comprehensive manner using hypervariable analysis, which is
based on the observation that gene expression for a majority of
genes is relatively stable among replicates in untreated cells. Any
measurable variation in this large set of genes by micro array
analysis reflects the combined effects of intrinsic normal biologic
variation and extrinsic technological variation in an unmanipulated
cell. Genes that were impacted by exposure to CSCs, and whose mRNA
expression varied over time in a statistically significant manner,
which was greater than this normal biologic and technical
variation, are termed "hypervariable" (HV).
[0616] Signals from independent samples can vary on a global-basis
and, preferably, are adjusted to a common standard. Adjustment of
expression levels in compared samples was performed as described.
(See Dozmorov, et al. Bioinformatics 19:204-211, 2003, expressly
incorporated by reference in its entirety). Briefly, compared
samples were first normalized using low level noise signals
(commonly referred to as additive noise (AN). The parameters of the
AN were calculated from non-expressed genes whose signal values
exhibited a normal distribution. The mean and standard deviation
(SD) of the AN signals was obtained by nonlinear curve fitting
after exclusion of expressed genes from the distribution.
Expression values from a given chip were then normalized such that
the AN distribution had a mean of 0 and a SD of 1. Genes expressed
3 SD above the mean of AN are defined as expressed genes and used
for further analysis. A second scaling step was then performed on
expressed genes that were scaled to a common standard through a
robust linear regression analysis.
[0617] Genes responsive to CSCs were also identified using an
analysis of temporally induced gene expression changes. This
procedure utilized an internal standard, denoted "the reference
group" to define the levels of technologic and normal biologic
variance in the experiment so that these values can be used to
define stimuli-induced variation in a statistically robust manner.
The majority of genes in the control group were not sensitive to
temporal changes. The reference group was therefore composed of a
group of genes that were statistically expressed significantly
above the mean of AN in control samples, whose residuals
approximate a normal distribution based on the Kolmogorov-Smirnov
criterion, and that have low variability of expression over time as
determined by an F-test. Variance in the reference group is due
only to technical variation and normal biologic variation and
therefore the distribution of expression of the reference group can
be used to identify genes that vary due to experimental conditions
in a manner that is statistically significantly higher than the
technologic and normal biologic variance of the system using an
F-test. Genes identified using these procedures are denoted
"hypervariable genes" or "HV-genes".
[0618] F-means cluster analysis of HV-genes co-expression involved
groupings of genes that varied in expression over time in a similar
manner, based on the technologic and normal biologic variation in
the system, in a given cluster. The reference group defined above
is once again used as a reference to define statistically
significant thresholds for clustering parameters used in an F-test.
In this manner, the variance of the system is used to define the
number of clusters thus removing the subjective nature of most
clustering methods. The method is not without some subjective
criterion as genes can belong to multiple clusters. In this method,
a given gene is placed into the largest cluster such that the
broadest biologic phenomena of the system, that is those involving
the largest number of genes, can be distinguished. To do this,
clustering is begun by defining a simple parameter for each
HV-gene. This parameter, denoted connectivity, is equal to the
number of genes that vary in expression in a similar manner as a
given gene. Clusters are nucleated starting with genes of highest
connectivity. Genes of lower connectivity will be included in a
given cluster if their expression varies over time in a manner
similar to the gene used to nucleate the cluster, i.e. if their
deviations of expression over time do not exceed the variation of
the residuals in the reference group based on an F test.
[0619] F-clustering was used to identify the kinetic behavior of
genes for each stimulus. Correlation coefficient analysis was used
to identify genes that behave in a similar manner among groups. In
this type of analysis, a Pearson correlation coefficient is used
for clustering of genes with similar time-dependent behavior among
groups. A correlation threshold was established using a Monte-Carlo
simulation experiment such that the chances of identifying a false
positive or false negative selection is <1. Matrices of
correlation coefficients are calculated for these clusters and are
represented in a graphical output termed a connectivity mosaic such
that patterns of correlated and non-correlated behavior of genes
can be identified by visual inspection.
[0620] Discriminant function analysis (DFA) is a method that
identifies a subset of genes whose expression values can be
linearly combined in an equation, denoted a root, whose overall
value is distinct for a given characterized group. DFA therefore,
allows the genes that maximally discriminate among the distinct
groups analyzed to be identified. (See Moore et al. Genet Epidemiol
23: 57-69, (2002), expressly incorporated by reference in its
entirety). In the experiments described herein, a variant of the
classical DFA, named the Forward Stepwise Analysis, was used for
selection of the set of genes whose expression maximally
discriminates among experimentally distinct groups. The Forward
Stepwise Analysis was built systematically. Specifically, at each
step all variables were reviewed to identify the one that most
contributes to the discrimination between groups. This variable was
included in the model, and the process proceeds to the next step.
The statistical significance of discriminative power of each gene
was also characterized by partial Wilk's Lambda coefficients (see
Cho et al., Optimal approach for classification of acute leukemia
subtypes based on gene expression data. Biotechnol Prog 18:
847-854, 2002), expressly incorporated by reference in its
entirety, which are equivalent to the partial correlation
coefficients generated by multiple regression analyses. The Wilk's
Lambda coefficient used a ratio of within group differences and the
sum of within plus between group differences. Its value ranged from
1.0 (no discriminatory power) to 0.0 (perfect discriminatory
power).
[0621] Of the 21,349 genes and open reading frames (ORFs) on the
high-density array used in these experiments, a combined total of
4,894 (22.9%) were classified as HV after CSC treatment (see FIG.
34A). Individually, the expression of 3,665 genes/ORFs was
modulated by CSC-A contact (i.e., 17.2% of all the genes/ORFs on
the array), and the expression of 3,668 genes/ORFs was modulated by
CSC-B contact (17.2%). These genes were hypervariable in at least
one time point during the 12-hour exposure period to CSC-A and
CSC-B respectively (see FIG. 36, Table 7). The observation that the
expression of a large number of genes was altered in a significant
manner during the 12 h treatment demonstrated a significant impact
by CSCs on steady-state levels of mRNAs in NHBE cells. A majority
of the HV genes (i.e., 2,439) were common to both CSC-treated
groups, providing evidence that the two CSCs affected cells largely
in a similar manner. However, unique non-overlapping sets of HV
genes were also identified after treatment with CSC-A (i.e., 1226
genes) and CSC-B (i.e., 1229 genes), which demonstrate that each
tobacco product has a specific quantitative and/or qualitative
difference in the chemical constituents comprising the two CSCs and
the cellular responses thereto.
TABLE-US-00013 TABLE 7 Genes Common to CSC-A and CSC-B exposed
cells, which are associated with a tobacco- related disease GenBank
Gene accession no. Abbreviation Gene description Disease NM_001613
ACTA2 Actin, alpha 2, smooth muscle, aorta Lung Cancer NM_005181
CA3 Carbonic anhydrase III, muscle specific Lung Cancer NM_005199
CHRNG Cholinergic receptor, nicotinic, gamma polypeptide Lung
Cancer NM_002594 PCSK2 Proprotein convertase subtilisin/kexin type
2 (PC2) Lung Cancer NM_004624 VIPR1 Vasoactive intestinal peptide
receptor 1 (VPAC1) Lung Cancer NM_004448 ERBB2 V-erb-b2
erythroblastic leukemia viral oncogene Lung (HER2/NEU) homolog 2
Cancer NM_024083 ASPSCR1 Alveolar soft part sarcoma chromosome
region, Lung candidate 1 Cancer NM_003872 NRP2 Neuropilin 2 Lung
Cancer U33749 TITF1 Thyroid transcription factor 1 Lung Cancer
NM_002639 SERPINB5 Serine (or cysteine) proteinase inhibitor, clade
B Lung (ovalbumin), member 5, (maspin) Cancer AF135794 AKT3 V-akt
murine thymoma viral oncogene homolog 3 Lung (protein kinase B,
gamma) Cancer NM_001618 ADPRT ADP-ribosyltransferase (NAD+; poly
(ADP- Lung ribose) polymerase) PARP1 Cancer NM_016434 TNFRSF6B
Tumor necrosis factor receptor superfamily, Lung member 6b, decoy
Cancer NM_003072 SMARCA4 SWI/SNF related, matrix associated, actin
Lung (BRG1) dependent regulator of chromatin, subfamily a, Cancer
member 4 NM_004061 CDH12 Cadherin 12, type 2 (N-cadherin 2) Lung
Cancer U28749 HMGIC High-mobility group (nonhistone chromosomal)
Lung protein isoform I-C Cancer NM_002592 PCNA Proliferating cell
nuclear antigen Lung Cancer NM_033215 PPP1R3F Protein phosphatase
1, regulatory (inhibitor) Lung subunit 3F (PPP1R3F), mRNA Cancer
NM_006218 PIK3CA Phosphoinositide 3-kinase, catalytic, alpha Lung
polypeptide Cancer NM_005506 CD36L2 CD36 antigen (collagen type I
receptor, Lung thrombospondin receptor)-like 2 (lysosomal Cancer
integral membrane NM_004994 MMP9 Matrix metalloproteinase 9 Lung
Cancer NM_003810 TNFSF10 Tumor necrosis factor (ligand)
superfamily, Lung member 10 (TRAIL) Cancer NM_002961 S100A4 S100
calcium binding protein A4 (calcium protein, Lung calvasculin,
metastasin, murine placental homolog) Cancer NM_007084 SOX21 SRY
(sex determining region Y)-box 21 Lung Cancer NM_003682 MADD
MAP-kinase activating death domain (DENN) Lung Cancer BC002712 MYCN
V-myc myelocytomatosis viral related oncogene, Lung neuroblastoma
derived (avian) Cancer NM_004353 SERPINH1 Serine (or cysteine)
proteinase inhibitor, clade H), Oral member 1, HSP47 Cancer
NM_000640 IL13RA2 Interleukin 13 receptor, alpha 2 Asthma NM_002046
GAPD Glyceraldehyde-3-phosphate dehydrogenase Asthma NM_021804 ACE2
Angiotensin I converting enzyme (peptidyl- Coronary dipeptidase A)
2 Heart Disease NM_017614 BHMT2 Betaine-homocysteine
methyltransferase 2 Coronary Heart Disease NM_020974 CEGP1 CEGP1
protein Coronary Heart Disease NM_018641 C4S0 Chondroitin
4-O-sulfotransferase 2 Coronary Heart Disease NM_006874 ELF2
E74-like factor 2 (ets domain transcription factor), Coronary NERF
Heart Disease *The sequences of the genes above are available from
GenBank using the referenced Gene ID No. and said sequences are
hereby expressly incorporated by reference in their entireties.
[0622] The 1229 genes that were induced by exposure to CSC-B but
not CSC-A were also analyzed with the commercially-available
microarray data analysis software Genespring (version 7.2, Agilent
Technologies), which identifies genes that are associated with a
tobacco-related disease. Of the 1229 unique genes that were induced
by exposure to CSC-B but not CSC-A, a total of 33 genes were
identified as being associated with cancer (see Table 8).
TABLE-US-00014 TABLE 8 Genes modulated by contact with CSC-B but
not CSC-A, which are associated with a tobacco-related disease
GeneBank # Name Description NM_00359 CUL4A Cullin 4A NM_00405 CDR1
Cerebellar degeneration-related protein (34 kD) NM_00521 CSF1R
Colony stimulating factor 1 receptor, formerly McDonough feline
sarcoma viral (v-fms) oncogene homol NM_00626 TFDP2 Transcription
factor Dp-2 (E2F dimerization partner 2) NM_01225 SNW1
SKI-interacting protein NM_00482 NTN1 Netrin 1 NM_00284 RAP1A
RAP1A, member of RAS oncogene family AF308602 NOTCH1 Notch homolog
1, translocation-associated (Drosophila) Lysosomal-associated
membrane NM_01438 LAMP3 protein 3 NM_00371 PPAP2A Phosphatidic acid
phosphatase type 2A NM_00164 ARHA Ras homolog gene family, member A
NM_01633 LOC51191 Cyclin-E binding protein 1 NM_01865 ERBB2IP Erbb2
interacting protein NM_01242 SETDB1 SET domain, bifurcated 1
AF156165 DCTN4 Dynactin 4 (p62) NM_00205 FOXO1A Forkhead box O1A
(rhabdomyosarcoma) AF163473 PPP2R1B Protein phosphatase 2 (formerly
2A), regulatory subunit A (PR 65), beta isoform NM_03328 PML
Promyelocytic leukemia AK024486 GLTSCR2 Glioma tumor suppressor
candidate region gene 2 NM_00343 ZNF151 Zinc finger protein 151
(pHZ-67) U18018 ETV4 Ets variant gene 4 (E1A enhancer binding
protein, E1AF) NM_00523 EWSR1 Ewing sarcoma breakpoint region 1
BC013971 HOXA10 Homeo box A10 AJ420488 EEF1A1 Eukaryotic
translation elongation factor 1 alpha 1 NM_00548 ST5 Suppression of
tumorigenicity 5 NM_00578 HNRPA3 Heterogeneous nuclear
ribonucleoprotein A3 NM_00094 RARA Retinoic acid receptor, alpha
NM_00675 N33 Putative prostate cancer tumor suppressor NM_00228 JUN
V-jun sarcoma virus 17 oncogene homolog (avian) AL110274 ALDH1A2
Aldehyde dehydrogenase 1 family, member A2 NM_01428 RBX1 Ring-box 1
NM_01787 FLJ20429 Hypothetical protein FLJ20429 NM_00437 BCR
Breakpoint cluster region *The sequences of the genes above are
available from GenBank using the referenced Gene ID No. and these
sequences are hereby expressly incorporated by reference in their
entireties.
[0623] Notably, it was discovered that CSC-B induced expression of
the proto/oncogenes Cullin 4A, C-jun, Hoxa10, and PPP2R1B, whereas
CSC-A did not. Cullin 4A has been described in non-small cell lung
cancer (see Singhal et al., Cancer Biol. Ther. 2(3):291-298
(2003)); C-jun has been found to be amplified or over expressed in
small cell lung cancer (see Cook et al., Curr. Probl. Cancer
17(2):69-141 (1993)); Hoxa10 has been found to be amplified or over
expressed in leukemia (see Calvo et al., Proc. Natl. Acad. Sci. USA
97(23):12776-12781 (2000)); and altered expression of PPP2R1B is
involved in lung and colorectal carcinomas (see Calin et al.,
Oncogene 19(9):1191-1195 (2000); all of these references are
expressly incorporated by reference in their entireties).
Accordingly, these results demonstrate that the tobacco product
comprising chemically modified tobacco (Brand A cigarette), which
was used to generate CSC-A, has a reduced potential to contribute
to a tobacco-related disease as compared to the tobacco product
(Brand B cigarette) used to generate CSC-B because CSC-A induces
expression of fewer genes associated with a tobacco-related disease
(e.g., 33 fewer genes associated with cancer). Notably, the tobacco
product used to generate CSC-A (Brand A) does not induce key genes
that have been associated with cancer in humans (e.g., the
proto/oncogenes Cullin 4A, C-jun, Hoxa10, and PPP2R1B); whereas the
tobacco product used to generate CSC-B (Brand B) induces expression
of these proto/oncogenes. Further, these results demonstrate that
the methods described herein can be used to effectively identify a
tobacco product that is less likely or more likely to contribute to
a tobacco related disease (e.g., cancer). That is, this example
demonstrates that the approaches described herein can be used to
identify a reduced risk tobacco product, which can be a tobacco
product that is less likely to contribute to a tobacco-related
disease because it modulates fewer genes associated with a
tobacco-related disease (e.g., cancer) or induces fewer
modifications to a gene product, which are associated with a
tobacco-related disease, as compared to a second tobacco product.
To confirm these data, more experiments were conducted on the
tobacco product used to generate CSC-A (Brand A) to determine
whether it was in fact less likely to contribute to a
tobacco-related disease (e.g., cancer), as compared to the tobacco
product used to generate CSC-B (Brand B). These experiments are
discussed in the following example.
Example 6
[0624] This example describes experiments that were conducted on
mice to demonstrate that the tobacco product used to generate CSC-A
(Brand A) is a reduced risk tobacco product in that it was less
likely to contribute to a tobacco-related disease, as compared to a
conventional tobacco product of the same class (e.g., "full flavor"
cigarette), Brand B, which was used to generate CSC-B in the
previous examples. In summary, the response of previously initiated
SENCAR mice to repeated topical applications of Brand-A or Brand-B
Cigarette Smoke Condensates (CSC-A or CSC-B), was tested over a
period of 24 consecutive weeks. One week after a single initiating
dose of 50 .mu.g 7,12-dimethylbenzanthracene (7,12-DMBA), female
SENCAR mice were exposed to the following three-times-per-week
treatment regimen: Negative-Initiation Control (0.1 ml acetone
promotion); Positive Control (1 .mu.g TPA promotion); Test (Brand-A
CSC promotion, low-dose [10 mg] and high-dose [20 mg]); or Test
(Brand-B CSC promotion, low-dose [10 mg] and high-dose [20 mg]).
The condensates and positive control articles were dissolved in
acetone and applied three times a week to the shaved dorsal skin of
female SENCAR mice. In addition, a vehicle control group was
initiated and promoted with acetone only. The effects of treatment
with the various articles on survival and group mean body weights
did not appear to be significantly affected by the Test CSC's
during the duration of the study phase.
[0625] The extent of tumor promotion by the cigarette smoke
condensates was quantitated by the incidence of tumor-bearing
animals per group, the multiplicity of tumors per animal, and the
latency period until the appearance of tumors. All quantitative
scoring was based on gross tumor detection, gross tumor numbers,
and gross characterization of tumors which was shown to be accurate
by histopathologic examination. The response to the Test CSCs was
evident in 13-87% incidence of DMBA-initiated animals exhibiting
actual tumors in the effective animals of those groups after 25
weeks compared to a 3% incidence (a single animal) exhibiting
actual tumors in the Negative Control (DMBA-Initiated) group. There
were no incidences of animals exhibiting actual tumors in the
acetone-initiated group.
[0626] The SENCAR mouse is an acceptable short-term in vivo model
for evaluating the promoting potential of a cigarette on
multi-stage epidermal carcinogenesis. This assay system takes
advantage of a mouse strain that is extremely sensitive to the
two-stage induction of skin tumors. SENCAR mice were bred for
increased sensitivity to skin tumor initiation and promotion. The
strain originated from Rockland all-purpose mice which were inbred
for sensitivity to skin tumor initiation by DMBA and promotion by
12-0-tetradecanoyl-phorbol-13-acetate (TPA) in 1959. In 1971, these
susceptible mice were outbred with Charles River CD-1 mice to
produce hybrid vigor. These mice have been bred for use in skin
carcinogenesis studies of up to 12 months duration.
[0627] Accordingly, the SENCAR mouse skin painting bioassay was
utilized to determine the relative promoting potential of various
cigarette smoke condensate (CSC) preparations applied topically for
24 consecutive weeks. The mice in Groups, as described below, were
initiated with a single application of 50 .mu.g
7,12-dimethylbenzanthracene (DMBA). One week after initiation, the
animals of each group received three topical applications per week
of either acetone (Negative Controls), TPA (Positive Control), or
one of two dose levels of cigarette smoke condensates (CSC) from
the Test cigarettes. The mice in Group 1 were initiated with
acetone vehicle rather than DMBA and received acetone promotion
thereafter.
[0628] Late in the quarantine period, the animals were weighed and
randomly distributed into nine study groups using a computerized
randomization program. This program insured that no statistically
significant differences in the group mean body weights existed
between the study groups at study start. Animals with body weights
that were .+-.20% of the mean body weight of the animal pool were
assigned to the study. Following assignment to a group (as listed
in TABLE 9), each animal was identified by a uniquely numbered tail
tattoo. A color-coded card which listed the study number, animal
number, group designation and treatment was displayed on each
cage.
TABLE-US-00015 TABLE 9 No. Group Animal of Test Article No. No.
Test Group Animals No. 1 1-30 Negative-Vehicle Control, 30 Not
Acetone Initiation and Applicable Acetone Promotion (0.1 ml each) 2
31-60 Negative-Initiation Control, 30 Not DMBA Initiation (50
.mu.g) Applicable Acetone Promotion (0.1 ml) 3 61-80 Positive
Control, DMBA Initiation 20 Not (50 .mu.g) Applicable TPA promotion
(1 .mu.g) 4 81-120 Low Dose Brand A, DMBA 40 AA49LY Initiation (50
.mu.g) Brand A CSC Promotion (10 mg) 5 121-160 High Dose Brand A,
DMBA 40 AA49LY Initiation (50 .mu.g) Brand A CSC Promotion (20 mg)
8 241-280 Low Dose Brand B, DMBA 40 AA52CE Initiation (50 .mu.g)
Brand B CSC Promotion (10 mg) 9 281-320 High Dose Brand B, DMBA 40
AA52CE Initiation (50 .mu.g) Brand B CSC Promotion (20 mg)
[0629] The Test cigarette smoke condensates at 100 and 200 mg total
tar content/ml were collected and prepared by Arista Laboratories
at a frequency of approximately every 8 weeks. Upon receipt, the
CSC samples were stored at <-20.degree. C. until further
sub-aliquoted by BioReliance (5.0 ml per vial for both the low and
high doses) and stored at .ltoreq.-20.degree. C. The dose
preparations, as received from Arista Laboratories, were divided
into 26 tightly sealed amber vials, with an expiration date of
approximately 13 weeks and stored at .ltoreq.-20.degree. C. This
allowed the use of one vial per dosing day and two backups which
could be used in case of spillage. All dosing solutions were used
within eight weeks of preparation. The Positive Control article
(TPA) was diluted with acetone to produce the desired concentration
of 10.0 .mu.g/ml once (prior to initiation of dosing) and delivered
to the animal laboratory and stored at room temperature (an extra
vial was stored at .ltoreq.-20.degree. C.).
[0630] The mice from Groups 2-9 received a single topical
application of DMBA (50 .mu.g/0.1 ml acetone/animal) as an
initiator on Day 1 of the study. The mice from Group 1 received a
single topical application of acetone vehicle (0.1 ml) as an
initiator. After one week, the animals were dosed topically three
times a week (Monday, Wednesday and Friday except for Holidays) for
24 consecutive weeks with the appropriate Vehicle Control, Positive
Control or Test article.
[0631] The dorsal application site (approximately 2.times.3 cm) was
shaved 3 days prior to the single application of the initiator, and
at least once a week thereafter, at least one day prior to
application of the appropriate dosing solution or vehicle. Shaving
was performed on all animals with an Oster Model 76059 small animal
electric clippers (Oster Co., Racine, Wis.) using a narrow
blade.
[0632] The animals were weighed at study initiation and at weekly
intervals for the next 11 weeks (12 total data collection points),
and once every four weeks thereafter and at terminal sacrifice. The
animals were observed twice daily (including weekends and holidays)
for mortality and moribundity, once in the morning before 10:00
a.m. and once in the afternoon after 2:00 p.m. (at least six hours
apart). Clinical observations performed cage-side to detect
abnormalities other than skin tumor responses were made once daily
for the first 5 weeks of the study (Days 1-35) and hands on once
every two weeks thereafter (beginning on Day 36). Clinical signs
noted at times other than the scheduled observation timepoints were
recorded on the Unscheduled Observations Sheet.
[0633] On Day 1 and at weekly intervals thereafter, the mice were
examined grossly for the presence of skin tumors. Pertinent
information such as date of observation, lesion location,
morphology, and type were recorded for each lesion at each
observation time. At necropsy, all representative skin from the
application site, skin from an untreated area, and other lesions
taken for histopathologic evaluation were indicated on the necropsy
data sheet. Lesions were identified in a manner which allowed
correlation of the individual lesion-specific histopathologic
findings with data collected during the in-life phase of the
study.
[0634] A tissue mass (in vivo) was considered to be a tumor
(papilloma) when that mass attained a 2 mm diameter and protruded
from the surface of the skin. The date at which a 2 mm diameter was
attained was recorded and represented the end of the "tumor latency
period" for that animal and the tumor was scored as a latent
papilloma. If a latent tumor remained countable for three (3)
consecutive weeks, it was considered an actual tumor. Such a tumor
remained in the total count of actual tumors for that animal even
if it subsequently decreased in size, disappeared, or the animal
died or was sacrificed early. The record of skin lesion data served
to differentiate papillomas from carcinomas and latent tumors from
actual tumors. In vivo differentiation of papillomas and carcinomas
was made on the basis of palpation, evidence of subcutaneous
invasion, and ulceration.
[0635] Group 3 (the positive control) served as a qualitative
indicator of the test system's response to a known and chemically
defined initiator (DMBA) and promotor (TPA). Considering the time
course and magnitude of the response in SENCAR mice, treated as
described above, collection of skin lesion data in the positive
control was discontinued after 90-100% of the animals in the group
exhibited tumors and the mean number of tumors per animal was at
least 8. Since this group was not counted through the entire study,
it was not included in any group comparisons noted below.
[0636] The number and location of skin papillomas (benign tissue
masses having attained a diameter >2 mm and protruding from the
surface) and carcinomas (malignant tissue masses with gross
evidence of invasive growth and tissue necrosis due to growth
outstripping vascular supply) were documented weekly. The
reliability of gross diagnoses of tumors was confirmed by
representative histopathologic examination of individually
identified and historically tracked skin lesions. Tumor data for
specific groups were calculated based on the appearance of tumors
of either type. The following parameters were recorded or
calculated for all groups (with the exception of Group 3, Positive
Control): [0637] 1. Date of tumor appearance for all tumors on all
mice. [0638] 2. Date of appearance of latent and actual tumors.
[0639] 3. Date of death or sacrifice for each mouse. [0640] 4. Time
interval from Day 1 of the study until the date of the appearance
of; (1) latent papillomas and carcinomas and, (2) actual papillomas
or carcinomas on each mouse. [0641] 5. Latency for all latent or
actual tumors (i.e., this was defined as the time from Day 1 to the
time a mass qualified as a latent tumor and subsequently as an
actual tumor). Three methods for numerically scoring latency were
used: [0642] a. The time elapsed until the appearance of the first
tumor of a specific type in a group. [0643] b. The mean time
elapsed until the appearance of all first tumors of a specific type
from all animals in a group developing one or more such tumors.
[0644] c. Time elapsed to attain 50% of the maximum incidence of
animals in a group with one or more tumors of a specific type.
[0645] 6. Percent of mice developing one or more latent and/or
actual tumors (Incidence) equals: [0646] Number of mice with at
least one latent and/or actual tumor.times.100 Number of mice
surviving at the time the first non-positive control group shows a
tumor [0647] 7. Tumors per tumor-bearing animal=Number of total or
specific-type tumors Number of animals bearing that type of
tumor
[0648] Group means and standard deviations were calculated for body
weights and skin tumor data. A Fisher's Exact test was performed to
analyze the percent of surviving animals in each group which
developed latent and/or actual tumors and percent of animals
started on study which developed actual tumors. Analysis of
Variance tests (ANOVA) were performed in order to determine if
differences in group means existed for the selected parameters. If
a significant F ratio was obtained (p<0.05), a Dunnett's t-test
was used for pair-wise comparisons of treatment test CSC groups to
the Negative Control (non-Initiated DMBA) and test CSC groups with
each other.
[0649] Incidence of Tumor-Bearing Animals
[0650] Statistical analysis of the incidence of animals bearing
actual tumors (Fisher's Exact Test, p<0.05) indicated a
significant increase in both the low- and high-dose groups
receiving CSC-B when compared to the negative vehicle control
group. Of the groups receiving the CSC-A, only the high-dose
exhibited a significantly increased number of animals bearing
actual tumors when compared to the negative vehicle control group.
When comparing the incidence of animals bearing actual tumors in
the low-dose CSC treatment groups to each other, a significant
increase was noted in the groups that received CSC-B when compared
to the group that received CSC-A. The same results were obtained
when making the same comparisons in the groups receiving the
high-dose CSC treatment. These findings are presented in TABLE
10.
TABLE-US-00016 TABLE 10 Statistical Results of Analysis of Percent
of Animals Bearing Actual Tumors Percent of Animals Bearing Group
Treatment Actual Tumors.sup.a,b 1 Negative Vehicle Control 0% 4
Low-Dose Brand A 13% 5 High-Dose Brand A 40% 8 Low-Dose Brand B 53%
9 High-Dose Brand B 78% .sup.aRepresents the percent of animals
started on study that developed at least one actual tumor.
.sup.bSignificantly increased when compared to the group indicated
in the superscript (Fisher's exact test, p < 0.05).
[0651] Statistical analysis of the incidence of animals bearing
actual and/or latent tumors (Fisher's Exact Test, p<0.05)
comparing the CSC treatment groups to the negative vehicle control
indicated the same results as the analyses of animals bearing
actual tumors discussed above.
[0652] Tumor Multiplicity
[0653] Statistical analysis of the number of actual tumors
(papillomas and carcinomas combined) per animal, after 24 weeks,
revealed significant increases (ANOVA, p.ltoreq.0.05) in the groups
treated with the high-dose CSC-B when compared to the negative
control group (acetone-initiated Group 1). The number of actual
tumors per animal in the group treated with the high-dose CSC-A
group was statistically comparable to the negative vehicle control
group. Analysis of the number of actual tumors per animal in the
low-dose CSC treatment groups indicated the group treated with the
low-dose Brand B CSC exhibited a statistically significantly
increased number of actual tumors when compared to the negative
control group. Group means, standard deviations, and statistical
results are presented in TABLE 11.
TABLE-US-00017 TABLE 11 Statistical Results of Analysis Number of
Actual Tumors per Animal Mean Number of Actual Group Treatment
Tumors per Animal .sup.a 1 Negative Vehicle Control 0.00 .+-. 0.00
4 Low-Dose Brand A 1.03 .+-. 3.90 5 High-Dose Brand A 2.58 .+-.
8.05 8 Low-Dose Brand B 3.80 .+-. 7.22 9 High-Dose Brand B 7.46
.+-. 7.86 .sup.a Significantly increased when compared to the group
indicated in the superscript.
[0654] When comparing the high-dose CSC treatment groups against
each other, a statistically significantly increased number of
actual tumors per animal was noted in the high-dose groups treated
with the Brand B CSC when compared to the high-dose Brand A group.
No statistically significant differences in the numbers of actual
tumors were noted in the low dose CSC treatment groups when
compared to each other. Statistical analysis of the number of
actual and/or latent tumors (ANOVA, p.ltoreq.0.05) indicated the
same results as the analyses of the number of actual tumors per
animal, as discussed above. Results are presented in the following
Table.
TABLE-US-00018 TABLE 12 Statistical Results of Analysis Number of
Latent and Actual Tumors per Animal Mean Number of Actual Group
Treatment Tumors per Animal .sup.a 1 Negative Vehicle Control 0.00
.+-. 0.00 4 Low-Dose Brand A 1.20 .+-. 4.33 5 High-Dose Brand A
2.75 .+-. 8.13 8 Low-Dose Brand B 4.73 .+-. 8.35 .sup.1 9 High-Dose
Brand B 8.49 .+-. 8.70 .sup.1,5 .sup.a Significantly increased when
compared to the group indicated in the superscript.
[0655] Latency Period Until Appearance of Tumors
[0656] Mean latency per group when defined as the time elapsed
until the appearance of the first actual tumor per animal was 18
weeks in the low-dose of both CSC-A and CSC-B treatment groups. In
the high-dose CSC treatment groups, mean actual tumor latency was
19 and 15 weeks in the groups treated with CSCs obtained from
Brands A and B, respectively.
[0657] Thus, the promotional capacity of the Brand A CSC was
statistically comparable to the negative vehicle control group in
terms of the incidences of tumor-bearing animals (at the low-dose
level) and the number of tumors per animal (both dose levels).
Statistical analysis comparing the groups that received the CSCs to
each other revealed significant increases in the high-dose Brand B
group when compared to the high-dose Brand A group in terms of
percent of animals bearing designated tumor types and the number of
those tumors per animal. Also, at the high-dose level, the Brand A
CSC mean latency period (until the appearance of the first tumor
per animal) was longer than the latency period of the Brand B CSC
treatment groups. The data provided in this example confirm that
the in vitro methods described herein, which utilize cell cultures
that are contacted with CS, CSC, TS, TSC or TPM (see Examples 4, 5,
and 8-13), accurately identify a tobacco product that has less
potential to contribute to a tobacco-related disease than another
tobacco product. The data provided in this example also confirm
that the in vitro methods described herein (see Examples 4, 5, and
8-13), can be used to develop tobacco products that have a reduced
potential to contribute to a tobacco-related disease and provide
further evidence, in particular, that Brand A is a reduced risk
tobacco product, as compared to Brand B.
[0658] Subsequent to exposure in vivo, the human body attempts to
detoxify, neutralize, and eliminate cigarette smoke toxins through
the action of Phase I and Phase II enzymes functioning in various
metabolic pathways. During this detoxification process, however, a
number of pro-carcinogenic compounds in tobacco smoke are
bioactivated into reactive electrophiles that have potent
carcinogenic potential in exposed cells. Thus, in order to dissect
the full biological potential of complex chemical mixtures, such as
a cigarette smoke condensate, it is desirable to evaluate the
pattern of gene expression after tobacco smoke condensate exposure
in an environment that contains a mixture of enzymes that mimic the
detoxification process in mammalian cells. The S9 microsomal
fraction from Aroclor 1254-treated rats, provides a set of enzymes
that mimic the detoxification process in mammalian cells.
Accordingly, experiments were conducted in the presence of the S9
microsomal fraction, as described in the following example, to
elucidate how the genetic fingerprint of particular tobacco
products shift in the presence of a mixture of enzymes that mimic
the detoxification process in mammals.
Example 7
S9 Microsomal Fraction Experiments
[0659] NHBE cells were exposed to cigarette smoke condensate (CSC)
in conjunction with an S9 microsomal fraction so as to identify the
effect detoxification enzymes have on the pattern or level of gene
expression. As a control to discriminate the effects of the S9
microsomal fraction on gene expression, alone, some experiments
were conducted on NHBE cells in the presence of the S9 microsomal
fraction in the absence of contact with a tobacco condensate. As
described above, an HV analysis was performed on microarray results
obtained from cells treated only with the S9 microsomal fraction
for 2, 4, 8, and 12 hours.
[0660] Several interesting observations emerged from this analysis.
First, the expression of 1680 (7.9%) genes became HV sometime
during the 12-hour exposure period with the S9 microsomal fraction
(see FIG. 34B). Second, FIG. 34B also shows that 1297 of these 1680
genes were also HV in one or both CSC treatments, which is not
surprising since all three treatment conditions (i.e., CSC-A,
CSC-B, and S9) had the same concentration of S9 microsomal
fraction. Third, even though the CSCs and the S9 microsomal
fraction induce a HV state in a large common set of genes, CSCs and
the S9 microsomal fraction did not affect these genes in similar
ways indicating differential kinetic effects between the S9
microsomal fraction alone and the S9 microsomal fraction in
conjunction with CSCs.
[0661] Subsequent to determining that the complex mixture of toxins
and carcinogens in CSCs had a broad impact on the transcriptome of
NHBE cells, it was contemplated that a sustained treatment to CSCs
(e.g., over a 12-hour period) would also allow detection, not only
of alterations such as induction and suppression, but of gene
induction/suppression with transient, sustained, or periodic
characteristics. Accordingly, the kinetic effects of gene
expression profiles generated from cells treated with CSC-A, CSC-B,
or S9 microsomal fraction from 0-12 hours using F-cluster analysis
were defined, which is a statistically robust method for defining
clusters of genes with similar expression patterns over time. These
experiments are described in the following example.
Example 8
Gene Expression Kinetics in CSC-Treated Cells
[0662] In this analysis, the normal variance of the system was
calculated and used to identify a statistical threshold for cluster
selection at which groups of genes were likely to cluster by
chance. This threshold was then used for further analysis to ensure
the statistical robustness of the clustering process. The biologic
significance of the cluster is related to cluster size, as the
largest clusters identified represent synchronous changes in the
greatest number of cellular processes. (See Spellman et al., Mol
Biol Cell 9: 3273-3297, 1998). Specifically, larger clusters
represent, in a statistically robust manner, the most significant
experimentally induced processes in these cells. When F-cluster
analysis was applied to the total HV set of 4894 genes/ORFs, 306
clusters were defined by statistical analysis, the majority of
which contained less than 50 member genes. Cluster numbers were
arbitrarily assigned from -150 to 150, with the corresponding
positive and negative numbers representing complementary gene
expression patterns (e.g., steady increase in expression over time
compared to a steady decrease in expression).
[0663] In each of the three treatment conditions, clusters
containing 50 or more genes were chosen for further
characterization because this cutoff generated a sufficient number
of large clusters that adequately represented the major kinetic
changes caused by each treatment (see FIGS. 2 A-C and TABLE 13). As
predicted, gene expression changes induced by CSCs were complex,
with the majority of clusters in CSC-treated cells being
multi-modal (see FIGS. 2A and B). For example, in CSC-A-treated
cells, genes in clusters 1, 3, 7, 12, 15, and 22 were up-regulated
within the first two hours, began to return to baseline, then were
once again induced late in the experiment, indicating that initial
treatment effected gene expression and some secondary effect (e.g.,
a CSC metabolite or the action of early gene expression changes,
reinitiated a cellular response). (See FIG. 35A). While genes
within each of these clusters showed early increases in expression
(within the first 2 h of treatment), indicating that CSC-A
treatment had immediate effects on cells, Clusters 18, 30, 35, and
39 showed a later increase in gene expression (i.e., .gtoreq.4 h).
FIG. 35B shows that in CSC-B treated cells, cluster analysis
revealed that gene expression peaks primarily between 4-8 hours, as
opposed to a 2 hour peak in CSC-A treated cells, providing evidence
that some of the effects of CSC-B treatment were delayed with
respect to those of CSC-A (e.g., see clusters 4, 5, 9, 10, 16, and
32). These data are in distinct contrast to the major clusters of
genes in S9-only treated cells, which displayed simple kinetics,
i.e., expression decreasing or increasing continuously over time
(see FIG. 35C). Although 66% of HV genes affected by CSC-A and
CSC-B were identical (see FIG. 34), it is clear from FIG. 35 that
the expression kinetics for these genes were nevertheless distinct
for the two different CSCs. This is evidenced by the fact that the
predominant coordinated behavior in CSC-A-treated cells is
represented by the largest cluster (i.e., cluster 1), that contains
1063 HV genes and whose expression peaked at 2 hours
post-treatment. This is in contrast to CSC-B-treated cells in which
case the predominant behavior of genes is represented by cluster 2,
which contains 1,036 genes and whose expression peaked at 4-8
hours, indicating that some of the effects of CSC-B treatment are
delayed with respect to those of CSC-A.
TABLE-US-00019 TABLE 13 HV Genes Specific for CSC-A and CSC-B
Treatment GenBank accession Gene no. abbreviation Gene description
AB032985 NXPH3 Neurexophilin 3 AB046848 KIAA1628 KIAA1628 protein
AB058772 SEMA6C Sema domain, transmembrane domain (TM), and
cytoplasmic domain, (semaphorin) 6C AF178532 BACE2 Beta-site
APP-cleaving enzyme 2 BC015737 Homo sapiens, ninjurin 2, clone
MGC:22993 IMAGE:4907813 BC015929 NR1D2 Nuclear receptor subfamily
1, group D, member 2 BC017732 STRBP Spermatid perinuclear RNA
binding protein M23326 TRDV3 T cell receptor delta variable 3
NM_000341 SLC3A1 Solute carrier family 3 (cystine, dibasic and
neutral amino acid transporters, activator of cystine), member 1
NM_000663 ABAT 4-aminobutyrate aminotransferase NM_000922 PDE3B
Phosphodiesterase 3B, cGMP-inhibited NM_000981 RPL19 Ribosomal
protein L19 NM_001383 DPH2L1 Diptheria toxin resistance protein
required for diphthamide biosynthesis-like 1 (S. cerevisiae)
NM_002046 GAPD Glyceraldehyde-3-phosphate dehydrogenase NM_002757
MAP2K5 Mitogen-activated protein kinase kinase 5 NM_002890 RASA1
RAS p21 protein activator (GTPase activating protein) 1 NM_003286
TOP1 Topoisomerase (DNA) I NM_003408 ZFP37 Zinc finger protein 37
homolog (mouse) NM_004057 CALB3 Calbindin 3, (vitamin D-dependent
calcium binding protein) NM_004066 CETN1 Centrin, EF-hand protein,
1 NM_004083 DDIT3 DNA-damage-inducible transcript 3 NM_004282 BAG2
BCL2-associated athanogene 2 NM_004846 EIF4EL3 Eukaryotic
translation initiation factor 4E- like 3 NM_004939 DDX1 DEAD/H
(Asp-Glu-Ala-Asp/His) box polypeptide 1 NM_005476 GNE
UDP-N-acetylglucosamine-2-epimerase/N- acetylmannosamine kinase
NM_005619 RTN2 Reticulon 2 NM_007217 PDCD10 Programmed cell death
10 NM_007275 FUS1 Lung cancer candidate NM_012192 FXCl Fracture
callus 1 homolog (rat) NM_012288 KIAA0057 TRAM-like protein
NM_013366 APC2 Anaphase-promoting complex subunit 2 NM_013401
RAB3IL1 RAB3A interacting protein (rabin3)-like 1 NM_014395 DAPP1
Dual adaptor of phosphotyrosine and 3- phosphoinositides NM_015057
KIAA0916 KIAA0916 protein NM_017491 WDR1 WD repeat domain 1
NM_017581 CHRNA9 Cholinergic receptor, nicotinic, alpha polypeptide
9 NM_020122 PCMF Potassium channel modulatory factor NM_020685
HT021 HT021 NM_021120 DLG3 Discs, large (Drosophila) homolog 3
(neuroendocrine-dlg) NM_031310 PLVAP Plasmalemma vesicle associated
protein
[0664] Accordingly, these experiments demonstrated that not only do
different tobacco products induce different genes, gene expression
patterns, and kinetics of gene expression but different tobacco
products have a different impact on a cell or a tobacco consumer.
That is, the procedures described above can be used to obtain a
genetic signature, pattern, or profile for a plurality of tobacco
products and, because some of the modulated genes are associated
with the induction or repression of a tobacco-related disease, this
data can be compared and/or analyzed to identify a tobacco product
with a reduced potential to contribute to a tobacco-related
disease.
[0665] Since clusters with a large number of member genes reflect
predominant biological behavior patterns that are likely to be
functionally interrelated, it was contemplated that the cluster 1
set of 1063 genes from CSC-A-treated cells and the cluster 2 set of
1036 genes from CSC-B-treated cells corresponded to important
biological phenomena common to the two CSCs. If this were correct,
then despite the fact that CSC-A and CSC-B treatments modulate
genes in a temporally distinct manner, the two clusters should
contain many of the same genes. To demonstrate this point, the
experiments in the following example were conducted.
Example 9
Analysis of Cluster 1 and Cluster 2 in CSC-Treated Cells
[0666] Upon analysis of cluster 1 and cluster 2 in CSC-treated
cells, it was found that a set of 554 genes (approximately 50% of
the genes in each cluster) were present in both cluster 1 (from
CSC-A) and cluster 2 (from CSC-B). A total of 330 genes from this
set of 554 genes (59.5%) have known functions while the remaining
224 are ORFs.
[0667] Functional classification of these 330 genes common to
cluster 1 and cluster 2 indicates that 10% have functional roles in
proliferation, 12.4% in transcription, 4.5% in apoptosis, and 5.1%
in damage/repair responses. In addition, as shown in TABLE 7, 34
(10%) of the identified genes are documented as having roles in
diseases that are associated with/long-term tobacco exposure (e.g.,
lung cancer, coronary heart disease, and asthma).
[0668] In clear contrast to both CSC-A and CSC-B treated cells, the
S9 microsomal fraction-treated cells show a pronounced tendency
towards suppression of gene expression. An F-clustering analysis of
the S9 microsomal fraction data (shown in FIG. 35C) resulted in
only four clusters that contained 50 or more genes. Clusters 2, 5,
and 44 all show decreases in gene expression level with a nadir at
4-8 h. Cluster 18 contains genes that show an increase in gene
expression levels, but whose expression peaks at 12 h, which is
notably different from the robust early gene responses elicited by
treatment with both CSCs. Additional evidence that the overall
effects of S9 microsomal fraction and CSC exposure on gene
expression levels are quite distinct was obtained when traditional
hierarchical clustering algorithms were used to compare the overall
differences in HV gene expression in each treatment group over the
entire 12-hour time course. FIG. 36 shows the results of this
analysis for the common subset of genes that were HV in all three
treatment groups (i.e., the 873 genes denoted in FIG. 34). Notably,
the expression data for these 873 genes partition into two separate
groups with S9-treated cells being clearly distinguishable from
CSC-A and CSC-B treated cells, which are similar to each other. The
data further indicate that the S9 microsomal fraction exerts a
largely suppressive effect on the transcriptome of NHBE cells in
contrast to a predominant inductive effect of CSC-A and CSC-B.
[0669] As discussed above, tobacco smoke condensates induce a range
of temporally distinct alterations to the homeostatic transcriptome
of the NHBE cells, which were unique in that they were
qualitatively and quantitatively dissimilar from the effects of
exposure to a S9 microsomal fraction. In an attempt to define a
biological context for these data, correlation analyses was used to
identify genes whose expression changes were highly correlated in
CSC-A and CSC-B treated cells but not in S9-treated cells. This was
achieved using a Monte Carlo analysis to establish a statistical
threshold above which correlated behavior was unlikely to have
occurred by chance. By this approach, gene expression levels were
randomized maintaining the same mean and standard deviation. A
correlation coefficient was then identified above which no genes
were correlated in the randomized data sets. The probability that
genes that correlate in experimental data sets above this threshold
would occur by chance is <1/total number of genes analyzed. The
following example describes these experiments in greater
detail.
Example 10
Defining CSC-Specific Toxicological Effects
[0670] The evidence provided in FIGS. 2 and 3 indicated that the
effect of exposure to CSC was significantly different than exposure
to an S9 microsomal fraction. Using the Monte Carlo analysis, as
shown in TABLE 13, forty HV genes were identified as having a
modulation of gene expression that was correlated in CSC-A and
CSC-B treated cells but not in S9-treated cells. The similarities
between the two tobacco-treated sample groups can be visualized by
applying a correlation coefficient analysis to the genes within a
given treatment, representing this visually in a correlation
mosaic, and comparing the visual pattern of the mosaic to other
such mosaics generated using data from different treatments. The
correlation coefficients of these genes were presented in a
correlation mosaic map (see FIG. 37) in which genes with a highly
correlated behavior were denoted by a grey pixel, and genes with
highly negatively correlated behavior by a black pixel. This mosaic
provided a way to assess the similarities of expression behavior of
the correlated genes in CSC-A, CSC-B, and S9-treated cells by
visual inspection.
[0671] The highly correlated expression characteristics of the
CSC-impacted genes identified by this analysis indicated that these
genes were likely to participate in pathways relevant to the
effects specific to CSC exposure and not to exposure to the S9
microsomal fraction. These pathways were more clearly defined using
PathwayAssist.TM. software (Stratagene, La Jolla, Calif.), a
commercially available visualization engine that scans and assesses
documented literature and available standardized databases in order
to filter, classify, and prioritize proteins in terms of their
functional relationships to known biological pathways. The results,
provided in FIG. 38, highlight the fact that this set of genes
encodes proteins that play key roles in pathways that are relevant
to the documented pathological effects of cigarette smoke. For
example, several of the genes listed in TABLE 13 are implicated in
lung oncogenesis (e.g., FUS1, GAPD, & semaphorin), in various
types of dysfunctions in lung cells involving apoptosis (e.g.,
PDE3B, PDCD10), in cell cycle control (e.g., MAP2K5, RASA1, APC2,
RASA1), in DNA topology and DNA repair (e.g., TOP1, DDIT3), and in
cellular stress (e.g., BAG2). In addition, several genes are
involved in neuro signaling (e.g., neurexophilin, KIAA1628),
neuroregeneration (e.g., semaphorin), neuropathology (e.g., BACE2,
ABAT, DLG3), and inflammation (e.g., NINJ2, TRDV3, SLC3A1).
[0672] The induction of a range of neuroendocrine-related genes is
interesting in light of the fact that many small cell lung cancers
and some non-small cell lung cancers exhibit a variety of
pathological and molecular features of pulmonary endocrine cells,
and can be stimulated by an autocrine/paracrine array of
neuroendocrine peptides. Accordingly, expression of neuroendocrine
markers has been shown to be useful in the differential diagnosis
of lung cancers. The gene set shown in TABLE 13 also includes
CHRNA9, a human nicotinic acetylcholine receptor expressed in
several tissues including inner ear hair cells, brain, and in
activated fibrosarcoma cells and whose relevance to nicotine
signaling in primary lung cells is as yet uncharacterized.
[0673] Using a similar approach, as described for the analysis of
CSC exposure in TABLE 13 and FIG. 37, the global effects of the
exposure to the S9 microsomal fraction were assessed by first
identifying the subset of HV genes that were correlated among all
three treatment groups and then assuming that the effect on these
genes was due to the S9 microsomal fraction solely, since their
expression characteristics did not change when the S9 microsomal
fraction was combined with contact to a CSC. As described above, a
Monte Carlo analysis was performed to define a statistically robust
correlation coefficient unlikely to occur by chance. Using this
threshold, the probability of identifying a gene correlated in all
three groups by chance was <1/total number of genes analyzed,
thereby confirming the high statistical specificity of this
method.
[0674] As shown in TABLE 14, a set of 52 genes was identified and
the probable function of these genes was assessed using
PathwayAssist.TM. software (see FIG. 39). Many of the genes
appeared to have roles in modulating apoptosis (e.g., AVEN, LIG1,
PTEN, etc.) indicating that the predominant cellular response to
chronic S9 microsomal fraction exposure is to activate apoptotic
programs. A second group of S9-modulated genes modulates cellular
surface chemistry, adhesion, and cellular differentiation (e.g.,
SIAT4B, KRT10, CDSN and EXT2). These results indicate that the
inclusion of S9 microsomal fractions in toxicogenetic experiments
significantly modulates cellular physiology, which may complicate
and bias the results assessing the effects of CSCs or any other
type of complex hydrocarbon mix requiring metabolic activation.
TABLE-US-00020 TABLE 14 Genes Specific for S9 Treatment GenBank
Gene accession no. abbreviation Gene description NM_001303 COX10
COX10 homolog, cytochrome c oxidase assembly protein AK056540 Homo
sapiens cDNA FLJ31978, weakly similar to Probable
hexosyltransferase NM_016013 LOC51103 CGI-65 protein NM_031916 ASP
AKAP-associated sperm protein NM_000947 PRIM2A Primase, polypeptide
2A (58kD) NM_006927 SIAT4B Sialyltransferase 4B NM_006441 MTHFS
5,10-methenyltetrahydrofolate synthetase NM_002699 POU3F1 POU
domain, class 3, transcription factor 1 NM_002954 RPS27A Ribosomal
protein S27a AK055508 FLJ11785 Rad50-interacting protein 1
NM_024636 FLJ23153 Likely ortholog of mouse tumor
necrosis-alpha-induced adipose-related protein BC011231 Homo
sapiens, Similar to angiotensinogen NM_007052 NOX1 NADPH oxidase 1
NM_000234 LIG1 Ligase I, DNA, ATP-dependent NM_032553 FKSG79
Putative purinergic receptor NM_000025 ADRB3 Adrenergic, beta-3-,
receptor AF023203 Homo sapiens homeobox protein Og12 U50536 Human
BRCA2 region, mRNA sequence CG011 NM_000421 KRT10 Keratin 10
(epidermolytic hyperkeratosis; keratosis palmaris et plantaris)
NM_001264 CDSN Corneodesmosin NM_000355 TCN2 Transcobalamin II;
macrocytic anemia NM_000401 EXT2 Exostoses (multiple) 2 NM_014214
IMPA2 Inositol(myo)-1(or 4)-monophosphatase 2 NM_003797 EED
Embryonic ectoderm development AF319523 Homo sapiens RT-LI mRNA,
complete sequence AF074331 PAPSS2 3'-phosphoadenosine
5'-phosphosulfate synthase 2 AF189011 RNASE3L Putative ribonuclease
III BC009752 Homo sapiens, Similar to sex comb on midleg-like 1
(Drosophila) NM_000691 ALDH3A1 Aldehyde dehydrogenase 3 family,
memberA1 NM_006006 ZNF145 Zinc finger protein 145 (expressed in
promyelocytic leukemia) NM_005831 NDP52 Nuclear domain 10 protein
L26584 RASGRF1 Ras protein-specific guanine nucleotide-releasing
factor 1 NM_014182 HSPC160 HSPC160 protein NM_004963 GUCY2C
Guanylate cyclase 2C (heat stable enterotoxin receptor) AB023223
STXBP- Tomosyn TOM NM_018919 PCDHGA6 Protocadherin gamma subfamily
A, 6 NM_002968 SALL1 Sal-like 1 (Drosophila) NM_003587 DDX16 DEAD/H
(Asp-Glu-Ala-Asp/His) box polypeptide 16 AK024449 PP2135 PP2135
protein AB034205 LUC7A Cisplatin resistance-associated
overexpressed protein BC011589 OSM Oncostatin M NM_006597 HSPA8
Heat shock 70kD protein 8 NM_004384 CSNK1G3 Casein kinase 1, gamma
3 AK057672 Homo sapiens cDNA FLJ33110 fis NM_016344 PRO1900 PRO1900
protein NM_018651 ZFP Zinc finger protein NM_004717 DGKI
Diacylglycerol kinase, iota NM_006479 PIR51 RAD51-interacting
protein AK024250 Homo sapiens cDNA FLJ14188 fis NM_001382 DPAGT1
Dolichyl-phosphate N- acetylglucosaminephosphotransferase 1
NM_020371 AVEN Cell death regulator aven NM_006311 NCOR1 Nuclear
receptor co-repressor 1
[0675] Discriminant Function Analysis (DFA) is a form of
multivariate analysis that identifies subsets of dependent
variables that characterize a system made up of related groups. In
this kind of gene expression analysis, a linear equation is
calculated, denoted a root, whose overall value is distinct for a
given characterized group. Accordingly, DFA identifies genes most
characteristic of a given state. DFA analysis was conducted on the
genes that were correlated after CSC treatment but not correlated
after S9 treatment, as described in the following example.
Example 11
Refined Analysis of CSC-Correlated Genes Using Discriminant
Function Analysis (DFA)
[0676] The set of 40 genes that were correlated after CSC
treatments (see Table 13 and FIG. 37) but not correlated after S9
microsomal fraction treatment were further analyzed using DFA. Of
the 40 CSC-correlated genes, 11 were identified by DFA as being
most highly distinct among CSC and S9 treated cells (Table 15).
Interestingly, a significant number of these genes were associated
with oncogenesis. For example, this gene set included 3 putative
proto-oncogenes including (1) MAP2K5, the over-expression of which
is associated with increased proliferative and invasive potential
of metastatic prostate cancer and is reported to be a potent
survival molecule in APO-MCF-7 breast carcinoma cells; (2) DDIT3, a
C/EBP transcriptional regulator involved in growth arrest induced
by DNA damage that is a common breakpoint in human myxoid
liposarcomas; and (3) BAG2, a BCL-2-binding apoptosis suppressor
that is over-expressed in human cervical, breast and lung cancer
cell lines. In addition, three putative tumor suppressor genes were
also identified in this gene set. These were FUS1, RASA1, and
FPH2L1. FUS1 can inhibit tumor cell growth by inducing apoptosis,
and was first identified in a search for potential tumor
suppressors within a critical homozygous deletion region at 3p21.3
common in lung cancers. RASA1 as a key member of the GAP1 family of
GTPase-activating proteins plays a key role in the Ras signaling
pathway. DPH2L1 is a BRCA1-induced gene that maps within a region
of 17p13.3, which is deleted in 80% of all ovarian epithelial
malignancies. DPH2L1 was identified by exon trapping in this region
and was implicated as a tumor suppressor as its expression is
reduced or undetectable in ovarian tumors and tumor cell lines. In
addition, a nicotinic cholinergic receptor, CHRNA9, and two
putative neural growth factors, NxpH3, a neuropeptide-like neural
signaling molecule, and NINJ2, a gene up-regulated in damaged nerve
cells that upregulates neurite outgrowth, were also identified in
this gene set. The impact on neural growth factors is not
surprising in light of the fact that many lung cancers express
neuroendocrine features and are also stimulated by an
autocrine/paracrine system of neuroendocrine peptide hormones.
[0677] A graphical representation of the DFA results for the three
treatment conditions at all time points was generated. The spatial
organization of the elements in this representation provided a
measure of the overall variance among groups (see FIG. 40). The
genes used for this analysis were correlated in CSC exposed cells
and not correlated in S9-treated cells. A correlation coefficient
of 0.8 was used as a threshold for defining similarity. The
expression of these genes should therefore be similar in
CSC-treated cells. Indeed the two CSC groups were more closely
associated than either CSC group was to the S9 microsomal
fraction-treated group. Of note, the samples from the CSC groups
did not overlap, indicating that the two CSC treatments elicit
somewhat distinct responses even in genes highly correlated in
their behavior in each CSC group.
TABLE-US-00021 TABLE 15 Discriminant Function Analysis of
CSC-Correlated Genes GenBank Gene accession no. abbreviation Gene
description M23326 TRDV3 T cell receptor delta variable 3 NM_002757
MAP2K5 Mitogen-activated protein kinase kinase 5 NM_004083 DDIT3
DNA-damage-inducible transcript 3 NM_004282 BAG2 BCL2-associated
athanogene 2 NM_007275 FUS1 Lung cancer candidate NM_003408 ZFP37
Zinc finger protein 37 homolog (mouse) NM_002046 GAPD
Glyceraldehyde-3-phosphate dehydrogenase NM_017581 CHRNA9
Cholinergic receptor, nicotinic BC015737 NINJ2 Ninjurin 2 AB032985
NXPH3 Neurexophilin 3 NM_002890 RASA1 RAS p21 protein activator
NM_001383 DPH2L1 Diptheria toxin resistance protein
[0678] FIG. 41 shows the result of a functional analysis of the
gene set in TABLE 14 using Pathway Assist. Not surprisingly, the
major cellular processes affected by these genes were subset of the
processes affected by the parent gene set, as illustrated in FIG.
38.
[0679] Four post-treatment expression characteristics were
established for each gene on the array: (1) whether or not the gene
was expressed above background at each time-point; (2) whether or
not the gene showed hypervariability (i.e. change greater than
normal) of expression in one, two, or all three treatment
conditions over the 12 h treatment period; (3) what was the
specific pattern of gene expression over the 12 h treatment period;
and (4) whether or not the gene expression pattern in each
condition correlated with its behavior under the two other
conditions from 0-12 h. Several interesting observations emerged
from this analysis. Significantly, treatment of NHBE cells with
CSCs from two American brands of cigarettes altered the expression
of approximately 3600 genes and ORFs (or 17% of the array) sometime
during the 12-hour exposure (see FIGS. 1 and 2). These data provide
evidence that due to their chemical complexity and temporal
requirement for metabolic activation, CSCs should have a broad and
dynamic effect on the homeostatic transcriptome of the NHBE cell.
In addition to the quantitative similarities in gene alterations
induced by the different CSCs, there were also qualitative
similarities in that both CSCs affected a large common block of
genes, which is not surprising given the relatively comparable
types of blended tobaccos used in most American cigarette
brands.
[0680] Several approaches were employed to discriminate and cluster
genes that became hypervariable after CSC treatment so as to
develop a robust and accurate statistical estimate of functional
significance for these perturbations. For example, as shown in FIG.
38, CSCs affected networks of genes that intersect critical
signaling pathways such as apoptosis, transcription, and cell cycle
regulation, which are known to play key roles in specific diseases
such as cancer, chronic inflammation, and impaired neural
development, and which both epidemiological and functional studies
conclude can be caused by chronic cigarette smoking. The relevance
of these pathways to smoking-related diseases is further supported
by a limited body of published data in which other cell types or
tissues exposed to either smoke, CSC, or a specific substance in
CSC (e.g., benzo[a]pyrene, nicotine, etc.) were assessed using
low-density arrays (see Nadadur et al., Chest 121: 83S-84S, 2002;
Nordskog et al. Cardiovasc Toxicol 3: 101-117, 2003; Zhang et al.
Physiol Genomics 5: 187-192, 2001; Gebel et al. Carcinogenesis
25:169-178, 2003).
[0681] The sensitivity and accuracy of the methodologies used
herein to identify genes impacted by CSCs was further shown by the
fact that the set of HV genes in CSC-treated cells included many of
the genes and/or gene families that have been identified using
various global expression analyses (e.g., Serial Analysis of Gene
Expression, Differential Display, and microarrays) and concluded to
be of importance in the development and/or maintenance of lung
cancers. These include erb-B2, matrix metalloproteinase 9 (MMP9),
the heterogeneous nuclear ribonucleoprotein (hnRNP) family, the
Fus1 lung cancer candidate, glutathione S-transferase pi, the
.beta.-retinoic acid receptor, chromogranin B, RAB5,
death-associated protein kinase 1 (DAPK), various cancer/testis
antigens [MAGE genes], and others. For the first time, however, the
present disclosure demonstrates that expression of these genes is
altered in normal bronchial epithelial cells exposed to CSCs for
only a short period of time, which provides evidence that one or
more of these genes are an early indicator of tobacco-related
cellular damage. In addition, the data herein identify a large
number of genes and gene families that had not yet been associated
with the induction or maintenance of pulmonary neoplasms or to
other tobacco-related diseases involving the cardiovascular and
immune systems. Accordingly, many of the genes identified using the
approaches described herein are particularly useful biomarkers of
the pathogenesis of these diseases.
[0682] The highly correlated expression characteristics of the
CSC-impacted genes shown in TABLE 7 and FIG. 38, for example,
highlight several genes that appear to play prominent roles in
tobacco-related diseases. Both DPH2L1 and Fus1 are putative tumor
suppressor genes associated with ovarian and lung cancer,
respectively. Fus1 is found at a homozygous deleted region of
chromosome 3p21 in lung tumors, and its forced expression in lung
carcinoma cells suppresses cell growth in vitro and growth and
metastases of tumors in vivo by mechanisms involving G1-arrest and
induction of apoptosis. The RASA1 is a component of the GAP1 family
of GTPase-activating proteins, which can suppress proliferation
signals by enhancing the weak intrinsic GTPase activity of normal
RAS p21 protein and maintaining it in its inactive GDP-bound form.
It is contemplated that Ras acts as a major nexus for multiple
signaling pathways that control a diverse range of functions, but
many of the subtleties of Ras functioning in individual cell types
remain unclear. It is also though that Ras plays an important role
in tumor cell survival. The MAP2K5 is a novel mitogen activated
protein kinase implicated in the regulation of cell proliferation.
Over-expression of MAP2K5 can, in cooperation with other effectors,
transform rodent cells, and function as a potent survival molecule
in breast cancer cells. MAP2K5 represents a potential therapeutic
target in prostate cancer as over-expression of MAP2K5 can induce
proliferation, motility, and invasion. Interestingly, MAP2K5 also
dramatically up-regulates the expression of matrix
metalloproteinase-9 (MMP9) in prostate cancers. As shown in TABLE
7, MMP9 was hypervariable in both CSC-treatment groups. The matrix
metalloproteinases (MMPs) are a large family of extracellular
matrix degrading enzymes believed to play central roles in
degradation, remodeling, and repair of basement membranes.
Inappropriate or over-expression of these proteins appear to a
critical determinant in tumor invasion and metastasis of a number
of neoplasms including those of the lung. For example, MMP9
potentiates pulmonary metastasis formation, and high serum levels
of MMP9 in patients with non-small-cell lung cancer (NSCLC)
correlated with significantly shorter survival than patients with
low serum levels of this protein.
[0683] In addition to a common set of affected genes, each
individual CSC also altered the expression of a relatively large
gene set that was unique to each CSC. That is, it was discovered
that each tobacco smoke condensate was associated with a unique
genetic fingerprint. The impact on these unique gene sets may be
due to qualitative and/or quantitative differences in the
constellation of chemical constituents in the two CSCs. It is
interesting to note that despite the fact that both Brand A and
Brand B are similar types of cigarettes (i.e., `full-flavor`) as
determined by FTC criteria, there are measurable differences in the
quantities of nicotine, tar, as well as, toxins and carcinogens
between Brand-A and Brand-B cigarettes. It is contemplated that the
differences in one or more of these substances directly correlates
with the observed differences in gene induction and level of
expression. Moreover, it is contemplated that each unique gene set
affected by CSC-A and CSC-B ultimately influences different
cellular pathways and results in different biological
consequences.
[0684] Several basic assumptions of the emerging field of
toxicogenomics are that there are reasonable similarities in gene
expression patterns induced by multiple members of one specific
class of toxicants, and subtle differences in these gene expression
patterns may distinguish distinct chemical-specific `gene
signatures` of exposure (Afshari et al., Cancer Res 59: 4759-4760,
1999; Neumann et al. Biotechnol Adv 20: 391-419, 2002). For the
first time, the approaches described herein provide one with the
ability to identify a unique genetic fingerprint or signature for a
plurality of tobacco products by contacting NHBE cells or another
cell type of the lung, mouth or oral cavity with a tobacco smoke
condensate or tobacco smoke from said plurality of tobacco
products, identifying the genes expressed as a result of the
contact in each individual tobacco product, as well as the level of
expression of each, comparing the fingerprint or component thereof
(e.g., a specific gene or set of genes or level of expression of a
specific gene or set of genes) of the plurality of tobacco products
that are being analyzed (or to a database containing genetic
fingerprints of tobacco products), identifying differences in the
fingerprint or component thereof between the products that are
being analyzed, and associating the difference in the fingerprint
or component thereof to an increased or decreased risk, proclivity,
or potential to acquire a tobacco-related disease (e.g., lung
cancer).
[0685] Another significant discovery made in the experiments
described above, as shown in FIG. 35, is that the majority of
CSC-affected genes do not return to baseline within the 12-hour
treatment period, especially for CSC-B-affected genes. This
observation is not simply due to the fact that the cells were
chronically exposed to the CSCs for the entire 12-hours, as is
discussed-infra. It is contemplated that many of the affected genes
require a significant amount of time to return to baseline even
after exposure is terminated. Accordingly, a current pack-a-day
smoker who averages >150 cigarette puffs/day may alter the
homeostatic expression of a large number of genes that cannot
return to a baseline state during a typical day. This chronically
perturbed state (either increased or decreased compared to
baseline) of one or more of these genes may ultimately be
etiologically involved in various pathological states caused by
exposure to cigarette smoke. Evidence of this is provided by the
fact that in subjects who quit smoking there is both short-term
improvement in the functioning of a number of affected organ
systems (e.g., lung, cardiovascular structures, kidneys, etc.) and
a long-term decline in incidence and mortality from various
diseases affecting these systems. Presumably, this reversal of
smoking-related damage at the tissue and population levels reflects
a corresponding reversal at a molecular and cellular level.
[0686] For example, chronic inflammatory processes in smokers play
fundamental roles in the pathogenesis of atherosclerosis, and
increased plasma and tissue levels of several biomarkers associated
with inflammation such as various cytokines (e.g., IL-1.beta.,
TNF-.alpha.), pro-atherogenic enzymes (e.g., lipoprotein lipase)
and cell adhesion molecules (e.g., VCAM-1) are associated with
future cardiovascular risk, while smoking cessation leads to
decreased expression of many pro-inflammatory biomolecules and a
concomitant reduction in cardiovascular risk. It is also possible
that the altered expression of one or more genes in the habitual
smoker becomes attenuated with time as an adaptive response to the
stress of chronic activation, and this phenomenon may have
unanticipated long-term biological consequences for the smoker.
[0687] Another unexpected finding of this study was that the S9
metabolic enzyme fraction significantly influenced gene expression
in NHBE cells. S9-exposed cells are traditionally considered a
negative control for toxicogenetic experiments performed to
establish environmental and occupational exposure guidelines. The
fact that gene alterations were observed as early as 2 hours
post-S9 exposure has interpretive implications for standard
toxicological assays that routinely measure biological and genetic
effects of control and test substances after 4 hours of exposure.
This observation is particularly relevant as the global shift
towards advanced genomic and proteomic technologies transforms the
field of toxicology from one relying on the induction of gross
genetic abnormalities such as mutations and structural/numerical
chromosomal abnormalities to one where altered expression of panels
of genes and proteins are used to determine risk to the human
population. In order to clearly establish the potential toxicity or
efficacy of an environmental substance, drug, or chemopreventive
agent, it is important to show that control substances or vehicles
used in the methodology cause minimal disruption of the
physiologically normal transcriptome. Furthermore, since S9 can
induce a range of alterations in gene expression levels independent
of any test substance, it is possible that one or more S9-induced
effects can be synergistic or antagonistic with the test
substances. For example, FIG. 36 shows that many of the same genes
that are down-regulated in S9-treated cells are up-regulated in
CSC-treated cells despite the fact that CSCs contain the same
concentration of S9 enzymes. Alternatively, the effects of S9 can
be mitigated by the test substance. Evidence for this is strongly
supported by the data, which shows that a number of genes whose
steady-state mRNA level were found to be altered only by S9 were
not found to be altered when cells were exposed to S9 in context
with either CSC-A or CSC-B. In this scenario, the direct effects of
S9, which can be directly cytotoxic to cells in cultures, may be
attenuated when sequestered and modified through contact with
substances in CSCs.
[0688] Although the analysis of normal human bronchial epithelial
cells (NHBE cells) contacted with tobacco smoke condensates,
described above, provide several ways to identify the genes that
are modulated in response to human exposure to tobacco smoke,
another approach involves analysis of cells of the mouth, oral
cavity, trachea, and lungs, either normal or immortalized cell
lines (e.g., human bronchial cells (e.g., BEP2D or 16HBE140 cells),
human bronchial epithelial cells (e.g., HBEC cells, 1198, or 1170-I
cells), normal human bronchial epithelial cells (NHBE cells), BEAS
cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer
(NSCLC) cells or human alveolar cells (e.g., H460, H1792, SK-MES-1,
Calu, H292, H157, H1944, H596, H522, A549, and H226) tongue cells
(e.g., CAL 27), and mouth cells (e.g., Ueda-1)), which are
contacted with cigarette smoke. Accordingly, as described in the
following example, several experiments were conducted to evaluate
the genes that were expressed, as well as the expression levels,
when NHBE cells were exposed to tobacco smoke.
Example 12
Microarray Analysis in CS Experiments
[0689] Once the NHBE cells were contacted with tobacco smoke or
with air ("mock exposure"), as described in Example 4, the cDNA of
NHBE cells that were either mock exposed or tobacco smoke exposed
was prepared for microarray analysis as follows. Cells were
harvested for total RNA extraction after either mock or smoke
treatment. The RNA from each Petri dish was used for a separate
microarray chip, which resulted in a total of 18 microarrays (ten
from Experiment 1 and eight from Experiment 2). The medium was
aspirated and the dishes were rinsed twice with 1 mL prewarmed PBS
per dish. After the second rinse, 1 mL of cold TRIzol.RTM.
(Invitrogen Corp., Carlsbad, Calif.) was added to each dish. NHBE
cell lysates were prepared and the RNA was extracted according to
the manufacturer's protocol. The RNA pellet was frozen and stored
at -80.degree. C.
[0690] Prior to cDNA synthesis, the RNA was resuspended in
diethylpyrocarbonate-treated water. RNA integrity was assessed
using capillary gel electrophoresis (Agilent BioAnalyzer, Agilent
Technologies, Palo Alto, Calif.) to determine the ratio of 28s:18s
rRNA in each sample. A threshold of 1.0 was used to define samples
of sufficient quality and only these samples were used for
microarray studies. The RNA quality of all samples was extremely
high with no ratios less than 1.8. Fluorescently labeled cDNA was
synthesized and purified as previously described. (See Jarvis et
al. Arthritis Res Ther, 6: R15-R32, 2004, expressly incorporated by
reference in its entirety).
[0691] A commercially available, genome-scale oligonucleotide
library containing gene-specific 70-mer oligonucleotides
representing 21,329 human genes was used for microarray production
(QIAGEN Inc., Valencia, Calif.). Oligonucleotides were spotted onto
Corning.RTM. UltraGAPS.TM. amino-silane coated slides, which were
then rehydrated with water vapor, snap dried at 90.degree. C.
Oligonucleotide DNAs were covalently fixed to the surface of the
glass using 300 mJ of ultraviolet radiation at a 254 nm wavelength.
Unbound free amines on the glass surface were blocked for 15 min
with moderate agitation in a solution of 143 mM succinic anhydride
dissolved in 1-methyl-2-pyrolidinone, 20 mM sodium borate, pH 8.0.
Slides were rinsed for 2 min in distilled water, immersed for 1 min
in 95% ethanol, and dried with a stream of nitrogen gas.
[0692] Hybridization was performed in an automated liquid delivery,
air-vortexed, hybridization station for 9 hr at 58.degree. C. under
an oil-based cover slip (Ventana Medical Systems, Inc. Tucson,
Ariz.). Microarrays were washed at a final stringency of
0.1.times.SSC. Microarrays were scanned using a simultaneous dual
color, 48-slide scanner (Agilent Technologies). Fluorescent
intensity was quantified using Imagene.TM. software (BioDiscovery,
Marina del Rey, Calif.).
[0693] Adjustment of expression levels in compared samples was
performed as previously described. (See Dozmorov, et al.
Bioinformatics., 19: 2004-211, 2003; Knowlton, N., et al.
Bioinformatics., 20: 3687-3690, 2004; and Dozmorov, et al.
Bioinformatics., 5:53, 2004, each of which is incorporated by
reference in its entirety). To determine differentially expressed
genes, the analysis was confined to the set of genes that were
expressed above background in at least one condition (i.e., 4
and/or 24 hours post-exposure, CS-treated or mock-treated). For
each experiment, replicates from each condition were averaged and
genes that were under- or over-expressed ("modulated") in response
to tobacco smoke treatment (e.g., cigarette smoke (CS)) by 1.5-fold
or more at either or both time points were identified. Genes
exhibiting similar expression behavior in both experiments were
determined.
[0694] Quantitative Reverse Transcriptase PCR (qRT-PCR)
[0695] To determine the level of expression, RNA was
reverse-transcribed using an Omniscript RT.TM. kit according to
manufacturer's instructions (Qiagen, Valencia, Calif.) and the
resultant cDNA subsequently purified using the Montage PCR 96-well
cleanup plate (Millipore, Billerica, Mass., USA). The qRT-PCR
amplifications were performed on an ABI.RTM.PRISM 7700 sequence
detection system using SYBR.RTM.Green I dye assay chemistry. A 15
uL PCR reaction for each gene of interest was prepared consisting
of 7.5 uL of 2.times.SYBR.RTM.Green PCR mix (Applied Biosystems
Inc., Foster City, Calif.), 4.9 .mu.l of H20, 0.6 .mu.l (30 pmoles)
of gene-specific forward and reverse primers, and 2 .mu.l (1 ng) of
cDNA template. All samples were run in triplicate with the
appropriate single qRT-PCR controls (no reverse transcriptase and
no template). Cycling conditions used for all amplifications were
one cycle of 95.degree. C. for 10 minutes and 40 cycles of
95.degree. C. for 15 seconds and 60.degree. C. for 1 minute.
Following the qRT-PCR, dissociation curve analysis was performed to
determine if the desired single gene product was produced.
[0696] Gene Expression Alterations Induced by CS Exposure
[0697] In order to determine the broad impact of a brief transient
exposure to cigarette smoke (CS) on the transcriptome of NHBE
cells, monolayer cultures of NHBE cells were treated in logarithmic
phase of growth for 15 minutes with whole smoke from a leading
representative brand of American cigarettes, and then assessed for
global alterations in their transcriptome at 4 h and 24 h
post-exposure. Furthermore, in an attempt to unambiguously define a
set of genes consistently impacted by CS, this experiment was
performed twice and then the focus was restricted to only those
individual genes whose RNA levels similarly deviated by 1.5 fold or
greater in the two experiments (either overexpressed or
underexpressed in response to CS treatment). By assessing global
RNA changes at 4 and 24 h post-exposure, the temporal relationships
of those genes whose RNA levels were altered a) by 4 hours and that
returned to baseline by 24 hours; b) by 4 hours and did not return
to baseline by 24 hours; and c) only by 24 hours could be
observed.
[0698] Approximately 10% of the 21,329 human genes represented on
the array were expressed above background in mock-treated cells.
This amount of expression presumably represents the typical
transcriptome of unstressed NHBE cells in vitro, and agrees well
with published data on the human airway transcriptome of healthy
nonsmokers. Interestingly, CS-treated NHBE cells also expressed
approximately 10% of the total gene complement, suggesting that
brief CS-exposure does not induce a major quantitative
reorganization of the normal transcriptome of lung cells.
[0699] Of the 21,329 genes on the array, a set of 364 genes
exhibited similar changes in expression level in both experiments
(See TABLE 16). A subset of 298 genes that were overexpressed
1.5-fold or more in both experiments was compared to mock-treated
cells. Of this set of 298 up-regulated genes, 184 were up-regulated
exclusively at 4 h post cigarette smoke exposure, while 69 were
up-regulated exclusively at 24 h post-exposure, and 45 were
up-regulated at both time points. The number of genes that were
under-expressed at least 1.5-fold in cells exposed to cigarette
smoke was 66, with 35 down-regulated exclusively at 4 h post
CS-exposure, 30 down-regulated exclusively at 24 h post-exposure,
and one down-regulated at both time points. Further confirmation
that the entire set of 364 up and down-regulated genes accurately
reflect a reliable genetic response to cigarette smoke exposure is
evidenced by the fact that a majority of the genes exhibited
remarkably consistent expression behaviors in both experiments.
TABLE-US-00022 TABLE 16 Genes Upregulated by Cigarette Smoke Fold
Fold In- In- crease crease at at Gene ID Gene Name Description 4 h
24 h NM_004261 SEP 15 15 kDa selenoprotein 1.71 1.29 NM_000859
HMGCR 3-hydroxy-3- 2.25 1.33 methylglutaryl- Coenzyme A reductase
AK025736 HMGCS1 3-hydroxy-3- 1.02 1.63 methylglutaryl- Coenzyme A
synthase 1 (soluble) NM_002526 NT5 5' nucleotidase (CD73) 1.45 1.69
NM_001109 ADAM8 A disintegrin and 1.17 2.72 metalloproteinase
domain 8 NM_005891 ACAT2 Acetyl-Coenzyme A 1.44 1.77
acetyltransferase 2 (acetoacetyl Coenzyme A thiolase) NM_006409
ARPC1A Actin related protein 2/3 2.01 1.79 complex, subunit 1A (41
kD) NM_018445 LOC55829 AD-015 protein 1.64 2.02 NM_001284 AP3S1
Adaptor-related protein 2.18 1.27 complex 3, sigma 1 subunit
NM_000485 APRT Adenine 1.56 1.63 phosphoribosyltransferase
NM_007002 ADRM1 Adhesion regulating 1.68 1.61 molecule 1 NM_006829
APM2 Adipose specific 2 1.96 2.34 NM_001667 ARL2 ADP-ribosylation
2.06 0.80 factor-like 2 NM_000693 ALDH1A3 Aldehyde dehydrogenase
0.82 2.88 1 family, member A3 NM_001635 AMPH Amphiphysin
(Stiff-Mann 1.78 2.16 syndrome with breast cancer 128kD
autoantigen) NM_001657 AREG Amphiregulin 1.96 0.33 (schwannoma-
derived growth factor) NM_001145 ANG Angiogenin, ribonuclease, 1.61
1.10 RNase A family, 5 NM_000700 ANXA1 Annexin A1 1.39 1.82
NM_005139 ANXA3 Annexin A3 1.34 1.71 NM_001154 ANXA5 Annexin A5
2.40 2.43 NM_004034 ANXA7 Annexin A7 2.10 1.64 NM_016476 ANAPC11
APC11 anaphase 1.68 1.30 promoting complex subunit 11 homolog
(yeast) NM_016085 APR-3 Apoptosis related 1.44 0.84 protein APR-3
NM_005721 ACTR3 ARP3 actin-related protein 1.63 1.72 3 homolog
(yeast) NM_017900 AKIP aurora-A kinase interacting 2.07 5.18
protein M90355 BTF3L2 Basic transcription 1.87 1.47 factor 3, like
2 NM_004281 BAG3 BCL2-associated 3.85 1.58 athanogene 3 NM_001196
BID BH3 interacting domain 1.54 1.05 death agonist NM_003860 BCRP1
Breakpoint cluster region 1.99 1.52 protein, uterine leiomyoma,
1-barrier to autointegration factor NM_014567 BCAR1 Breast cancer
anti-estrogen 1.00 1.88 resistance 1 NM_021096 CACNA1I Calcium
channel, voltage- 1.68 2.75 dependent, alpha 1I subunit NM_005186
CAPN1 Calpain 1, (mu/I) large 1.62 1.11 subunit NM_001750 CAST
Calpastatin 1.47 1.76 NM_013376 SEI1 CDK4-binding protein 2.46 1.87
p34SEI1 NM_015965 GRIM19 Cell death-regulatory 2.16 2.23 protein
GRIM19 NM_016041 F-LAN-1 CGI-101 protein 1.51 1.58 NM_016038
LOC51119 CGI-97 protein 1.78 2.34 BC002971 CCT5 Chaperonin
containing 1.81 1.74 TCP1, subunit 5 (epsilon) NM_006429 CCT7
Chaperonin containing 2.85 3.21 TCP1, subunit 7 (eta) NM_000647
CCR2 Chemokine (C-C motif) 0.69 3.35 receptor 2 NM_012111 C14orf3
Chromosome 14 open 1.88 1.15 reading frame 3 AK026450 C20orf162
Chromosome 20 open 1.16 1.49 reading frame 162 NM_007096 CLTA
Clathrin, light 1.96 2.01 polypeptide (Lca) BC010039 CLP
Coactosin-like protein 1.54 1.24 NM_016451 COPB Coatomer protein
complex, 1.82 1.79 subunit beta NM_007263 COPE Coatomer protein
complex, 2.58 2.98 subunit epsilon NM_004645 COIL Coilin 1.21 1.79
AL162070 CORO1C Coronin, actin binding 2.00 1.59 protein, 1C
NM_000389 CDKN1A Cyclin-dependent kinase 4.69 1.38 inhibitor 1A
(p21, Cip1) NM_000099 CST3 Cystatin C (amyloid 2.11 1.54 angiopathy
and cerebral hemorrhage) NM_001554 CYR61 Cysteine-rich, angiogenic
2.44 0.67 inducer, 61 NM_007274 HBACH Cytosolic acyl coenzyme A
1.61 2.28 thioester hydrolase NM_020189 DC6 DC6 protein 1.64 1.73
NM_004396 DDX5 DEAD/H (Asp-Glu-Ala- 2.01 4.10 Asp/His) box
polypeptide 5 (RNA helicase, 68kD) NM_001357 DDX9 DEAD/H
(Asp-Glu-Ala- 1.44 1.53 Asp/His) box polypeptide 9 (RNA helicase A,
nuclear DNA helicase II-leukophysin AB040961 DTX2 Deltex homolog 2
1.76 1.62 (Drosophila) NM_007326 DIA1 Diaphorase (NADH) 1.84 2.06
(cytochrome b-5 reductase) NM_020548 DBI Diazepam binding inhibitor
1.69 1.84 (GABA receptor modulator, acyl- Coenzyme A binding
protein) NM_ 013253 DKK3 Dickkopf homolog 3 1.64 0.84 (Xenopus
laevis) NM_004405 DLX2 Distal-less homeo box 2 29.27 2.13 AL080156
DKFZP434J DKFZP434J214 protein 2.97 1.43 214 NM_014045 DKFZP564C
DKFZP564C1940 protein 1.79 1.73 1940 NM_001539 DNAJA1 DnaJ (Hsp40)
homolog, 2.11 1.85 subfamily A, member 1 NM_006145 DNAJB1 DnaJ
(Hsp40) homolog, 4.99 1.57 subfmaily B, member 1 NM_004419 DUSP5
Dual specificity 1.97 0.47 phosphatase 5 NM_001946 DUSP6 Dual
specificity 2.08 2.29 phosphatase 6 NM_014390 p100 EBNA-2
co-activator 2.00 1.02 (100kD) NM_005451 ENIGMA Enigma (LIM domain
1.21 2.34 protein) NM_004092 ECHS1 Enoyl Coenzyme A 1.60 1.23
hydratase, short chain, 1, mitochondrial NM_004431 EPHA2 EphA2 2.37
1.93 NM_016357 EPLIN Epithelial protein lost in 1.74 1.63 neoplasm
beta BF541376 ESTs, Weakly similar 2.71 4.50 to FRHUL ferritin
light chain [H. sapiens] NM_003757 EIF3S2 Eukaryotic translation
1.83 1.47 initiation factor 3, subunit 2 (beta, 36kD) NM_003755
EIF3S4 Eukaryotic translation 2.12 2.40 initiation factor 3,
subunit 4 (delta, 44kD) NM_001417 EIF4B Eukaryotic translation 2.33
2.41 initiation factor 4B NM_004095 EIF4EBP1 Eukaryotic translation
1.69 1.26 initiation factor 4E binding protein 1 NM_005243 EWSR1
Ewing sarcoma breakpoint 2.02 1.33 region 1 NM_005245 FAT FAT tumor
suppressor 1.87 0.77 homolog 1 (Drosophila) NM_004104 FASN Fatty
acid synthase 1.24 1.60 AK054816 FTH1 Ferritin, heavy 2.07 3.32
polypeptide 1 NM_001457 FLNB Filamin B, beta (actin 1.05 1.90
binding protein 278) NM_014164 FXYD5 FXYD domain-containing 1.24
1.67 ion transport regulator 5 AL365404 GPR108 G protein-coupled
2.00 1.17 receptor 108 NM_007278 GABARAP GABA(A) receptor- 1.55
1.75 associated protein NM_001520 GTF3C1 General transcription 8.72
0.41 factor IIIC, polypeptide 1 (alpha subunit, 220kD) AK024486
GLTSCR2 Glioma tumor suppressor 2.63 1.85 candidate region gene 2
NM_001498 GCLC Glutamate-cysteine ligase, 8.96 1.40 catalytic
subunit NM_002061 GCLM Glutamate-cysteine ligase, 2.85 1.56
modifier subunit NM_004446 EPRS Glutamyl-prolyl-tRNA 1.76 0.73
synthetase NM_002064 GLRX Glutaredoxin 3.12 2.31 (thioltransferase)
NM_002083 GPX2 Glutathione peroxidase 2 3.71 9.99
(gastrointestinal) NM_000637 GSR Glutathione reductase 1.57 1.54
NM_002087 GRN Granulin 1.36 1.58 L24498 GADD45A Growth arrest and
DNA- 2.81 0.61 damage-inducible, alpha NM_006644 HSP105B Heat shock
105kD 2.83 1.02 NM_002157 HSPE1 Heat shock 10kD protein 1 1.92 1.34
(chaperonin 10) NM_005345 HSPA1A Heat shock 70kD 5.77 1.30 protein
1A NM_006597 HSPA8 Heat shock 70kD protein 8 1.48 4.56 NM_004134
HSPA9B Heat shock 70kD protein 2.23 1.39 9B (mortalin-2) NM_016292
TRAP1 Heat shock protein 75 1.57 1.05 NM_002133 HMOX1 Heme
oxygenase 55.83 2.81 (decycling) 1 NM_004712 HGS Hepatocyte growth
factor- 1.21 1.64 regulated tyrosine kinase substrate NM_001533
HNRPL Heterogeneous nuclear 1.50 0.89 ribonucleoprotein L AK057120
HMG1 High-mobility 1.72 0.79 group (nonhistone chromosomal) protein
1 AF130111 HDAC3 Histone deacetylase 3 1.92 1.38 NM_001536 HRMT1L2
HMT1 hnRNP 1.83 1.16 methyltransferase- like 2 (S. cerevisiae)
AK023395 Homo sapiens 1.82 1.39 cDNA FLJ13333 fis, clone
OVARC1001828 AK054711 Homo sapiens 1.57 0.76 cDNA FLJ30149 fis,
clone BRACE2000280, weakly similar to MNN4 PROTEIN AK055071 Homo
sapiens 1.36 1.64 cDNA FLJ30509 fis, clone BRAWH2000595 AK056736
Homo sapiens 1.18 4.26 cDNA FLJ32174 fis, clone PLACE6001064
AK024927 Homo sapiens 1.83 0.89 cDNA: FLJ21274 fis, clone COL01781
AK055564 Homo sapiens 1.00 1.50 cDNA: FLJ22182 fis, clone HRC00953
AK026181 Homo sapiens 4.30 1.72
cDNA: FLJ22528 fis, clone HRC12825 AK026902 Homo sapiens 1.76 1.09
cDNA: FLJ23249 fis, clone COL04196 AL512727 Homo sapiens 2.01 2.48
mRNA-cDNA DKFZp547P042 (from clone DKFZp547P042) AL117595 Homo
sapiens 2.71 1.30 mRNA-cDNA DKFZp564C2063 (from clone
DKFZp564C2063) AL050378 Homo sapiens 1.37 1.70 mRNA-cDNA
DKFZp586I1420 (from clone DKFZp586I1420)- partial cds AF041429 Homo
sapiens pRGR1 1.37 1.86 mRNA, partial cds AF118072 Homo sapiens
PRO1716 5.32 19.31 mRNA, complete cds AF065241 Homo sapiens
thioredoxin 1.20 1.80 delta 3 (TXN delta 3) mRNA, partial cds
BC010009 Homo sapiens, clone 1.49 1.93 IMAGE: 3355383, mRNA,
partial cds BC011880 Homo sapiens, Similar to 1.07 1.65
hypothetical protein, MGC: 7764, clone MGC: 20548 IMAGE: 3607345,
mRNA, comple BC017001 Homo sapiens, Similar 26.36 5.69 to RIKEN
cDNA 1700127B04 gene, clone IMAGE: 4425440, mRNA, partial cds
BC007307 Homo sapiens, 1.89 1.59 Similar to zinc finger protein
268, clone IMAGE: 3352268, mRNA, partial cds NM_014029 HSPC022
HSPC022 protein 1.33 3.77 NM_014047 HSPC023 HSPC023 protein 1.64
1.98 AF161415 HSPC030 HSPC030 protein 4.27 1.52 NM_016099 Loc51125
HSPC041 protein 1.46 1.08 NM_014168 HSPC133 HSPC133 protein 1.58
1.41 NM_014182 HSPC160 HSPC160 protein 1.28 2.58 AL139112 Human DNA
sequence 1.88 2.68 from clone GS1-103B18 on chromosome Xq27.1-27.3
Contains ESTs, STSs and GSSs. Con AL354915 Human DNA sequence 1.38
2.01 from clone RP11-392A19 on chromosome 13. Contains ESTs, STSs
and GSSs. Contains a NM_000182 HADHA Hydroxyacyl-Coenzyme A 2.39
1.22 dehydrogenase/ 3-ketoacyl-Coenzyme A thiolase/enoyl- Coenzyme
A hydratase (trif NM_016404 HSPC152 Hypothetical protein 1.59 1.30
NM_016623 BM-009 Hypothetical protein 1.53 1.08 NM_015932 HSPC014
Hypothetical protein 1.31 1.56 NM_015343 HSA011916 Hypothetical
protein 1.79 1.22 AF103803 H41 Hypothetical protein 1.63 2.00
NM_014886 YR-29 Hypothetical protein 1.53 1.44 NM_018437 EDAG-1
Hypothetical protein 1.46 1.94 EDAG-1 NM_018306 FLJ11036
Hypothetical protein 2.07 2.12 FLJ11036 NM_032813 FLJ14624
Hypothetical protein 1.80 2.88 FLJ14624 NM_022842 FLJ22969
Hypothetical protein 3.39 31.88 FLJ22969 NM_031207 HT036
Hypothetical 1.26 2.55 protein HT036 NM_024508 MGC10796
Hypothetical protein 1.46 1.84 MGC10796 AK027859 MGC11266
Hypothetical protein 2.46 2.14 MGC11266 NM_032771 MGC12217
Hypothetical protein 1.56 1.02 MGC12217 BC014850 MGC13071
Hypothetical protein 1.74 1.98 MGC13071 NM_032899 MGC14128
Hypothetical protein 1.15 6.78 MGC14128 NM_024040 MGC2491
Hypothetical protein 2.69 2.86 MGC2491 NM_024038 MGC2803
Hypothetical protein 1.59 1.48 MGC2803 NM_031943 IFP38 IFP38 2.11
1.95 NM_052815 IER3 Immediate early response 3 2.94 1.54 NM_016545
IER5 Immediate early response 5 9.20 1.18 NM_005542 INSIG1 Insulin
induced gene 1 2.02 2.62 NM_021999 ITM2B Integral membrane 1.84
1.06 protein 2B NM_006147 IRF6 Interferon regulatory 2.30 1.09
factor 6 NM_000576 IL1B Interleukin 1, beta 0.98 3.03 Z17227 IL10RB
Interleukin 10 1.74 1.68 receptor, beta NM_004508 IDI1
Isopentenyl-diphosphate 1.89 2.68 delta isomerase NM_005354 JUND
Jun D proto-oncogene 1.67 1.25 NM_006854 KDELR2 KDEL
(Lys-Asp-Glu-Leu) 2.03 1.42 endoplasmic reticulum protein retention
receptor 2 NM_000421 KRT10 Keratin 10 (epidermolytic 1.87 1.68
hyperkeratosis-keratosis palmaris et plantaris) NM_000224 KRT18
Keratin 18 1.22 1.81 NM_005555 KRT6B Keratin 6B 1.44 2.26 NM_014815
KIAA0130 KIAA0130 gene product 1.31 4.73 NM_000899 KITLG KIT ligand
1.35 2.21 NM_001730 KLF5 Kruppel-like factor 5 2.34 1.01
(intestinal) NM_003937 KYNU Kynureninase 3.31 3.29 (L-kynurenine
hydrolase) NM_005558 LAD1 Ladinin 1 1.44 2.29 NM_016201 LCCP Leman
coiled-coil protein 1.89 1.09 NM_015925 LISCH7 Liver-specific
bHLH-Zip 1.29 1.64 transcription factor NM_014463 LSM3 Lsm3 protein
1.85 1.98 NM_004995 MMP14 Matrix metalloproteinase 2.20 2.57 14
(membrane-inserted) NM_005916 MCM7 MCM7 minichromosome 1.60 1.07
maintenance deficient 7 (S. cerevisiae) NM_006428 MAAT1
Melanoma-associated 1.99 1.43 antigen recognised by cytotoxic T
lymphocytes NM_006636 MTHFD2 Methylene tetrahydrofolate 1.81 0.68
dehydrogenase (NAD+ dependent), methenyltetrahydrofolate
cyclohydrolase NM_004528 MGST3 Microsomal glutathione S- 1.73 1.76
transferase 3 NM_022818 MAP1A/1B Microtubule-associated 2.18 0.95
LC3 proteins 1A/1B light chain 3 NM_014341 MTCH1 Mitochondrial
carrier 1.81 1.69 homolog 1 NM_014161 MRPL18 Mitochondrial
ribosomal 3.58 1.63 protein L18 NM_021134 MRPL23 Mitochondrial
ribosomal 1.58 1.23 protein L23 NM_017446 MRPL39 Mitochondrial
ribosomal 1.74 1.13 protein L39 NM_021210 MUM2 MUM2 protein 1.20
1.61 NM_004529 MLLT3 Myeloid/lymphoid 1.15 2.41 or mixed-lineage
leukemia (trithorax homolog, Drosophila)- translocated to, 3
NM_033546 MLC-B Myosin regulatory 1.95 1.89 light chain AB032945
MYO5B Myosin VB 1.50 1.74 NM_017534 MYH2 Myosin, heavy polypeptide
1.66 0.90 2, skeletal muscle, adult NM_002473 MYH9 Myosin, heavy
polypeptide 1.82 2.60 9, non-muscle NM_002356 MARCKS Myristoylated
alanine-rich 0.22 2.70 protein kinase C substrate NM_000903 NQO1
NAD(P)H dehydrogenase, 2.64 2.77 quinone 1 NM_004541 NDUFA1 NADH
dehydrogenase 1.27 1.88 (ubiquinone) 1 alpha subcomplex, 1 (7.5kD,
MWFE) NM_004548 NDUFB10 NADH dehydrogenase 1.63 1.29 (ubiquinone) 1
beta subcomplex, 10 (22kD, PDSW) NM_004547 NDUFB4 NADH
dehydrogenase 1.63 2.11 (ubiquinone) 1 beta subcomplex, 4 (15kD,
B15) NM_002494 NDUFC1 NADH dehydrogenase 1.70 1.17 (ubiquinone) 1,
subcomplex unknown, 1 (6kD, KFYI) NM_014328 NESCA Nesca protein
1.52 1.23 BC010285 NET1 Neuroepithelial cell 0.78 2.28 transforming
gene 1 NM_000271 NPC1 Niemann-Pick disease, 2.31 1.39 type C1
NM_006096 NDRG1 N-myc downstream 1.50 1.95 regulated gene 1
NM_006164 NFE2L2 Nuclear factor (erythroid- 3.80 1.23 derived
2)-like 2 NM_003489 NRIP1 Nuclear receptor 0.94 1.63 interacting
protein 1 NM_017838 NOLA2 Nucleolar protein family A, 1.83 1.94
member 2 (H/ACA small nucleolar RNPs) NM_002820 PTHLH Parathyroid
hormone-like 1.66 2.59 hormone NM_020992 PDLIM1 PDZ and LIM domain
1 1.56 1.60 (elfin) NM_002574 PRDX1 Peroxiredoxin 1 1.68 1.80
NM_003713 PPAP2B Phosphatidic acid 1.22 1.84 phosphatase type 2B
NM_002631 PGD Phosphogluconate 4.37 23.25 dehydrogenase NM_002632
PGF Placental growth factor, 3.61 1.79 vascular endothelial growth
factor-related Protein NM_002658 PLAU Plasminogen activator, 1.69
1.78 urokinase NM_014287 PM5 PM5 protein 1.55 1.54 NM_003819 PABPC4
Poly(A) binding protein, 1.62 1.25 cytoplasmic 4 (inducible form)
NM_000937 POLR2A Polymerase 1.23 1.65 (RNA) II (DNA directed)
polypeptide A (220kD) NM_001198 PRDM1 PR domain containing 1, 7.04
3.20 with ZNF domain NM_002583 PAWR PRKC, apoptosis, 1.96 1.50 WT1,
regulator NM_000917 P4HA1 Procollagen-proline, 2- 1.08 1.51
oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha
polypeptide I NM_053024 PFN2 Profilin 2 1.73 1.17 AB051437 ProSAP2
Proline rich synapse 2.30 1.25 associated protein 2 (rat) NM_002778
PSAP Prosaposin (variant 1.70 2.72 Gaucher disease and variant
metachromatic leukodystrophy) NM_000963 PTGS2 Prostaglandin- 6.51
0.98 endoperoxide synthase 2 (prostaglandin G/H synthase and
cyclooxygenase)
BC013908 PSMC1 Proteasome 1.68 1.13 (prosome, macropain) 26S
subunit, ATPase, 1 NM_002806 PSMC6 Proteasome 1.64 1.25 (prosome,
macropain) 26S subunit, ATPase, 6 NM_002815 PSMD11 Proteasome
(prosome, 1.77 1.35 macropain) 26S subunit, non-ATPase, 11
NM_002812 PSMD8 Proteasome 2.17 3.03 (prosome, macropain) 26S
subunit, non-ATPase, 8 NM_002797 PSMB5 Proteasome (prosome, 2.82
3.28 macropain) subunit, beta type, 5 NM_002799 PSMB7 Proteasome
(prosome, 1.36 1.74 macropain) subunit, beta type, 7 NM_014330
PPP1R15A Protein phosphatase 7.10 0.88 1, regulatory (inhibitor)
subunit 15A NM_004156 PPP2CB Protein phosphatase 1.67 1.11 2
(formerly 2A), catalytic subunit, beta isoform NM_006808 SEC61B
Protein translocation 1.44 1.57 complex beta NM_015714 G0S2
Putative lymphocyte 0.90 6.31 G0/G1 switch gene BC012513 ARHE Ras
homolog gene family, 2.39 0.99 member E NM_003979 RAI3 Retinoic
acid induced 3 1.05 3.46 NM_001666 ARHGAP4 Rho GTPase activating
2.49 1.96 protein 4 NM_001033 RRM1 Ribonucleotide reductase 1.54
0.87 M1 polypeptide NM_002950 RPN1 Ribophorin I 2.08 1.10 NM_001029
RPS26 Ribosomal protein S26 1.31 1.70 NM_002953 RPS6KA1 Ribosomal
protein 1.65 2.00 S6 kinase, 90kD, polypeptide 1 AB037819 RRBP1
Ribosome binding protein 3.68 2.68 1 homolog 180kD (dog) NM_014248
RBX1 Ring-box 1 1.30 2.13 NM_006743 RBM3 RNA binding motif 2.01
1.74 protein 3 NM_004902 RNPC2 RNA-binding region 1.61 0.75 (RNP1,
RRM) containing 2 NM_000687 AHCY S-adenosylhomocysteine 1.74 1.82
hydrolase AB051532 SEMA4B Sema domain, 1.11 1.77 immunoglobulin
domain (Ig), transmembrane domain (TM) and short cytoplasmic
domain, (se NM_003900 SQSTM1 Sequestosome 1 3.34 2.82 NM_001085
SERPINA3 Serine (or cysteine) 2.74 #DIV/ proteinase inhibitor, 0!
clade A (alpha-1 antiproteinase, antitrypsin), member 3 NM_030666
SERPINB1 Serine (or cysteine) 3.11 2.58 proteinase inhibitor, clade
B (ovalbumin), member 1 NM_000602 SERPINE1 Serine (or cysteine)
2.32 2.38 proteinase inhibitor, clade E (nexin, plasminogen
activator inhibitor type 1), NM_ 015966 SDBCAG84 Serologically
defined 1.86 1.45 breast cancer antigen 84 NM_006622 SNK
Serum-inducible kinase 3.02 1.13 AB000462 SH3BP2 SH3-domain binding
4.63 2.02 protein 2 NM_003134 SRP14 Signal recognition particle
1.58 1.45 14kD (homologous Alu RNA binding protein) NM_003145 SSR2
Signal sequence receptor, 1.64 1.79 beta (translocon-associated
protein beta) NM_007107 SSR3 Signal sequence receptor, 1.74 1.26
gamma (translocon- associated protein gamma) AF395440 HEJ1 Similar
to DNAJ 2.50 1.94 NM_005870 SAP18 Sin3-associated 1.50 1.21
polypeptide, 18kD NM_006109 SKB1 SKB1 homolog (S. pombe) 1.55 2.52
NM_015523 DKFZP566E Small fragment nuclease 2.04 1.55 144 NM_030981
RAB1B Small GTP-binding protein 1.53 1.16 NM_006518 SPRR2C Small
proline-rich 1.41 4.09 protein 2C NM_005628 SLC1A5 Solute carrier
family 1 1.87 0.82 (neutral amino acid transporter), member 5
NM_004207 SLC16A3 Solute carrier family 16 1.56 2.65
(monocarboxylic acid transporters), member 3 NM_018976 5LC38A2
Solute carrier family 38, 2.48 0.85 member 2 NM_014331 SLC7A11
Solute carrier 2.40 0.73 family 7, (cationic amino acid
transporter, y+ system) member 11 NM_003130 SRI Sorcin 0.92 1.80
NM_004599 SREBF2 Sterol regulatory element 1.47 1.03 binding
transcription factor 2 NM_006745 SC4MOL Sterol-C4-methyl 1.68 1.82
oxidase-like NM_006918 SC5DL Sterol-C5-desaturase 1.59 1.11 (ERG3
delta-5-desaturase homolog, fungal)- like NM_006819 STIP1
Stress-induced- 2.88 2.34 phosphoprotein 1 (Hsp70/Hsp90-organizing
protein) NM_006704 SGT1 Suppressor of G2 1.81 1.32 allele of SKP1,
S. cerevisiae, homolog of NM_002999 SDC4 Syndecan 4 (amphigl ycan,
1.21 1.71 ryudocan) NM_006289 TLN1 Talin 1 1.53 1.59 NM_015641 TES
Testis derived transcript 2.10 0.95 (3 LIM domains) NM_003217 TEGT
Testis enhanced gene 1.71 1.28 transcript (BAX inhibitor 1)
NM_003314 TTC1 Tetratricopeptide repeat 1.68 2.06 domain 1
NM_003329 TXN Thioredoxin 1.39 2.24 NM_003330 TXNRD1 Thioredoxin
reductase 1 7.66 2.72 NM_004238 TRIP12 Thyroid hormone receptor
1.73 1.43 interactor 12 NM_006755 TALDO1 Transaldolase 1 1.96 1.72
NM_003234 TFRC Transferrin receptor 1.51 3.15 (p90, CD71) NM_001064
TKT Transketolase (Wernicke- 1.60 1.44 Korsakoff syndrome)
NM_012459 TIMM8B Translocase of inner 1.32 1.57 mitochondrial
membrane 8 homolog B (yeast) NM_006470 TRIM16 Tripartite motif-
1.57 1.53 containing 16 NM_003449 TRIM26 Tripartite motif- 1.39
2.55 containing 26 NM_003289 TPM2 Tropomyosin 2 (beta) 2.13 1.79
NM_003404 YWHAB Tyrosine 3- 2.06 3.12 monooxygenase/ tryptophan 5-
monooxygenase activation protein, beta polypeptide NM_012321 LSM4
U6 snRNA-associated 1.61 0.95 Sm-like Protein M26880 UBC Ubiquitin
C 1.73 1.07 NM_014501 E2-EPF Ubiquitin carrier protein 1.83 1.41
NM_003334 UBE1 Ubiquitin-activating 1.91 1.67 enzyme E1 (A1S9T and
BN75 temperature sensitivity complementing) AL110132 UBE2V1
Ubiquitin-conjugating 1.80 1.66 enzyme E2 variant 1 BC007657 UBE2M
Ubiquitin-conjugating 1.58 1.80 enzyme E2M (UBC12 homolog, yeast)
NM_003364 UP Uridine phosphorylase 2.48 1.13 NM_003574 VAPA VAMP
(vesicle-associated 1.85 1.71 membrane protein)- associated protein
A (33kD) NM_012323 MAFF V-maf musculoaponeurotic 1.71 0.72
fibrosarcoma oncogene homolog F (avian) NM_002359 MAFG V-maf
musculoaponeurotic 1.85 1.41 fibrosarcoma oncogene homolog G
(avian) NM_002467 MYC V-myc myelocytomatosis 2.75 1.98 viral
oncogene homolog (avian) NM_006007 ZNF216 Zinc finger protein 216
2.01 1.29 NM_013360 ZNF222 Zinc finger protein 222 2.26 1.86
NM_004234 ZFP93 Zinc finger protein 93 0.75 1.64 homolog (mouse)
Genes Downregulated by Cigarette Smoke M4/ M24/ Gene ID Gene Name
Description S4 S24 NM_006856 ATF7 activating transcription 0.81
2.32 factor 7 NM_001143 AMELY amelogenin, Y-linked 1.61 1.03
NM_001657 AREG amphiregulin 0.50 2.95 (schwannoma- derived growth
factor) AB053314 ALS2CR12 amyotrophic lateral 2.01 1.12 sclerosis 2
(juvenile) chromosome region, candidate 12 AK023086 CDNA FLJ13024
fis, 1.56 1.05 clone NT2RP3000865 BI820294 CDNA FLJ26296 fis, 1.69
0.89 clone DMC07192, highly similar to Ig kappa chain V-III region
HAH precursor AK025253 CDNA FLJ42432 fis, 2.15 1.70 clone
BLADE2006412 NM_001271 CHD2 chromodomain helicase 1.10 1.62 DNA
binding protein 2 NM_006589 C1orf2 chromosome 1 open 1.56 0.87
reading frame 2 AK000796 C14orf129 chromosome 14 open 0.79 1.80
reading frame 129 NM_001934 DLX4 distal-less homeobox 4 1.29 2.08
NM_005509 DMXL1 Dmx-like 1 2.05 1.22 NM_004419 DUSP5 Dual
specificity 0.45 2.37 phosphatase 5 NM_003494 DYSF dysferlin, limb
1.19 2.31 girdle muscular dystrophy 2B (autosomal recessive)
NM_000145 FSHR follicle stimulating 1.58 1.29 hormone receptor
NM_005708 GPC6 glypican 6 1.78 1.51 NM_002053 GBP1 guanylate
binding 1.31 1.58 protein 1, interferon- inducible, 67kDa AB033063
HEG HEG homolog 0.88 1.97 NM_002129 HMGB2 High-mobility group box 2
0.69 2.78 NM_003542 HIST1H4F histone 1, H4f 1.57 1.92 NM_024598
FLJ13154 hypothetical protein 0.81 1.67 FLJ13154 NM_017933 FLJ20701
hypothetical protein 1.37 2.03 FLJ20701 NM_024037 MGC2603
hypothetical protein 1.59 0.74 MGC2603 BC016840 MGC34695
hypothetical protein 0.99 2.33 MGC34695 AK027858 MGC4248
hypothetical protein 1.53 0.92 MGC4248 NM_006903 PPA2 inorganic
0.63 1.64 pyrophosphatase 2 NM_000526 KRT14 keratin 14 1.09 2.13
(epidermolysis bullosa simplex, Dowling-Meara, Koebner) NM_000424
KRT5 keratin 5 (epidermolysis 1.48 1.86
bullosa simplex, Dowling- Meara/Kobner/Weber- Cockayne types)
NM_005554 KRT6A keratin 6A 1.53 1.17 NM_005556 KRT7 keratin 7 1.54
1.06 AK024583 LOC400078 1.60 1.19 (LOC387888), mRNA NM_005583 LYL1
lymphoblastic leukemia 1.73 1.17 derived sequence 1 AL137524 MRNA*
cDNA 1.03 1.67 DKFZp434H2218 (from clone DKFZp434H2218) AL117623
MRNA* cDNA 1.72 1.02 DKFZp564O2364 (from clone DKFZp564O2364)
NM_012334 MYO10 myosin X 2.08 1.13 AB007959 NHLH2 nescient helix
loop helix 2 1.08 1.53 NM_002520 NPM1 nucleophosmin (nucleolar 1.62
1.67 phosphoprotein B23, numatrin) NM_033014 OGN osteoglycin 1.21
1.62 (osteoinductive factor, mimecan) NM_024594 PANK3 pantothenate
kinase 3 1.88 1.42 AB029015 PLCL2 Phospholipase C-like 2 5.45 2.99
NM_018049 PLEKHJ1 pleckstrin homology 2.48 1.68 domain containing,
family J member 1 BC015542 PVR poliovirus receptor 1.54 0.98
NM_018936 PCDHB2 protocadherin beta 2 1.64 1.02 NM_000320 QDPR
quinoid dihydropteridine 1.24 1.81 reductase NM_000456 RAB5B RAB5B,
member RAS 2.56 2.22 oncogene family NM_007273 REA repressor of
estrogen 0.83 1.51 receptor activity NM_005978 S100A2 S100 calcium
binding 1.89 1.55 protein A2 NM_016372 TPRA40 seven transmembrane
1.57 0.84 domain orphan receptor NM_006456 SIAT7B sialyltransferase
7 0.77 2.21 ((alpha-N- acetylneuraminyl- 2,3-beta-
galactosyl-1,3)-N-acetyl galactosaminide alpha-2,6-
sialyltransferase) B NM_024624 SMC6L1 SMC6 structural 1.74 1.71
maintenance of chromosomes 6-like 1 (yeast) AL353933 SLC22A15
solute carrier family 1.85 1.07 22 (organic cation transporter),
member 15 AK027663 STC2 stanniocalcin 2 0.77 1.74 AK024451
Tangerine 1.56 1.30 DKFZp762C 186 NM_005480 TROAP trophinin
associated 1.77 1.12 protein (tastin) NM_002466 MYBL2 v-myb
myeloblastosis 1.01 1.63 viral oncogene homolog (avian)-like 2
NM_006385 ZNF211 Zinc finger protein 211 1.96 1.22 NM_005096 ZNF261
Zinc finger protein 261 1.68 1.53 NM_003430 ZNF91 Zinc finger
protein 91 1.47 1.53 (HPF7, HTF10) AC006033 1.52 1.21 AF111848 1.68
1.27 AK025272 8.36 4.55 AL137077 2.59 1.28 L24498 0.31 1.58
NM_003590 2.06 1.03 NM_005774 1.72 1.29 NM_014111 1.53 2.49
[0700] A typical example is shown in FIG. 42, which compares the
expression of the heat shock genes DnaJ (HSP40) A1/B1 at 4 and 24 h
in mock-treated and CS-treated cells in both experiments. The
figure shows not only a consistent temporal relationship in the two
experiments with both genes being up-regulated by 4 hrs and then
returning to baseline by 24 hrs, but also that there is a
consistent relative level of expression between the two genes
(i.e., 4 hr expression levels of B1 exceed that of A1 in both
experiments).
[0701] Confirmation of Differential Expression by qRT-PCR
[0702] The relative expression levels of 6 genes that were
determined by microarray analysis to be up-regulated in CS-treated
NHBE cells were reassessed by quantitative PCR using RNA from
samples taken at both 4 and 24 hr. This gene set included: ferritin
heavy polypeptide, nuclear factor (erythroid-derived 2)-like 2,
heat shock protein 70, heme oxygenase, thioredoxin reductase,
cyclooxygenase 2, and sequestosome 1. It was determined that
beta-actin expression levels in the normalized microarray data were
nearly identical among all the CS and mock-treated samples, so this
gene was used as an internal normalization standard in these
experiments. Quantitative PCR results were in strong qualitative
agreement with the microarray results, as all 6 genes were also
up-regulated by CS when assessed by qRT-PCR. Moreover, the qRT-PCR
results recapitulated the general trends of expression at both 4
and 24 hr that were observed by microarray (Table 17).
TABLE-US-00023 TABLE 17 Microarray data microarray Qper microarray
qRT-PCR Gene 4 hr/fold 4 hr/fold 24 hr/fold 24 hr/fold 1 change
change change change FTH1 2.3 2.6 3.4 3.5 HSPA1A 16.1 25.1 2.4 5.0
NFE2L2 3.8 3.47 1.23 1.21 TXNRD1 11.4 16.0 3.2 2.0 HMOX1 42.5 77.6
1.7 4.7 PTGS2 5.4 17.0 0 0 SQSTM1 3.9 7.7 2.6 3.3
[0703] Since the wide range of gases, toxins, free radicals, and
carcinogens present in tobacco smoke are believed to cause multiple
types of structural and chemical damage, the NHBE cells that are
exposed to tobacco smoke would presumably have to mount an
integrated biological and genetic response in an attempt to
prioritize and attenuate this damage. In an effort to understand
the type of response mounted by the NHBE cells after cigarette
smoke exposure, several databases were analyzed and genes that were
identified as being over-expressed or under-expressed in response
to exposure to cigarette smoke were grouped according to functional
similarities. The following example describes this effort in
greater detail.
Example 13
Functional Grouping of Genes Modulated in Response to CS
Exposure
[0704] Information from the Gene Ontology (GO) Consortium and from
the scientific literature was used to categorize the genes
identified as being modulated (i.e., over-expressed or
under-expressed) in response to cigarette smoke exposure. Of the
genes up-regulated by CS exposure that have known functions (235
out of 298 genes), four major groups of functionally related genes
were identified (Table 11). These four groups collectively
represent a large proportion (45%; 105 out of 235 genes) of the
differentially expressed genes with known function, indicating that
these genes are involved in biological pathways that are highly
responsive to CS-induced damage. In contrast, although 42 of the 66
genes that were under-expressed in response to CS have known
functions, they reflected multiple biological processes without a
clear dominance of specific function. As can be seen in Table 11,
the predominant pathways highlighted by the over-expressed gene set
indicate that the cell is responding to a sudden increase in
oxidative stress and the concentration of misfolded or damaged
proteins, while simultaneously attempting to modulate its cell
cycle and apoptotic controls. Unexpectedly, it was also observed
that a proportionally large group of CS-responsive genes are
related to the metabolism and cellular trafficking of
cholesterol.
TABLE-US-00024 TABLE 18 Fold Fold In- In- crease crease Gene ID
Symbol Description at 4 h at 24 h RESPONSE TO OXIDATIVE STRESS
BF541376 FTL ESTs, Weakly similar 2.71 4.50 to FRHUL ferritin light
chain [H. sapiens] AK054816 FTH1 Ferritin, heavy 2.07 3.32
polypeptide 1 NM_001498 GCLC Glutamate-cysteine ligase, 8.96 1.40
catalytic subunit NM_002061 GCLM Glutamate-cysteine ligase, 2.85
1.56 modifier subunit NM_002064 GLRX Glutaredoxin 3.12 2.31
(thioltransferase) NM_002083 GPX2 Glutathione peroxidase 2 3.71
9.99 (gastrointestinal) NM_000637 GSR Glutathione reductase 1.57
1.54 NM_002133 HMOX1 Heme oxygenase 55.83 2.81 (decycling) 1
NM_005354 JUND Jun D proto-oncogene 1.67 1.25 NM_004528 MGST3
Microsomal glutathione S- 1.73 1.76 transferase 3 NM_000903 NQO1
NAD(P)H dehydrogenase, 2.64 2.77 quinone 1 NM_006096 NDRG1 N-myc
downstream 1.50 1.95 regulated gene 1 NM_006164 NFE2L2 Nuclear
factor (erythroid- 3.80 1.23 derived 2)-like 2 NM_020992 PDLIM1 PDZ
and LIM domain 1 1.56 1.60 (elfin) NM_002574 PRDX1 Peroxiredoxin 1
1.68 1.80 NM_000687 AHCY S-adenosylhomocysteine 1.74 1.82 hydrolase
NM_003329 TXN Thioredoxin 1.39 2.24 NM_003330 TXNRD1 Thioredoxin
reductase 1 7.66 2.72 NM_012323 MAFF V-maf musculoaponeurotic 1.71
0.72 fibrosarcoma oncogene homolog F (avian) NM_002359 MAFG V-maf
musculoaponeurotic 1.85 1.41 fibrosarcoma oncogene homolog G
(avian) CELL GROWTH/PROLIFERATION/APOPTOSIS NM_001657 AREG
Amphiregulin 1.96 0.33 (schwannoma- derived growth factor)
NM_016085 APR-3 Apoptosis related 1.44 0.84 protein APR-3 NM_017900
AKIP aurora-A kinase interacting 2.07 5.18 protein NM_001196 BID
BH3 interacting domain 1.54 1.05 death agonist NM_005186 CAPN1
Calpain 1, (mu/I) large 1.62 1.11 subunit NM_013376 SEI1
CDK4-binding 2.46 1.87 protein p34SEI1 NM_015965 GRIM19 Cell
death-regulatory 2.16 2.23 protein GRIM19 NM_001554 CYR61
Cysteine-rich, angiogenic 2.44 0.67 inducer, 61 NM_004396 DDX5
DEAD/H (Asp-Glu- 2.01 4.10 Ala-Asp/His) box polypeptide 5 (RNA
helicase, 68kD) NM_013253 DKK3 Dickkopf homolog 3 1.64 0.84
(Xenopus laevis) NM_004419 DUSP5 Dual specificity 1.97 0.47
phosphatase 5 NM_001946 DUSP6 Dual specificity 2.08 2.29
phosphatase 6 NM_004431 EPHA2 EphA2 2.37 1.93 NM_005245 FAT FAT
tumor suppressor 1.87 0.77 homolog 1 (Drosophila) NM_002087 GRN
Granulin 1.36 1.58 L24498 GADD45A Growth arrest and DNA- 2.81 0.61
damage-inducible, alpha AF130111 HDAC3 Histone deacetylase 3 1.92
1.38 AF103803 H41 Hypothetical protein 1.63 2.00 NM_052815 IER3
Immediate early response 3 2.94 1.54 NM_016545 IER5 Immediate early
response 5 9.20 1.18 NM_000576 IL1B Interleukin 1, beta 0.98 3.03
NM_001730 KLF5 Kruppel-like factor 5 2.34 1.01 (intestinal)
NM_004529 MLLT3 Myeloid/lymphoid 1.15 2.41 or mixed-lineage
leukemia (trithorax homolog, Drosophila)- translocated to, 3
NM_002632 PGF Placental growth factor, 3.61 1.79 vascular
endothelial growth factor-related protein NM_002658 PLAU
Plasminogen activator, 1.69 1.78 urokinase NM_001198 PRDM1 PR
domain containing 7.04 3.20 1, with ZNF domain NM_002583 PAWR PRKC,
apoptosis, 1.96 1.50 WT1, regulator NM_014330 PPP1R15A Protein
phosphatase 7.10 0.88 1, regulatory (inhibitor) subunit 15A
NM_015714 G0S2 Putative lymphocyte 0.90 6.31 G0/G1 switch gene
NM_001666 ARHGAP4 Rho GTPase activating 2.49 1.96 protein 4
NM_006622 SNK Serum-inducible kinase 3.02 1.13 NM_006109 SKB1 SKB1
homolog (S. pombe) 1.55 2.52 NM_006704 SGT1 Suppressor of G2 allele
1.81 1.32 of SKP1, S. cerevisiae, homolog of NM_003217 TEGT Testis
enhanced gene 1.71 1.28 transcript (BAX inhibitor 1) NM_002467 MYC
V-myc myelocytomatosis 2.75 1.98 viral oncogene homolog (avian)
UBIQUITINATION/PROTEIN TURNOVER/HEAT SHOCK NM_001109 ADAM8 A
disintegrin and 1.17 2.72 metalloproteinase domain 8 NM_004281 BAG3
BCL2-associated 3.85 1.58 athanogene 3 BC002971 CCT5 Chaperonin
containing 1.81 1.74 TCP1, subunit 5 (epsilon) NM_006429 CCT7
Chaperonin containing 2.85 3.21 TCP1, subunit 7 (eta) NM_007278
GABARAP GABA(A) receptor- 1.55 1.75 associated protein NM_001539
DNAJA1 DnaJ (Hsp40) homolog, 2.11 1.85 subfamily A, member 1
NM_006145 DNAJB1 DnaJ (Hsp40) homolog, 4.99 1.57 subfmaily B,
member 1 AF395440 HEJ1 Similar to DNAJ 2.50 1.94 NM_006644 HSP105B
Heat shock 105kD 2.83 1.02 NM_002157 HSPE1 Heat shock 10kD protein
1 1.92 1.34 (chaperonin 10) NM_005345 HSPA1A Heat shock 70kD 5.77
1.30 protein 1A NM_006597 HSPA8 Heat shock 70kD protein 8 1.48 4.56
NM_004134 HSPA9B Heat shock 70kD 2.23 1.39 protein 9B (mortalin-2)
NM_016292 TRAP1 Heat shock protein 75 1.57 1.05 NM_006819 STIP1
Stress-induced- 2.88 2.34 phosphoprotein 1 (Hsp70/Hsp90-organizing
protein) NM_004995 MMP14 Matrix metalloproteinase 14 2.20 2.57
(membrane-inserted) BC013908 PSMC1 Proteasome (prosome, 1.68 1.13
macropain) 26S subunit, ATPase, 1 NM_002806 PSMC6 Proteasome
(prosome, 1.64 1.25 macropain) 26S subunit, ATPase, 6 NM_002815
PSMD11 Proteasome (prosome, 1.77 1.35 macropain) 26S subunit,
non-ATPase, 11 NM_002812 PSMD8 Proteasome (prosome, 2.17 3.03
macropain) 26S subunit, non-ATPase, 8 NM_002797 PSMB5 Proteasome
(prosome, 2.82 3.28 macropain) subunit, beta type, 5 NM_002799
PSMB7 Proteasome (prosome, 1.36 1.74 macropain) subunit, beta type,
7 NM_006808 SEC61B Protein translocation 1.44 1.57 complex beta
NM_014248 RBX1 Ring-box 1 1.30 2.13 NM_003900 SQSTM1 Sequestosome 1
3.34 2.82 NM_003134 SRP14 Signal recognition 1.58 1.45 particle
14kD (homologous Alu RNA binding protein) NM_003314 TTC1
Tetratricopeptide 1.68 2.06 repeat domain 1 NM_004238 TRIP12
Thyroid hormone receptor 1.73 1.43 interactor 12 M26880 UBC
Ubiquitin C 1.73 1.07 NM_014501 E2-EPF Ubiquitin carrier protein
1.83 1.41 NM_003334 UBE1 Ubiquitin-activating 1.91 1.67 enzyme E1
(A1S9T and BN75 temperature sensitivity complementing) AL110132
UBE2V1 Ubiquitin-conjugating 1.80 1.66 enzyme E2 variant 1 BC007657
UBE2M Ubiquitin-conjugating 1.58 1.80 enzyme E2M (UBC12 homolog,
yeast) NM_000859 HMGCR 3-hydroxy-3- 2.25 1.33 methylglutaryl-
Coenzyme A reductase AK025736 HMGCS1 3-hydroxy-3- 1.02 1.63
methylglutaryl- Coenzyme A synthase 1 (soluble) CHOLESTEROL/LIPID
METABOLISM NM_005891 ACAT2 Acetyl-Coenzyme A 1.44 1.77
acetyltransferase 2 (acetoacetyl Coenzyme A thiolase) NM_000700
ANXA1 Annexin A1 1.39 1.82 NM_007274 HBACH Cytosolic acyl coenzyme
1.61 2.28 A thioester hydrolase NM_020548 DBI Diazepam binding 1.69
1.84 inhibitor (GABA receptor modulator, acyl-Coenzyme A binding
protein) NM_004092 ECHS1 Enoyl Coenzyme A 1.60 1.23 hydratase,
short chain, 1, mitochondrial NM_004104 FASN Fatty acid synthase
1.24 1.60 NM_000182 HADHA Hydroxyacyl-Coenzyme A 2.39 1.22
dehydrogenase/3-ketoacyl- Coenzyme A thiolase/enoyl- Coenzyme A
hydratase NM_005542 INSIG1 Insulin induced gene 1 2.02 2.62
NM_004508 IDI1 Isopentenyl-diphosphate 1.89 2.68 delta isomerase
NM_000271 NPC1 Niemann-Pick disease, 2.31 1.39 type C1 NM_003713
PPAP2B Phosphatidic acid 1.22 1.84 phosphatase type 2B NM_002778
PSAP Prosaposin (variant 1.70 2.72 Gaucher disease and variant
metachromatic leukodystrophy) NM_004599 SREBF2 Sterol regulatory
1.47 1.03 element binding transcription factor 2 NM_006745 SC4MOL
Sterol-C4-methyl 1.68 1.82 oxidase-like NM_006918 SC5DL
Sterol-C5-desaturase 1.59 1.11 (ERG3 delta-5-desaturase homolog,
fungal)-like
[0705] In order to visualize any underlying temporal expression
patterns among these four functional classes a hierarchical
clustering of the genes was made (see FIG. 43). This cluster
analysis of the expression data shows two important points: 1) that
the four conditions (4 & 24 h mock-treated and 4 & 24 h
CS-treated) are clearly distinguishable by these functional groups
of genes; and 2) that the expression of the specific genes in the
four functional groups do not have strong temporal relationships
(i.e. they do not overwhelmingly cluster within either the 0-4
hours or 4-24 hour time frame). However, it is clear from FIG. 43
that the majority of the CS-responsive genes in these functional
groups exhibit a higher expression at 4 h post-exposure than at 24
h. Since the cells were treated for only 15 minutes and then
analyzed for a change in gene expression after 4 and 24 hrs, the
decrease in expression for many of these genes by 24 hrs indicates
that the cell is attempting to "reset" its transcriptome to
pre-exposure levels, which would not be an unexpected response to a
transient insult. However, the fact that the expression of many of
these genes remains increased over pre-exposure levels for up to 24
hrs also indicates that the biological ramifications of CS-exposure
can affect the cell for a long period of time after exposure to
tobacco smoke is terminated. Accordingly, it is plausible that many
of these genes may not return to homeostatic baseline in a habitual
smoker, which may have unforeseen pathological consequences.
[0706] A notable exception to most of the genes shown in FIG. 43
and TABLE 18, whose expression remain elevated up to 24 hrs
post-exposure, is a large block of genes in the protein
damage/turnover group, and which encode primarily heat shock and
heat shock-associated proteins. The expression of these heat shock
related genes is dramatically elevated at 4 hrs but returns to
baseline by 24 hrs, indicating that the processes that engage and
clear a buildup of CS-induced damaged and dysfunctional proteins
are rapid. Finally, there are a small subset of genes whose
expression levels are higher at 24 h than at 4 h, including
ferritin, NADH dehydrogenase, peroxiredoxin 1, and glutathione
peroxidase. Since each of these genes is involved in redox
reactions, it could signify that oxidative stress caused by CS
induces long-lived perturbations to redox homeostasis.
[0707] The four major functional groups of genes listed in Table 18
and shown in FIG. 43 show a well-organized attempt by the NHBE cell
to attenuate the damage caused by exposure to tobacco smoke. This
type of coordinated response provides evidence that functionally
related blocks of genes are transcriptionally regulated by the same
or similar transcriptional activators. In the full set of 298 genes
up-regulated by CS (see TABLE 16), there are 21 genes with gene
products that function as transcriptional regulators, including
v-myc, interferon regulatory factor 6, eukaryotic translation
initiation factor 4B, Kruppel-like factor 5, sterol regulatory
element binding transcription factor 2 (SREB2), and Nuclear factor
(erythroid-derived 2)-like 2 (NRF2). NRF2 is of particular interest
in this regard since studies of NRF2-knockout mice show that this
transcription factor activates over 200 genes in several functional
classes with the two most predominant being oxidative stress
response and protein turnover (Kwak et al., J. Biol. Chem. (2003)
278:8135-8145). As shown in Table 18, both of these classes of
genes are disproportionately activated by exposure of NHBE cells to
tobacco smoke. Specifically, of the 105 genes presented in Table
18, 33 are known to be under transcriptional control of NRF2, or to
act as cofactors for NRF2-regulated transcription (see FIG.
43).
[0708] In addition, it has been shown that the short-term exposure
of mice to cigarette smoke results in the induction of a set of 46
protective genes, all of which are under the control of NRF2
(Rangasamy et al., J. Clin. Invest. (2004) 114:1248-1259). In
concordance with this observation, the data show that despite only
brief exposure cells to CS in vitro, the RNA levels of 19 human
homologues of these 46 mouse genes (41%) are similarly induced,
indicating that the CS-related molecular events occurring in vitro
are very similar to those observed in vivo. This set of CS-induced
genes in both the mouse and NHBE cells includes those responsive to
oxidative stress (heme oxygenase, phosphogluconate dehydrogenase,
thioredoxin reductase, glutathione pathway genes, NADPH:quinone
reductase), protein damage (HSP40, mortalin, GADD45), and protein
turnover (ubiquitin C, proteasome subunits, sequestosome).
[0709] The fact that cigarette smoke, as well as various
constituents of cigarette smoke, can cause disruptions to the
genome, transcriptome, and proteome, allows one to develop a set of
relevant biomarkers that are useful for monitoring exposure to
tobacco toxins, detecting pre-malignant disease, improving
diagnosis and prognosis of current disease, developing new
treatment options, and testing risk reduction strategies for
current and former smokers. A number of studies assessing the
clinical usefulness of alterations in global gene and protein
expression patterns in malignant and normal human lung tissues have
recently shown that quantitative and/or qualitative changes in a
small number of expressed genes and proteins, in combination with
standard clinicopathological variables, may have prognostic and/or
diagnostic potential in patients with tobacco-related diseases.
Thus, elucidating the various molecular, genetic, and cellular
dysfunctions induced by tobacco smoke may not only reveal a useful
set of tobacco-specific biomarkers, but also result in a detailed
mechanistic understanding of how chronic tobacco exposure causes
disease.
[0710] In more embodiments, a second tobacco product (e.g., a
cigarette) is compared to a first tobacco product (e.g., a
cigarette) using the methods above so as to identify which of the
two tobacco products is less likely to contribute to a
tobacco-related disease. For example, a first population of
isolated human cells of the mouth, tongue, oral cavity, or lungs
(e.g., NHBE cells), is contacted with a CS from a first tobacco
product (e.g., a "reduced risk full flavor" cigarette) in an amount
and for a time sufficient to modulate expression of one or more
genes or to modify a gene product, and identification of the genes
that are modulated or modified gene product (e.g., phosphorylated)
or the level or amount of gene expression or modification can be
determined using any technique available that analyzes
transcription (e.g., microarray, genechip, qRT-PCR or
hybridization), protein production (e.g., ELISA, Western blot, or
other antibody detection techniques), modifications of proteins
(e.g., oxidation or phosphorylation), or the appearance or
disappearance of metabolites associated with genes that are
modulated in response to exposure to CS (e.g., cysteine,
glutathione, fragments of proteins or lipids or fatty acids). A
second population of isolated human cells of the mouth, tongue,
oral cavity, or lungs (e.g., NHBE cells), preferably the same type
of cell as used in the analysis of the first tobacco product, is
also contacted with a CS from a second tobacco product (e.g., a
cigarette) in an amount and for a time sufficient to modulate
expression of one or more genes or to modify a gene product.
Identification of a gene that is modulated or modified gene product
(e.g., phosphorylated) or the level or amount of gene expression or
modification can be accomplished using any technique available that
analyzes transcription (e.g., microarray, genechip, qRT-PCR or
hybridization), protein production (e.g., ELISA, Western blot, or
other antibody detection techniques), modifications of proteins
(e.g., oxidation or phosphorylation), or the appearance or
disappearance of metabolites associated with genes that are
modulated in response to exposure to CS (e.g., cysteine,
glutathione, fragments of proteins or lipids or fatty acids).
[0711] The data obtained from the analysis of the first tobacco
product can be compared to the data obtained from the analysis of
the second tobacco product so as to identify, for example, a
gene(s) that are induced in response to exposure to the first
tobacco product but not the second tobacco product or vice versa.
Additionally, the comparison will reveal that the level of
expression of one or more genes induced by both tobacco products
differs with respect to the two tobacco products or that the first
product has more, less, or no modification of a particular gene
product (e.g., phosphorylation), as compared to the second tobacco
product or vice versa. These data (e.g., the types of genes
expressed, the amount of expression, and modification) allow one to
develop a profile for each tobacco product analyzed (in this
example only two products are being compared but a plurality of
products can be compared using the same approach). These tobacco
product profiles can be recorded on a computer readable media and
databases containing this information can be created. Once a gene
is identified, it can be analyzed using PathwayAssist.TM. software
(Stratagene, La Jolla, Calif.), Genespring (version 7.2, Agilent
Technologies), or other similar software so as to determine whether
the gene contributes to a tobacco-related disease.
[0712] By analyzing the differences between the tobacco products
analyzed, (e.g., the types of genes expressed, the amount of
expression, and modifications), one can identify a tobacco product
that has less potential to contribute to a tobacco related disease
or that, for example, a first tobacco product has a reduced risk to
contribute to a tobacco-related disease, as compared to a second
tobacco product or vice versa. By one technique, for example, a
tobacco product that is less likely to contribute to a
tobacco-related disease is identified because it induces fewer
genes associated with a tobacco-related disease. A related approach
(using CSC) was employed to identify a tobacco product as having a
reduced potential to contribute to a tobacco-related disease, as
compared to a second tobacco product. (See Examples 4-6).
[0713] The methods provided herein can be used not only to identify
a tobacco product that has a reduced potential to contribute to a
tobacco-related disease, as compared to a second tobacco product,
but also to develop tobacco products that have a reduced potential
to contribute to a tobacco-related disease, as compared to a second
tobacco product. That is, by coordinating techniques (e.g.,
chemical or genetic modification) to modulate expression of genes
that produce various components in tobacco with the analytical
methods disclosed herein, one can rapidly determine whether the
modulation of a particular gene that produces a particular
component in tobacco results in a modulation of a gene in human
cells (e.g., NHBE cells) that results in a reduced potential to
contribute to a tobacco-related disease, as compared to the tobacco
prior to modulation of component-producing gene. The section below
describes these embodiments in greater detail.
[0714] Epidemiological Determinations
[0715] In still more embodiments, cells of the mouth, oral cavity,
trachea, or lung (e.g., NHBE cells) from a plurality of
individuals, preferably the same cell type, are independently
contacted with a tobacco composition (e.g., CS) in an amount and
for a time sufficient to induce damage of cellular genetic material
or modulate cell homeostasis. The fact that CS, as well as various
constituents of CS, can cause disruptions to the cell allows one to
develop a set of relevant biomarkers that are useful for monitoring
exposure to tobacco toxins, detecting pre-malignant disease,
improving diagnosis and prognosis of current disease, developing
new treatment options, testing chemopreventive compounds, and
testing risk reduction strategies for current and former smokers.
Accordingly, also provided herein are methods of detecting
pre-malignant disease, improving diagnosis and prognosis of current
disease, developing new treatment options, testing chemopreventive
compounds, and testing risk reduction strategies for current and
former smokers by determining the amount of induction of damage of
cellular genetic material or modulation of cell homeostasis to the
cells of a smoker or other tobacco consumer or a subject exposed to
a tobacco composition. The cells of different individuals can
respond differently to tobacco compositions and thereby have
different levels of risk of developing a tobacco-related disease.
The methods provided herein for determining a modulation of cell
homeostasis, or determining a marker indicative of modulation of
cell homeostatis, such as the methods of determining a modulation
of gene expression (e.g., transcriptome or proteome modulation), or
determining the amount of induction of damage of cellular genetic
material in cells contacted with a tobacco composition can be used
to assess a subject's level of risk of developing a tobacco-related
disease. Such methods can be generally performed in accordance with
the methods provided herein, where the cells of the subject can be
first contacted with smoke from the tobacco product in vivo (e.g.,
by the subject smoking a cigarette or side-stream smoke exposure),
and then the cells can be harvested using known methods (e.g., lung
lavage or cheek swab); alternatively, the cells of a subject can be
first harvested and optionally cultured, and then contacted with
smoke from the tobacco product in accordance with the methods
provided herein. Provided below are non-limiting exemplary methods
for testing tobaccos, tobacco products, compounds and the like; it
is understood that any of the methods provided herein for
monitoring a modulation of cell homeostasis can be used in the
examples provided below.
[0716] In one example, primary cultures of lung cells, bronchial
cells, cells of the mouth, pharynx, larynx, and tongue can be
generated from an individual to be tested and these cells are be
contacted with a tobacco composition (e.g., CS from a tobacco
product) so as to elucidate the individuals proclivity to acquire a
tobacco related disease. Certain patterns of amount of induction of
damage of cellular genetic material or modulation of cell
homeostasis to tobacco compositions can be associated with
individuals that do not develop a tobacco related disease and a
different pattern of amount of induction of damage of cellular
genetic material or modulation of cell homeostasis can be
associated with individuals that have developed a tobacco-related
disease. Analysis of the amount of induction of damage of cellular
genetic material or modulation of cell homeostasis of many of such
individuals allows the development of databases that provide an
expected type and amount of induction of damage of cellular genetic
material or modulation of cell homeostasis that is associated or
not associated with a tobacco-related disease. That is, this
information can be used to provide a baseline for an individual
that is not likely to acquire a tobacco-related disease (e.g., a
control level exemplified by non-tobacco users that do not develop
a tobacco-related disease) and a baseline for an individual that is
likely to acquire a tobacco related disease (e.g., a control level
exemplified by tobacco users that have developed a tobacco-related
disease). Accordingly, when a subject is analyzed for the
predilection to develop a tobacco-related disease, the amount of
induction of damage of cellular genetic material or modulation of
cell homeostasis can be evaluated and, by comparing the determined
values to that in one or both of the databases described above, the
analyzed subject can be identified as having a predilection for
developing a tobacco-related disease.
[0717] Additionally, a comparison of the induction of DNA damage
induced by conventional tobacco products and a tobacco product
containing a modified tobacco (e.g., a genetically modified
tobacco) is contemplated. By one approach, a first set of
biological samples (e.g., cells of the oral cavity (cheek or gum
swab) or lung cells (lung lavage)) are obtained from individuals
that are consumers of conventional tobacco products. These cells
are analyzed for double strand DNA breaks using one of the assays
described herein. Next, the individuals are provided a tobacco
product comprising a modified tobacco to consume exclusively (i.e.,
in replacement for the conventional product). After a period of
time has passed (e.g., 1, 2, 3, or 4 weeks or 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, or 12 months since the conversion from the
conventional tobacco product to the tobacco product containing the
modified tobacco), a second set of biological samples are taken
from the individual and are analyzed for the presence of double
strand DNA breaks. It will be determined that fewer double strand
breaks will be observed in the second set of biological samples
than the first set, which will provide evidence that the tobacco
product comprising the modified tobacco has a reduced potential to
contribute to a tobacco related disease (i.e., that said tobacco
product comprising the modified tobacco is a reduced risk tobacco
product).
[0718] Additionally, a reduction by a chemopreventive compound of
the induction of DNA damage induced by a tobacco product can also
be measured by the methods provided herein. By one approach, a
first set of biological samples (e.g., cells of the oral cavity
(cheek or gum swab) or lung cells (lung lavage)) are obtained from
individuals that are consumers of tobacco products. These cells are
analyzed for double strand DNA breaks using one of the assays
described herein. Next, the individuals are provided a candidate
chemoprotective compound to consume or use before, during, or after
use of the tobacco product. After a period of time has passed
(e.g., 1, 2, 3, or 4 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or
12 months) since the commencement of using the test chemoprotective
compound, a second set of biological samples are taken from the
individual and are analyzed for the presence of double strand DNA
breaks. It will be determined that fewer double strand breaks will
be observed in the second set of biological samples than the first
set, which will provide evidence that the test chemoprotective
compound can reduce the potential of tobacco to contribute to a
tobacco related disease.
[0719] Further provided herein are kits to be used in practicing
the above methods. In various embodiments such kits can comprise an
antibody that binds to phosphorylated but not unphosphorylated
H2AX, a reference smoke product, a detectably labeled second
antibody that specifically binds to the antibody that binds to
phosphorylated H2AX, and suitable cells, as provided herein
elsewhere.
[0720] Also provided herein are cells containing DNA having
double-stranded breaks produced by exposure to a tobacco smoke
product and, in particular, to genetically altered cells comprising
cells prepared by a method comprising the steps of: (a) exposing a
first cell population to a tobacco smoke product; (b) identifying
cells containing a greater degree of phosphorylated H2AX relative
to control cells; and (c) selectively collecting the cells
identified in step (b) to form the composition of genetically
altered cells. In preferred non-limiting embodiments, the cells
having a higher degree of phosphorylated H2AX are identified by an
immunofluorescence method and selectively collected, for example by
fluorescence activated cell sorting. To permit the identification
of genes associated with tobacco-induced diseases, also provided
herein are libraries prepared by cloning a plurality of nucleic
acid molecules prepared from the cells, the cells prepared
according to methods provided for forming cells containing DNA
having double-stranded breaks produced by exposure to a tobacco
smoke product, herein into a plurality of vector molecules. The
following section describes several types of modified tobacco that
can be used used with the methods described herein.
[0721] Analysis of Changes in Cell Homeostasis: Changes in
transcriptome or proteome
[0722] High-density microarrays can be used to elucidate how cells
of the oral cavity, mouth, tongue, trachea, bronchi, and lung mount
a multigenic response to cigarette smoke and the major classes of
smoke constituents (e.g., vapor and particulate phases). Using
microarray technology and/or Reverse Transcriptase Polymerase Chain
Reaction (e.g., qRT-PCR), gene expression patterns and levels of
gene expression in short-term cultures of normal human bronchial
epithelial (NHBE) cells exposed to cigarette smoke and cigarette
smoke condensates were analyzed. It was found that subtle
alterations to the `homeostatic transcriptome` are useful in
defining the major signaling pathways activated upon exposure to
chronic, but low level, doses of carcinogenic mixtures such as that
which occur daily in an individual smoker. This type of analysis is
especially relevant for complex bioactive mixtures, such as
cigarette smoke (CS), cigarette smoke condensate (CSC), tobacco
smoke (TS), tobacco smoke condensate (TSC), and total particulate
matter (TPM) since assessing the specific effects of individual
components of such mixtures does not reflect the true impact on a
cell or the body due to the synergistic or antagonistic
interactions that occur with the entirety of the components that
are normally present. Moreover, because the contemplated methods
described herein analyze human cells of the mouth, oral cavity,
trachea, and lungs, either normal or immortalized cell lines (e.g.,
human bronchial cells (e.g., BEP2D or 16HBE140 cells), human
bronchial epithelial cells (e.g., HBEC cells, 1198, or 1170-I
cells), normal human bronchial epithelial cells (NHBE cells), BEAS
cells (e.g., BEAS-2B), NCI-H292 cells, non-small cell lung cancer
(NSCLC) cells or human alveolar cells (e.g., H460, H1792, SK-MES-1,
Calu, H292, H157, H1944, H596, H522, A549, and H226) tongue cells
(e.g., CAL 27), and mouth cells (e.g., Ueda-1)), which are
contacted with cigarette smoke or smoke condensates (as opposed to
exposure to a single agent with a well-defined mechanism of
toxicity), one can identify unique genomic responses and cellular
damage over time. That is, novel genes and gene expression patterns
are identified using the methods described herein because the vapor
and particulate components of tobacco smoke contain numerous
substances that immediately and directly damage a range of
biomolecules, as well as, other substances whose toxicity is
activated only after biotransformation by cellular enzymes into
reactive nucleophiles that then attack various cellular
elements.
[0723] Although it is known that cigarette smoke, as well as
various smoke components, can cause numerous disruptions to the
genome (see Chujo et al., Lung Cancer 38: 23-29, 2002; Wistuba, et
al. Semin Oncol 28: 3-13, 2001), transcriptome (see Bhattacharjee,
et al. Proc Natl Acad Sci USA 98: 13790-13795, 2001 and Garber et
al., Proc Natl Acad Sci USA 98: 13784-13789, 2001), and proteome
(see Hanash, et al. Dis Markers 17: 295-300, 2001); relatively
little is known about the effects of cigarette smoke condensates
(CSC) and cigarette smoke (CS) exposure on the overall impact on
steady state mRNA levels, transcriptional regulation, protein
production, and protein modification in normal cells of the oral
cavity, mouth, tongue, trachea, bronchi, and lung. Accordingly,
experiments were conducted to identify a set of biomarkers that
could be used to monitor exposure to tobacco toxins, detect
pre-malignant disease, improve diagnosis and prognosis of current
tobacco-related disease, develop patient-specific treatment
options, test risk reduction strategies for current and former
smokers, and identify and develop tobacco products that have a
lower potential to contribute to a tobacco-related disease (e.g., a
tobacco product that has a lower carcinogenic potential than a
conventional tobacco product, a reduced risk tobacco product). More
particularly, as described herein, several approaches to identify a
gene expression pattern or fingerprint from cells of the oral
cavity, mouth, tongue, trachea, bronchi, and lung (normal or
immortal), which have been exposed to tobacco smoke or a tobacco
smoke condensate have been discovered and the information generated
by practicing these methods can be used in diagnostics, therapeutic
and prophylactic procedures, as well as, approaches to identify and
develop less harmful tobacco products. In addition, elucidating the
various molecular, genetic, cellular, and systemic effects of
cigarette smoke provides a detailed mechanistic understanding of
how chronic tobacco exposure ultimately causes disease.
[0724] Several studies assessing the clinical usefulness of
alterations in global gene and protein expression patterns in
malignant and normal human lung tissues have shown that
quantitative and/or qualitative changes in a small number of
expressed genes and proteins, in combination with standard
clinicopathological variables, have prognostic and/or diagnostic
potential for patients with tobacco-related diseases. A direct
cause and effect relationship between any of these documented
molecular events and cell exposure to tobacco smoke is unclear,
however. Thus, it was decided to examine the effects of tobacco
constituents on the transcriptome of normal lung cells in a
controlled in vitro environment.
[0725] Several methods described herein analyze the transcriptome
of cells of the oral cavity, mouth, tongue, trachea, bronchi, and
lung after exposure to a smoke or smoke condensate using
high-density microarrays, qRT-PCR, or another conventional nucleic
acid or protein detection method such as ELISA or Western blot. The
data show that exposure of such cells (e.g., normal human bronchial
epithelial cells (NHBE cells) to cigarette smoke or cigarette smoke
condensates results in a modulation of a specific set of genes
whose expression levels varied over the normal variability of gene
expression in these cells. Accordingly, these genes can be used to
monitor tobacco-induced changes to the transcriptome. By sorting
these genes into biologically functional classes, dominant
biochemical pathways known to be relevant to tobacco-related
disease were identified. In addition, it was surprising to learn
that treatment with an S9 microsomal fraction, a step common in
many toxicological studies, has a broad impact on gene expression
in normal lung cells that is distinctly different from the impact
of tobacco exposure.
[0726] Accordingly, some embodiments concern the identification of
a gene or a plurality of genes from cells of the oral cavity,
mouth, tongue, trachea, bronchi, and lung (e.g., NHBE cells), which
are modulated (e.g., up-regulated or down-regulated expression) in
response to contact with a cigarette smoke (CS), a cigarette smoke
condensate (CSC), tobacco smoke (TS), tobacco smoke condensate
(TSC), or total particulate matter (TPM). In some embodiments, a
gene expression pattern, fingerprint, or signature is obtained,
which is an identification of a specific plurality of genes or set
of genes that are modulated (i.e., up-regulated or down-regulated)
after contact with CS, CSC, TS, TSC or TPM. The plurality of genes
that are affected can be any combination or subset of genes that
are identified as being influenced by exposure to CS, CSC, TS, TSC
or TPM. In some embodiments, the plurality of affected genes are a
subset of suppressor genes. In some embodiments, the plurality of
genes that are affected by exposure to CS, CSC, TS, TSC or TPM are
a subset of genes affecting cholesterol regulation and production.
In some embodiments, the subset of genes that are affected genes
are involved in oxidative stress, cell proliferation, apoptosis,
protein turn-over, heat shock, ubquitination, or endoplasmic
reticulum stress.
[0727] Several approaches to conduct a gene expression analysis
that involve the use of NHBE cells are provided herein, whereby
said cells are contacted with a CS, CSC, TS, TSC or TPM and a gene,
pattern of gene expression or a fingerprint from said CS, CSC, TS,
TSC or TPM-treated cells is obtained. The gene expression data
generated by the approaches described herein can be recorded onto a
recordable media (e.g., a hard drive, memory, cache, floppy,
CD-ROM, DVD-ROM) and can be analyzed using various statistical
approaches to determine whether said data identifies a genetic
modulation event (e.g., an up-regulation or down-regulation of
expression) that is statistically relevant. Statistically relevant
genetic modulation events that occur in the cells that were
contacted with a CS, CSC, TS, TSC or TPM can then be used to
identify a molecular pathway that is involved in a tobacco-related
disease. Accordingly, the approaches described herein can be used
to identify a marker for a tobacco-related disease and to determine
whether this marker is modulated (e.g., a marker gene is
up-regulated or down-regulated) in response to exposure to a
particular CS, CSC, TS, TSC or TPM.
[0728] Furthermore, this data can be used to create a genetic
profile for a particular tobacco product, which allows one to
empirically determine the components of a given tobacco product's
smoke (or tobacco per se) that contribute to a gene expression
event in a human cell that is associated with a tobacco-related
disease. Accordingly, by using the approaches described herein, one
can identify specific tobacco products, as well as, growing,
harvesting, curing, processing, and blending practices that have a
reduced potential to contribute to a genetic modulation that is
associated with a tobacco-related disease. That is, the approaches
described herein can be used to identify and develop reduced risk
cigarettes. Still further, the markers for tobacco-related disease,
and the genetic profiles identified by using the approaches
described herein can be used to diagnose, provide a prognosis or
otherwise identify an individual at risk of acquiring a
tobacco-related disease and the effect of tobacco smoke on a
subject at a molecular level. The section below describes several
methods that can be used to identify genes that are modulated after
exposure to CS, CSC, TS, TSC or TPM and to identify and develop
tobacco products that have a reduced risk of contributing to a
tobacco-related disease.
[0729] Tobacco Products that have a Reduced Potential to Contribute
to a Tobacco-Related Disease
[0730] More embodiments concern methods to identify components of a
tobacco product that contribute to a tobacco-related disease, the
selective removal or inhibition of production of these components,
and the determination that the removal of the component(s)
modulates expression of a gene that is associated with a
tobacco-related disease in a manner that reduces the potential for
the tobacco product to contribute to a tobacco related disease. It
is contemplated that particular components of tobacco products are
the factors that modulate expression of genes in human cells that
contribute to tobacco-related disease. It is further contemplated
that modification of genes that contribute to the production of
these toxic components in tobacco (e.g., genetic engineering or
chemical treatment) will, concomitantly, result in a modulation of
gene expression in human cells that come in contact with the smoke
from said modified tobacco, which is less likely to contribute to a
tobacco-related disease than the tobacco prior to modification of
the component-producing gene. Accordingly, by selectively removing
the components that induce the genetic events that contribute to
tobacco-related disease in a human, one can develop tobacco
products that are less likely to contribute to a tobacco-related
disease.
[0731] By one approach, for example, CS is generated using a
smoking machine from a first tobacco product that has been
genetically modified to have a reduced amount of a compound. A
first population of NHBE cells is contacted with said CS obtained
from the modified tobacco, as described in Examples 4, 12, and 13.
As described in these examples, the RNA is isolated and analyzed by
microarray or qRT-PCR or both and a pattern of gene expression and
gene product modification events are obtained. Programs such as
PathwayAssist.TM. software (Stratagene, La Jolla, Calif.) and/or
Genespring (version 7.2, Agilent Technologies) can be used to
determine the identity of the genes that are modulated and their
relationship to a tobacco-related disease.
[0732] A second population of NHBE cells is then contacted with CS
generated from the parental variety of tobacco. That is, the
parental variety of tobacco is the unmodified tobacco variety used
to generate the modified tobacco variety, wherein the unmodified
tobacco retains the component that was removed or inhibited in the
modified tobacco. As above, the RNA is isolated and analyzed by
microarray or qRT-PCR or both and a pattern of gene expression and
gene product modification events are obtained. Programs such as
PathwayAssist.TM. (Stratagene, La Jolla, Calif.) and/or Genespring
(version 7.2, Agilent Technologies) can be used to determine the
identity of the genes that are modulated and their relationship to
a tobacco-related disease.
[0733] A comparison of the data obtained from the analysis of the
first and second tobacco products will reveal that the modified
tobacco modulates fewer genes associated with a tobacco-related
disease than the parental, unmodified tobacco. The data will also
show that the modified tobacco product induces expression of fewer
proto/oncogenes. By this approach, one can effectively identify the
contribution of individual components of a tobacco product to a
tobacco-related disease. This combinatorial approach can be used to
develop tobacco products that are less likely to contribute to a
tobacco-related disease and reduced risk tobacco products
identified by these methods are aspects of the invention. Further,
tobacco products prepared by these approaches can be prepared
according to good manufacturing processes (GMP) (e.g., suitable for
or accepted by a governmental regulatory body, such as the Federal
Drug Administration (FDA), and containers that house said tobacco
products can comprise a label or other indicia, with or without
structure-function indicia, which reflects approval of said tobacco
product from said regulatory body. The example below describes this
approach in greater detail.
Example 14
[0734] This example provides several approaches that can be used to
obtain tobacco and tobacco products that have a reduced potential
to contribute to a tobacco-related disease. Generally, these
methods involve a two-tiered analysis involving first, an analysis
of a parent strain of tobacco that has a component or compound that
contributes to a tobacco related disease and second, an analysis of
a progeny of the parent strain of tobacco that has been modified to
modulate (i.e., up-regulate or down-regulate) expression of a gene
that induces a cascade that contributes to a tobacco-related
disease.
[0735] Accordingly, by one approach, a first tobacco (e.g., Burley
21 LA) that comprises a compound that contributes to a
tobacco-related disease (e.g., nicotine) is provided. Next,
preferably, smoke is obtained from said first tobacco (e.g., CS),
however a smoke condensate from the first tobacco can also be
obtained. Once the smoke or smoke condensate has been prepared from
the first tobacco, a first isolated population of cells, preferably
human cells of the mouth, tongue, trachea, bronchi, or lungs (e.g.,
NHBE cells) is contacted with said smoke or smoke condensate from
said first tobacco. The contact can be made in a smoking chamber,
for example, for less than, equal to, or more than, 5 seconds, 20,
seconds, 45 seconds, 1 minute, 5 minutes, 10 minutes, 15, minutes,
20 minutes, 30 minutes, 45 minutes, 1 hour, two hours, three hours.
Subsequent to the contact with the smoke or smoke condensate, a
first gene that is modulated (up-regulated or down-regulated) in
said first population of cells in response to said contact with
said smoke or smoke condensate from said first tobacco is
identified (e.g., an proto/oncogene). The identification of the
first gene can be accomplished using an oligonucleotide array,
microarray, qRT-PCR, nucleic acid detection (e.g., hybridization),
protein detection (e.g., antibody detection, ELISA or Western
blot), or detection of a metabolite (e.g., protein fragment or
cysteine) or a modified gene product (e.g., oxidized or
phosphorylated protein or amino acid). The first gene identified as
being modulated (e.g., up-regulated or down-regulated) in response
to contact with the smoke or smoke condensate of the first tobacco
is then analyzed for its contribution to a tobacco-related disease.
The correlation of many of the genes that are identified by the
approach above to a tobacco-related disease can be accomplished by
simply reviewing available literature or by employing commercially
available software that identifies the association of a particular
gene with a tobacco-related disease (e.g., PathwayAssist.TM.,
available from Stratagene, La Jolla, Calif. and/or Genespring
(version 7.2, available from Agilent Technologies).
[0736] Next, a second tobacco that is, preferably, the same variety
and grown under the same conditions as the first tobacco is
provided. The second tobacco has been modified to reduce expression
of a second gene, a gene that contributes to the production of a
compound or component present in the first tobacco (e.g., a gene
involved in nicotine synthesis, such as QPTase or PMTase). The
modification of the second gene can be accomplished by genetic
engineering or chemical treatment. Several approaches to modify
tobacco to reduce the amount of nicotine are known. (See e.g., U.S.
patent application Ser. No. 10/729,121, WO0067558A1, WO9428142A1,
WO05000352A1, WO05018307A1, WO03086076A1, and WO0218607A2, all of
which are hereby expressly incorporated by reference in their
entireties).
[0737] By one approach, the second tobacco is genetically modified
to reduce expression of QPTase, as described above (e.g., Vector
21-41). RNAi constructs that comprise fragments of a gene involved
in nicotine synthesis have also been used to reduce the amount of
nicotine and TSNA in tobacco, as described above. By one approach,
for example, the RNAi construct provided in FIG. 1 was used to
generate a reduced nicotine and TSNA tobacco. By another approach,
the RNAi construct provided in FIG. 2 was used to generate a
reduced nicotine and TSNA tobacco. More details on the preparation
of these RNAi constructs and the methods used to create transgenic
tobacco having a reduced amount of nicotine and TSNAs is provided
in the section that follows and Example 15.
[0738] Once the modified second tobacco is obtained, preferably a
genetically modified second tobacco (e.g., a second tobacco that
has been genetically modified to reduce the amount of nicotine),
smoke or a smoke condensate is obtained from said second tobacco.
Then, a second isolated population of cells, preferably the same
cell type as analyzed above (e.g., NHBE cells) is contacted with
the smoke or smoke condensate from the second tobacco, preferably
for the same amount of time as the cells that were contacted with
the first tobacco. Subsequent to the exposure of the second
population of cells to the second tobacco, an approach to identify
the modulation of gene expression in said second population of
cells is employed, preferably the same approach that was used to
analyze the first population of cells after exposure to the smoke
or smoke condensate of the first tobacco product (e.g., an
oligonucleotide array, microarray, qRT-PCR, nucleic acid detection
(e.g., hybridization), protein detection (e.g., antibody detection,
ELISA or Western blot), or detection of a metabolite (e.g., protein
fragment or cysteine) or a modified gene product (e.g., oxidized or
phosphorylated protein or amino acid).
[0739] A modulation (up-regulation or down-regulation) in
expression of a first gene that contributes to a tobacco-related
disease in said second population of cells, as compared to the
amount of expression of the same gene induced by the first tobacco,
will be observed. This difference in expression of a gene that is
related to a tobacco-related disease provides strong evidence that
the modification in the second tobacco has resulted in a tobacco
that has a reduced potential to contribute to a tobacco-related
disease. That is, said (modified) second tobacco has a reduced risk
to contribute to a tobacco-related disease, as compared to the
first (unmodified) tobacco.
[0740] Conventional techniques in cultivation of said second
tobacco, harvesting, curing, blending, and processing are then
employed so as to generate a tobacco product (e.g., snuff, chew,
tobacco leaf, cigarette, pipe tobacco, cigar, or lozenge) and said
tobacco product can be identified as a product that has a reduced
potential to contribute to a tobacco-related disease as compared to
a tobacco product comprising said first tobacco.
[0741] It will be appreciated that the promoters used in the
above-described vectors can either be constitutive or regulatable.
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.
Regulatable promoters are promoters for which the desired level of
expression can be controlled. An example of a regulatable 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.
[0742] 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.
[0743] 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 hairpin structure, or two antiparallel RNA strands
that form a double-stranded RNA, wherein both ends form a hairpin
structure. In some embodiments, synthetic interfering RNAs comprise
a plurality of RNA duplexes.
[0744] By way of example, tobacco having reduced amounts of
nicotine and TSNAs is generated from a tobacco plant that is
created by exposing at least one tobacco cell of a selected tobacco
variety, such as LA Burley 21, to a nucleic acid construct
comprising a promoter that is operable in a plant cell, wherein the
promoter controls the expression of a RNA comprising both strands
of a duplex interfering RNA. For example, the RNA that is expressed
comprises a first nucleotide sequence that is substantially similar
or identical to at least a portion of an mRNA or at least a portion
of the coding strand of a gene that is involved in nicotine
biosynthesis. This first nucleotide sequence is followed by a
non-complementary sequence that is involved in hairpin formation,
and then, a second nucleotide sequence that is complementary or
substantially complementary to at least a portion of the first
nucleotide sequence. The exposed tobacco cell is then transformed
with the nucleic acid construct. Cells that are successfully
transformed are selected using either negative selection or
positive selection techniques and at least one tobacco plant is
regenerated from transformed cells. The regenerated tobacco plant
or portion thereof is preferably analyzed to determine the amount
of nicotine and/or TSNAs present and these values can be compared
to the amount of nicotine and/or TSNAs present in a control tobacco
plant or portion thereof. Preferably the transformed and control
tobacco plants are of the same variety.
[0745] In some embodiments, a cDNA sequence encoding a plant
quinolate phosphoribosyl transferase (QPTase) is used (See Example
15). 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 an interfering RNA, which lowers the
amount of QPTase in tobacco roots. Other embodiments include the
expression of an interfering RNA, which lowers the amount of any
QPTase that may be present in tobacco leaves, stems and/or other
tobacco tissues.
[0746] Some embodiments also 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 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 biosynthesis. The
RNA duplex also comprises a second strand that is complementary or
substantially complementary to the first strand.
[0747] 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 and/or TSNA as compared to unmodified or control
tobacco cells and plants.
[0748] 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 TSNAs. These blended tobacco products can be used in tobacco
product cessation programs so as to slowly move a consumer from a
high nicotine and TSNA product to a low nicotine and TSNA product.
Some embodiments of the invention comprise a tobacco use cessation
kit, comprising two or more tobacco products with different levels
of nicotine and/or TSNAs. For example, a smoker can begin the
program smoking blended cigarettes having or delivering by FTC
methodology 1-2 mg of nicotine and 0.2 .mu.g of TSNA, gradually
move to smoking cigarettes having or delivering 0.75 mg of nicotine
and 0.1 .mu.g of TSNA, followed by cigarettes having or delivering
0.5 mg nicotine and 0.1 .mu.g TSNA, followed by cigarettes having
or delivering 0.1 mg nicotine and 0.05 .mu.g TSNA, followed by
cigarettes having or delivering 0.05 mg nicotine and no detectable
TSNA until the consumer decides to smoke only the cigarettes having
virtually no nicotine and TSNAs or quitting smoking altogether.
Accordingly, the blended cigarettes described herein provide the
basis for an approach to reduce the carcinogenic potential in a
human in a step-wise fashion. The components of the tobacco use
cessation kit described herein may include other tobacco products,
including but not limited to, smoking materials (e.g., cigarettes,
cigars, pipe tobacco), snuff, chewing tobacco, gum, and
lozenges.
[0749] 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. Such heterologous nucleic
acids can be copies of a sequence which is naturally found in the
cell being transformed, or fragments thereof. To produce a tobacco
plant having decreased QPTase levels, and a reduced amount of
nicotine and TSNAs, as compared to an untransformed or control
tobacco plant or portion thereof, a tobacco cell can be transformed
with an exogenous nucleic acid construct which encodes an
interfering RNA having an RNA duplex comprising a first strand that
is substantially similar or identical to at least a portion of the
coding strand of the full-length QPT cDNA sequence, a partial QPT
chromosomal sequence, a full-length QPT chromosomal sequence, or an
mRNA produced from the QPT gene. Alternatively, the tobacco cell
can be transformed with a synthetic or an in vitro transcribed
interfering RNA. In some embodiments of the present invention, the
interfering RNA and/or nucleic acid encoding the interfering RNA
are stably transformed. In certain embodiments, the nucleic acid
encoding the interfering RNA can be integrated in the cell genome.
In other embodiments, the interfering RNA and/or nucleic acid
encoding the interfering RNA are transiently transformed.
[0750] 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.
[0751] 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.
[0752] In some embodiments, the heterologous sequence utilized in
the methods of the present invention may be selected so as to
produce an interfering RNA product comprising a first strand that
is substantially similar or identical to the entire QTPase mRNA
sequence, or to a portion thereof, and a second strand that is
complementary to the entire QPTase 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.
[0753] Interfering RNAs employed in carrying out the present
invention include those comprising a first strand having sequence
similarity to the QPTase gene or a fragment thereof at least or
equal to 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, 800 or more consecutive nucleotides of the QTPase.
(See U.S. Pat. No. 6,586,661, which provides the sequence of the
QPTase gene and protein, herein expressly incorporated by reference
in its entirety). This definition is intended to encompass natural
allelic variations in QPTase proteins. Thus, nucleic acid sequences
that hybridize to nucleic acids of the QPTase gene under the
conditions provided supra may also be employed in carrying out
aspects of the invention. Multiple forms of the tobacco QPT enzyme
may exist. Multiple forms of an enzyme may be due to
post-translational modification of a single gene product, or to
multiple forms of the NtQPT1 gene.
[0754] Conditions that permit other nucleic acid sequences, which
code for expression of a protein having QPTase activity, to
hybridize to a QPTase gene or to other nucleic acid sequences
encoding a QPTase protein can be determined in a routine manner.
For example, hybridization of such sequences to nucleic acids
encoding the QPTase protein may be carried out under conditions of
reduced stringency or even stringent conditions (e.g., conditions
represented by a wash stringency of 0.3 M NaCl, 0.03 M sodium
citrate, 0.1% SDS at 60.degree. C. or even 70.degree. C.) herein in
a standard in situ hybridization assay. See J. Sambrook et al.,
Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring
Harbor Laboratory)). In general, such sequences will be at least
65% similar, 75% similar, 80% similar, 85% similar, 90% similar, or
even 95% similar or more, with the tobacco QPTase gene, or nucleic
sequences encoding the QPTase protein. 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.
[0755] Differential hybridization procedures are available which
allow for the isolation of cDNA clones whose mRNA levels are as low
as about 0.05% of poly(A)RNA. (See M. Conkling et al., Plant
Physiol. 93, 1203-1211 (1990)). In brief, cDNA libraries are
screened using single-stranded cDNA probes of reverse transcribed
mRNA from plant tissue (e.g., roots and/or leaves). For
differential screening, a nitrocellulose or nylon membrane is
soaked in 5.times.SSC and placed in a 96 well suction manifold; 150
.mu.L of stationary overnight culture is transferred from a master
plate to each well and vacuum applied until all liquid has passed
through the filter. Approximately, 150 .mu.L of denaturing solution
(0.5M NaOH, 1.5 M NaCl) is placed in each well using a multiple
pipetter and allowed to sit about 3 minutes. Suction is applied as
above and the filter removed and neutralized in 0.5 M Tris-HCl (pH
8.0), 1.5 M NaCl. It is then baked 2 hours in vacuo and incubated
with the relevant probes. By using nylon membrane filters and
keeping master plates stored at -70.degree. C. in 7% DMSO, filters
may be screened multiple times with multiple probes and appropriate
clones recovered after several years of storage.
IV. Use of Tobacco Products
[0756] Nicotine Reduction and/or Tobacco-Use Cessation Programs
Methods
[0757] 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 TSNAs. These
blended tobacco products can be used in nicotine reduction and/or
tobacco-use cessation programs so as to move a consumer from a high
nicotine and TSNA product to a low nicotine and TSNA product.
[0758] In some embodiments provided herein, a stepwise nicotine
reduction and/or tobacco-use cessation program can be established
using the low nicotine, low TSNA products described above. As an
example, the program participant initially determines 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, 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 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 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, concomitantly, the harmful
effect of nicotine yet retains the secondary factors of addiction,
including but not limited to, smoke intake, oral fixation, and
taste.
[0759] For example, a smoker can begin the program smoking blended
cigarettes having or delivering 5 mg of nicotine and 1.5 .mu.g of
TSNA, gradually move to smoking cigarettes with 3 mg of nicotine
and 1 .mu.g of TSNA, followed by cigarettes having or delivering 1
mg nicotine and 0.5 .mu.g TSNA, followed by cigarettes having or
delivering 0.5 mg nicotine and 0.25 .mu.g TSNA, followed by
cigarettes having or delivering 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 TSNAs or quitting
smoking altogether. Preferably, a three-step program is followed
whereby at step 1, cigarettes providing 0.6 mg nicotine and less
than 2 .mu.g/g TSNA are used; at step 2, cigarettes providing 0.3
mg nicotine and less than 1 .mu.g/g TSNA are used; and at step 3,
cigarettes providing 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 providing 0.6 mg nicotine
and less than 2 .mu.g/g TSNA are used; at step 2, cigarettes
providing 0.3 mg nicotine and less than 1 .mu.g/g TSNA are used;
and at step 3, cigarettes providing less than 0.05 mg nicotine and
less than 0.7 .mu.g/g TSNA are used. Accordingly, the blended
cigarettes described herein provide the basis for an approach to
reduce the carcinogenic potential in a human in a step-wise
fashion.
[0760] 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 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.
[0761] Embodiments, for example, include stepwise blends of tobacco
products, which are prepared with a variety of amounts of nicotine.
These stepwise blends are made to have reduced levels of TSNAs and
varying amounts of nicotine. As an example, cigarettes may deliver,
for example, 5 mg, 4, 3, 2, 1, 0.5, 0.1, or 0 mg of nicotine per
cigarette. More preferably, blended cigarettes provide less than
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%, and 0.6% nicotine.
[0762] In another aspect provided herein, 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 below. 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, embodiments provided
herein 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.
[0763] As an example, Virginia flue tobacco was blended with
genetically modified Burley (i.e., Burley containing a
significantly reduced amount of nicotine and TSNA) to yield a
blended tobacco that was incorporated into three levels of reduced
nicotine cigarettes: a step 1 cigarette providing 0.6 mg nicotine,
a step 2 cigarette providing 0.3 mg nicotine, and a step 3
cigarette providing less than 0.05 mg nicotine. The stepwise packs
of cigarettes are clearly marked as to their nicotine content, and
the step in the stepwise nicotine reduction program is 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.
[0764] 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.
[0765] In another aspect provided herein, individuals would choose
to obtain only cigarettes that provide 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. More discussion on Smoking Cessation Programs that use
reduced nicotine tobacco can be found in PCT/US2004/01695, which
designates the United States and was published in English, hereby
expressly incorporated by reference in its entirety.
[0766] In another aspect provided herein, 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 below.
[0767] 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 providing 0.6 mg
nicotine, step 2 cigarettes providing 0.3 mg nicotine, and step 3
cigarettes providing 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 provided 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 would 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.
[0768] 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.
[0769] 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.
[0770] 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.
[0771] 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.
[0772] 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.
[0773] 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.
[0774] 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.
[0775] 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.
[0776] 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.
[0777] 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.
[0778] 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.
[0779] 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.m/g, 4.0 .mu.m/g, 3.0
.mu.m/g, 2.0 .mu.m/g, 1.0 .mu.m/g, 0.5 .mu.m/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)).
[0780] 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)).
[0781] 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)).
[0782] 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 .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 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)).
[0783] 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)).
[0784] 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)).
[0785] 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)).
[0786] 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)).
[0787] 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).
[0788] 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.
[0789] 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.
[0790] 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
provided herein. All references cited herein are hereby expressly
incorporated by reference.
Sequence CWU 1
1
5614PRTNicotiana tabacum 1Asp Glu Val Asp1 21056DNANicotiana
tabacum 2atgtttagag ctattccttt cactgctaca gtgcatcctt atgcaattac
agctccaagg 60ttggtggtga aaatgtcagc aatagccacc aagaatacaa gagtggagtc
attagaggtg 120aaaccaccag cacacccaac ttatgattta aaggaagtta
tgaaacttgc actctctgaa 180gatgctggga atttaggaga tgtgacttgt
aaggcgacaa ttcctcttga tatggaatcc 240gatgctcatt ttctagcaaa
ggaagacggg atcatagcag gaattgcact tgctgagatg 300atattcgcgg
aagttgatcc ttcattaaag gtggagtggt atgtaaatga tggcgataaa
360gttcataaag gcttgaaatt tggcaaagta caaggaaacg cttacaacat
tgttatagct 420gagagggttg ttctcaattt tatgcaaaga atgagtggaa
tagctacact aactaaggaa 480atggcagatg ctgcacaccc tgcttacatg
ttggagacta ggaaaactgc tcctggatta 540cgtttggtgg ataaatgggc
ggtattgatc ggtgggggga agaatcacag aatgggctta 600tttgatatgg
taatgataaa agacaatcac atatctgctg ctggaggtgt cggcaaagct
660ctaaaatctg tggatcagta tttggagcaa aataaacttc aaataggggt
tgaggttgaa 720accaggacaa ttgaagaagt acgtgaggtt ctagactatg
catctcaaac aaagacttcg 780ttgactagga taatgctgga caatatggtt
gttccattat ctaacggaga tattgatgta 840tccatgctta aggaggctgt
agaattgatc aatgggaggt ttgatacgga ggcttcagga 900aatgttaccc
ttgaaacagt acacaagatt ggacaaactg gtgttaccta catttctagt
960ggtgccctga cgcattccgt gaaagcactt gacatttccc tgaagatcga
tacagagctc 1020gcccttgaag ttggaaggcg tacaaaacga gcatga
10563360DNANicotiana tabacum 3atgtttagag ctattccttt cactgctaca
gtgcatcctt atgcaattac agctccaagg 60ttggtggtga aaatgtcagc aatagccacc
aagaatacaa gagtggagtc attagaggtg 120aaaccaccag cacacccaac
ttatgattta aaggaagtta tgaaacttgc actctctgaa 180gatgctggga
atttaggaga tgtgacttgt aaggcgacaa ttcctcttga tatggaatcc
240gatgctcatt ttctagcaaa ggaagacggg atcatagcag gaattgcact
tgctgagatg 300atattcgcgg aagttgatcc ttcattaaag gtggagtggt
atgtaaatga tggcgataaa 3604241DNANicotiana tabacum 4cattttacca
tctttcgcca gaagtatgat cgagtcttaa tcaagtgaat aatgaacact 60ggtagtacaa
tcattggacc aagatcgagt cttaatcaag tgaataaata agtgaaatgc
120gacgtattgt aggagaattc tgcagtaatt atcataattt ccaattcaca
atcattgtaa 180aattctttct ctgtggtgtt tcgtacttta atataaattt
tcctgctgaa gttttgaatc 240g 2415628DNANicotiana tabacum 5tgatcaagtg
aacatcatca aagcaattaa agaagctgga aatatcaaga gatttcttcc 60ttcagaattt
ggatttgatg tggatcatgc tcgtgcaatt gaaccagctg catcactctt
120cgctctaaag gtaagaatca ggaggatgat agaggcagaa ggaattccat
acacatatgt 180aatctgcaat tggtttgcag atttcttctt gcccaacttg
gggcagttag aggccaaaac 240ccctcctaga gacaaagttg tcatttttgg
cgatggaaat cccaaagcaa tatatgtgaa 300ggaagaagac atagcgacat
acactatcga agcagtagat gatccacgga cattgaataa 360gactcttcac
atgagaccac ctgccaatat tctatccttc aacgagatag tgtccttgtg
420ggaggacaaa attgggaaga ccctcgagaa gttatatcta tcagaggaag
atattctcca 480gattgtacaa gagggacctc tgccattaag gactaatttg
gccatatgcc attcagtttt 540tgttaatgga gattctgcaa actttgaggt
tcagcctcct acaggtgtcg aagccactga 600gctatatcca aaagtgaaat acacaacc
6286439DNANicotiana tabacum 6ccacattggg gcttctgtta ctcaatgctt
cataaggttt ctcgtagctt tgctctcgtc 60attcaacaac ttcccgtcga gcttcgtgac
gccgtgtgca ttttctattt ggttcttcga 120gcacttgaca ctgttgagga
tgataccagc attcccaccg atgttaaagt acctattctg 180atctcttttc
atcagcatgt ttatgatcgt gaatggcatt tttcatgtgg tacaaaagag
240tacaaggttc tcatggacca gttccatcat gtttcaactg cttttctgga
gcttaggaaa 300cattatcagc aggcaattga ggatattacc atgaggatgg
gtgcaggaat ggcaaaattc 360atatgcaagg aggtggaaac aactgatgat
tatgacgaat attgtcacta tgtagctggg 420cttgttgggc taggattgt
4397850DNANicotiana tabacum 7gaggctgtaa atgatggcaa agacctccat
atttcagtaa ctatgccttc tattgaggtt 60ggcacagttg gtggtggaac tcaacttgca
tcacagtcag cttgcttgaa cttattagga 120gtgaaaggtg caaacaggga
ggcagcaggg tcaaatgcaa ggctcttggc cacaatagta 180gcaggttctg
ttcttgctgg tgagttatct ctcatgtctg ctatctcagc agggcagctg
240gttaagagtc acatgaaata caatagatct agcaaagatg ttactaagat
ttcctcttag 300taaggaaaaa gacaaattta ttatcccaac atcgtgtaca
tcaccatcct ttatggacca 360tcattattaa agaaatggat tacaataaaa
gtaggaataa aattttccaa ttagggagag 420caataagtaa agggtagacc
aaaaagttga aaaagtgtaa ggcattagtc atgtggagaa 480agatcaagaa
gaaaaagaca agcaaatcaa gggtggacgt ggatctgtat gtagtgttgt
540attctttcta tgaaggcatg tgaggaggta gggtcgtatt tttttctgag
ttcgtgtaaa 600aaaacctgca aatatttggt gaagatctac gaaaggtgtt
aggtgggatg gtgaccagtg 660gggttaactt gtaattcaac atttggttaa
tttcattcat gcgccaagga agataacccc 720ttttttttta aataatcttt
tctgttgtac tgtctttcgt ttgtttgtta attgtgacta 780gattgtaatt
tagagagaaa tggcatctca aactctttat gtttgctcag aaagtttgct
840tttgtatatg 8508780DNANicotiana tabacum 8agaagctacc tgccatgcac
cagatccatt gggatgctat aaagagattt accgggtgct 60gaagcctggt caatgtttcg
ctgtgtatga gtggtgcatg accgattctt acaaccccaa 120taacgaagag
cacaacagga tcaaggccga aattgagctc ggaaatggcc tccctgaggt
180tagattgaca acacagtgcc tcgaagcagc caaacaagct ggttttgaag
ttgtatggga 240caaggatctg gctgatgact cacctgttcc atggtacttg
cctttggata cgagtcactt 300ctcgctcagt agcttccgcc taacagcagt
tggcagactt ttcaccagaa atctggtttc 360ggcgcttgaa tacgtgggac
ttgctcctaa aggtagtcaa agggttcaag ctttcttaga 420gaaagctgca
gaaggtcttg tcggtggtgc caagaaaggg attttcacac caatgtactt
480cttcgtggtt cgcaagccca tttcagactc tcagtaatat ggagtttagt
cacttagctt 540tttgctttag ctagcaaatc tgtaagattc ttcgcacaga
actttacaca ttgaatatga 600ccgccctaat taaggtgact acagtttttg
gagggcgttg tgggtggagg gtttcttttt 660ctgtgttgct tgtctggcac
aatttgattt catgtcttgc tatttttgcc attgatgtcc 720ttgttctaag
atatatacct attgacaagc tcataaaggt gggcatttgc taatatatgg
7809885DNANicotiana tabacum 9aagaatatca cgttcttcgt tggcccagaa
gtgtcggccc atttctttaa ggccccagaa 60accgatctca gtcaacaaga ggtttatcag
ttcaatgtgc ctacttttgg ccctggtgtg 120gtttttgacg ttgattatac
tatcagacaa gagcaattta ggttctttac tgaatctttg 180agggtaaata
aattgaaggg atatgtggat cagatggtca tggaagctga ggagtacttc
240tcaaaatggg gtgatagtgg tgaagtggac ttgaagtatg aactggagca
tcttatcata 300ctgacagcta gtagatgtct gttgggagaa gaggttcgca
ataaactctt tgaggatgtc 360tctgctctct tccatgacct ggacaatggg
atgcttccta tcagtgtaat ctttccctac 420cttcccattc cagcccatcg
ccgtcgtgac aatgcccgca agaagctcgc ggagatcttt 480gcaaacatca
tagattctag aaaacgtaca ggcaaggcgg agagcgatat gttacaatgc
540ttcattgact ccaagtacaa agatgggcgg gcaacgacag agtctgagat
cacaggtctt 600ctgattgctg ctcttttcgc tgggcaacac accagttcca
tcacctccac ttgggcaggg 660gcataccttc tctgcaacaa caagtacatg
tctgccgtcg tagatgaaca gaagaatctg 720atgaagaaac atgggaataa
ggtcgatcat gacatccttt ccgagatgga agtcctctat 780agatgcataa
aggaagccct gagactccat cctccactga taatgcttct acgtagttcg
840catagtgatt tcactgttaa aaccagggaa ggaaaagagt atgat
885101701DNAArabidopsis thaliana 10atggttgtgt ttgggaatgt ttctgcggcg
aatttgcctt atcaaaacgg gtttttggag 60gcactttcat ctggaggttg tgaactaatg
ggacatagct ttagggttcc cacttctcaa 120gcgcttaaga caagaacaag
gaggaggagt actgctggtc ctttgcaggt agtttgtgtg 180gatattccaa
ggccagagct agagaacact gtcaatttct tggaagctgc tagtttatct
240gcatccttcc gtagtgctcc tcgtcctgct aagcctttga aagttgtaat
tgctggtgct 300ggattggctg gattgtcaac tgcaaagtac ctggctgatg
caggccacaa acctctgttg 360cttgaagcaa gagatgttct tggtggaaag
atagctgcat ggaaggatga agatggggac 420tggtatgaga ctggtttaca
tattttcttc ggtgcttatc cgaatgtgca gaatttattt 480ggagaacttg
ggatcaatga tcggttgcag tggaaggaac actccatgat ttttgctatg
540ccaagtaaac ctggagaatt tagtagattt gacttcccag atgtcctacc
agcaccctta 600aatggtattt gggctatttt gcggaacaac gagatgctga
catggccaga gaaaataaag 660tttgctattg gacttttgcc agccatggtc
ggcggtcagg cttatgttga ggcccaagat 720ggtttatcag tcaaagaatg
gatggaaaag cagggagtac ctgagcgcgt gaccgacgag 780gtgtttattg
ccatgtcaaa ggcgctaaac tttataaacc ctgatgaact gtcaatgcaa
840tgcattttga tagctttgaa ccggtttctt caggaaaaac atggttccaa
gatggcattc 900ttggatggta atcctccgga aaggctttgt atgccagtag
tggatcatat tcgatcacta 960ggtggggaag tgcaacttaa ttctaggata
aagaaaattg agctcaatga cgatggcacg 1020gttaagagtt tcttactcac
taatggaagc actgtcgaag gagacgctta tgtgtttgcc 1080gctccagtcg
atatcctgaa gctcctttta ccagatccct ggaaagaaat accgtacttc
1140aagaaattgg ataaattagt tggagtacca gttattaatg ttcatatatg
gtttgatcga 1200aaactgaaga acacatatga tcacctactc tttagcagaa
gtaaccttct gagcgtgtat 1260gccgacatgt ccttaacttg taaggaatat
tacgatccta accggtcaat gctggagcta 1320gtatttgcac cagcagagga
atggatatca cggactgatt ctgacatcat agatgcaaca 1380atgaaagaac
ttgagaaact cttccctgat gaaatctcag ctgaccaaag caaagctaaa
1440attctgaagt accatgtcgt taagactcca agatctgggt acaagaccat
cccaaactgt 1500gaaccatgtc gtcctctaca aagatcacct attgaaggat
tctacttagc tggagattac 1560acaaaacaga agtacttagc ttccatggaa
ggcgctgtcc tctctggcaa attctgctct 1620cagtctattg ttcaggatta
cgagctactg gctgcgtctg gaccaagaaa gttgtcggag 1680gcaacagtat
catcatcatg a 1701111701DNAArabidopsis thaliana 11atggttgtgt
ttgggaatgt ttctgcggcg aatttgcctt atcaaaacgg gtttttggag 60gcactttcat
ctggaggttg tgaactaatg ggacatagct ttagggttcc cacttctcaa
120gcgcttaaga caagaacaag gaggaggagt actgctggtc ctttgcaggt
agtttgtgtg 180gatattccaa ggccagagct agagaacact gtcaatttct
tggaagctgc tagtttatct 240gcatccttcc gtagtgctcc tcgtcctgct
aagcctttga aagttgtaat tgctggtgct 300ggattggctg gattgtcaac
tgcaaagtac ctggctgatg caggccacaa acctctgttg 360cttgaagcaa
gagatgttct tggtggaaag atagctgcat ggaaggatga agatggggac
420tggtatgaga ctggtttaca tattttcttc ggtgcttatc cgaatgtgca
gaatttattt 480ggagaacttg ggatcaatga tcggttgcag tggaaggaac
actccatgat ttttgctatg 540ccaagtaaac ctggagaatt tagtagattt
gacttcccag atgtcctacc agcaccctta 600aatggtattt gggctatttt
gcggaacaac gagatgctga catggccaga gaaaataaag 660tttgctattg
gacttttgcc agccatggtc ggcggtcagg cttatgttga ggcccaagat
720ggtttatcag tcaaagaatg gatggaaaag cagggagtac ctgagcgcgt
gaccgacgag 780gtgtttattg ccatgtcaaa ggcgctaaac tttataaacc
ctgatgaact gtcaatgcaa 840tgcattttga tagctttgaa cccgtttctt
caggaaaaac atggttccaa gatggcattc 900ttggatggta atcctccgga
aaggctttgt atgccagtag tggatcatat tcgatcacta 960ggtggggaag
tgcaacttaa ttctaggata aagaaaattg agctcaatga cgatggcacg
1020gttaagagtt tcttactcac taatggaagc actgtcgaag gagacgctta
tgtgtttgcc 1080gctccagtcg atatcctgaa gctcctttta ccagatccct
ggaaagaaat accgtacttc 1140aagaaattgg ataaattagt tggagtacca
gttattaatg ttcatatatg gtttgatcga 1200aaactgaaga acacatatga
tcacctactc tttagcagaa gtaaccttct gagcgtgtat 1260gccgacatgt
ccttaacttg taaggaatat tacgatccta accggtcaat gctggagcta
1320gtatttgcac cagcagagga atggatatca cggactgatt ctgacatcat
agatgcaaca 1380atgaaagaac ttgagaaact cttccctgat gaaatctcag
ctgaccaaag caaagctaaa 1440attctgaagt accatgtcgt taagactcca
agatctgtgt acaagaccat cccaaactgt 1500gaaccatgtc gtcctctaca
aagatcacct attgaaggat tctacttagc tggagattac 1560acaaaacaga
agtacttagc ttccatggaa ggcgctgtcc tctctggcaa attctgctct
1620cagtctattg ttcaggatta cgagctactg gctgcgtctg gaccaagaaa
gttgtcggag 1680gcaacagtat catcatcatg a 1701121701DNAArabidopsis
thaliana 12atggttgtgt ttgggaatgt ttctgcggcg aatttgcctt atcaaaacgg
gtttttggag 60gcactttcat ctggaggttg tgaactaatg ggacatagct ttagggttcc
cacttctcaa 120gcgcttaaga caagaacaag gaggaggagt actgctggtc
ctttgcaggt agtttgtgtg 180gatattccaa ggccagagct agagaacact
gtcaatttct tggaagctgc tagtttatct 240gcatccttcc gtagtgctcc
tcgtcctgct aagcctttga aagttgtaat tgctggtgct 300ggattggctg
gattgtcaac tgcaaagtac ctggctgatg caggccacaa acctctgttg
360cttgaagcaa gagatgttct tggtggaaag atagctgcat ggaaggatga
agatggggac 420tggtatgaga ctggtttaca tattttcttc ggtgcttatc
cgaatgtgca gaatttattt 480ggagaacttg ggatcaatga tcggttgcag
tggaaggaac actccatgat ttttgctatg 540ccaagtaaac ctggagaatt
tagtagattt gacttcccag atgtcctacc agcaccctta 600aatggtattt
gggctatttt gcggaacaac gagatgctga catggccaga gaaaataaag
660tttgctattg gacttttgcc agccatggtc ggcggtcagg cttatgttga
ggcccaagat 720ggtttatcag tcaaagaatg gatggaaaag cagggagtac
ctgagcgcgt gaccgacgag 780gtgtttattg ccatgtcaaa ggcgctaaac
tttataaacc ctgatgaact gtcaatgcaa 840tgcattttga tagctttgaa
ccggtttctt caggaaaaac atggttccaa gatggcattc 900ttggatggta
atcctccgga aaggctttgt atgccagtag tggatcatat tcgatcacta
960ggtggggaag tgcaacttaa ttctaggata aagaaaattg agctcaatga
cgatggcacg 1020gttaagagtt tcttactcac taatggaagc actgtcgaag
gagacgctta tgtgtttgcc 1080gctccagtcg atatcctgaa gctcctttta
ccagatccct ggaaagaaat accgtacttc 1140aagaaattgg ataaattagt
tggagtacca gttattaatg ttcatatatg gtttgatcga 1200aaactgaaga
acacatatga tcacccactc tttagcagaa gtaaccttct gagcgtgtat
1260gccgacatgt ccttaacttg taaggaatat tacgatccta accggtcaat
gctggagcta 1320gtatttgcac cagcagagga atggatatca cggactgatt
ctgacatcat agatgcaaca 1380atgaaagaac ttgagaaact cttccctgat
gaaatctcag ctgaccaaag caaagctaaa 1440attctgaagt accatgtcgt
taagactcca agatctgtgt acaagaccat cccaaactgt 1500gaaccatgtc
gtcctctaca aagatcacct attgaaggat tctacttagc tggagattac
1560acaaaacaga agtacttagc ttccatggaa ggcgctgtcc tctctggcaa
attctgctct 1620cagtctattg ttcaggatta cgagctactg gctgcgtctg
gaccaagaaa gttgtcggag 1680gcaacagtat catcatcatg a
1701131061DNANicotiana tabacum 13aatatgaaag gaaacatatt caatacattg
tagtttgcta ctcataatcg ctagaatact 60ttgtgccttg ctaataaaga tacttgaaat
agcttagttt aaatataaat agcataatag 120attttaggaa ttagtatttt
gagtttaatt acttattgac ttgtaacagt ttttataatt 180ccaaggccca
tgaaaaattt aatgctttat tagttttaaa cttactatat aaatttttca
240tatgtaaaat ttaatcggta tagttcgata ttttttcaat ttatttttat
aaaataaaaa 300acttacccta attatcggta cagttataga tttatataaa
aatctacggt tcttcagaag 360aaacctaaaa atcggttcgg tgcggacggt
tcgatcggtt tagtcgattt tcaaatattc 420attgacactc ctagttgttg
ttataggtaa aaagcagtta cagagaggta aaatataact 480taaaaaatca
gttctaagga aaaattgact tttatagtaa atgactgtta tataaggatg
540ttgttacaga gaggtatgag tgtagttggt aaattatgtt cttgacggtg
tatgtcacat 600attatttatt aaaactagaa aaaacagcgt caaaactagc
aaaaatccaa cggacaaaaa 660aatcggctga atttgatttg gttccaacat
ttaaaaaagt ttcagtgaga aagaatcggt 720gactgttgat gatataaaca
aagggcacat tggtcaataa ccataaaaaa ttatatgaca 780gctacagttg
gtagcatgtg ctcagctatt gaacaaatct aaagaaggta catctgtaac
840cggaacacca cttaaatgac taaattaccc tcatcagaaa gcagatggag
tgctacaaat 900aacacactat tcaacaacca taaataaaac gtgttcagct
actaaaacaa atataaataa 960atctatgttt gtaagcactc cagccatgtt
aatggagtgc tattgcctgt taactctcac 1020ttataaaata gtagtagaaa
aaatatgaac caaaacacaa c 106114711DNANicotiana tabacum 14gaattcaatg
gagaaggaaa atatttccag tgtaaacaca agtgaatgaa gagaagccaa 60aataatctct
atcattcaag ccttaggtgg agattaaaaa aattatttac tttcttatca
120aagtaatagg tgatcaacag ctttcgtaaa acgtcattag gagaatatta
taatctcttt 180tatgctgaag aacccacata aggaagatca taaaatacat
gactttcaga tgacttcttg 240gagctttatt tttaaagagt ggctagctgg
tcagcaaaga ggtgctcgtc agatatcata 300aaattttact attatttgtt
ttaagaggga gatggggcac acatgcttgt gacaaaagta 360agaggaagaa
aggagacaga agaggaaata gatttggggg gggggggggg ggtttcacaa
420tcaaagaaaa tttttaaaat ggagagagaa atgagcacac acatatacta
acaaaatttt 480actaataatt gcaccgagac aaacttatat tttagttcca
aaatgtcagt ctaaccctgc 540acgttgtaat gaatttttaa ctattatatt
atatcgagtt gcgccctcca ctcctcggtg 600tccaaattgt atttaaatgc
atagatgttt attgggagtg tacagcaagc tttcggaaaa 660tacaaaccat
aatactttct cttcttcaat ttgtttagtt taattttgaa a 711151278DNANicotiana
tabacum 15tgcgtcaaat ggataaacaa aaaaatagca taagttagtt ttgttactcg
agagttatgt 60attataaggt atagggaaat gactcaaaca taccactgaa cttaacgaaa
cgacgcatat 120atatactact taacttaacg aaaaaggggt gagagtggat
gggtgctggt aaataatgaa 180gggtttatat aacgtcacgt gtcaaaattc
gatagtagta gtttcgttag ttgtaatagc 240atatatggcc caaagttata
atatagataa tatgtttatg tccaactatt aacgagtgac 300atagacagtt
cattttgtga agttcaatga catatttgag ccctttccct tttattatct
360ccttttattt gttctaataa aagaatggca tttattatgt acatagacaa
ataactattt 420tctttggaat ataatttgtt tatatatttt aaaatcatgt
ctcaatttag tttgttttgt 480gcatatttca actattcaat tttgtccata
tatttattac cttcccccat ttacaagcat 540tgaaccgctt tgctcaccaa
aacttatgca cattgcaaaa atatcatgta aaggttttat 600gtatgctgta
attaaggtct gaactcatcg tgattttatt tttaggcttc attgaccact
660accaaactct ttgatgctac attttctaat tatattggag ttcgattata
tccgaattcg 720cgttgcgtag ggcccattcg agggaaaaca ctccctatca
aggatttttt catacccaga 780gctcgaactc aagacatctg gttaagggaa
gaacagtctc atccactgca ccatatcctt 840ttgtggtcaa caagtaaatt
ttatgtagaa ccaaaaacta tactcgaatt gataaaataa 900atggtgtaaa
atattgtttt ctttcttaca ttttggacag taaatatgta ggacaataat
960aattagcgtg gggtcttaag aaaattagca tagatttcca gaaattccaa
atcaaccggc 1020agttccaggt ttgaaaacta caactcattc cgacggttca
aacttcaaac catgcttgct 1080gactcggctt cttctttctt tttcaccaag
acagagcagt agtcacgtga cacccctcac 1140gtgcctcccc cctttatatt
tcagactgca accctacact ttcgctacat tcactaccat 1200attcttttca
ctaagcaatt ttctctccta cttttcttta aacccctttt ttctccccta
1260agccatggca tctagatc 1278161079DNANicotiana tabacum 16atcttattgt
ataaatatcc ataaacacat catgaaagac actttctttc acggtctgaa 60ttaattatga
tacaattcta atagaaaacg aattaaatta cgttgaattg tatgaaatct
120aattgaacaa gccaaccacg acgacgacta acgttgcctg gattgactcg
gtttaagtta 180accactaaaa aaacggagct gtcatgtaac acgcggatcg
agcaggtcac agtcatgaag 240ccatcaaagc aaaagaacta atccaagggc
tgagatgatt aattagttta aaaattagtt 300aacacgaggg aaaaggctgt
ctgacagcca ggtcacgtta tctttacctg tggtcgaaat 360gattcgtgtc
tgtcgatttt aattattttt ttgaaaggcc gaaaataaag ttgtaagaga
420taaacccgcc tatataaatt catatatttt ctctccgctt tgaattgtct
cgttgtcctc 480ctcactttca tcggccgttt ttgaatctcc ggcgacttga
cagagaagaa caaggaagaa 540gactaagaga gaaagtaaga gataatccag
gagattcatt ctccgttttg aatcttcctc 600aatctcatct tcttccgctc
tttctttcca aggtaatagg aactttctgg atctacttta 660tttgctggat
ctcgatcttg ttttctcaat ttccttgaga tctggaattc gtttaatttg
720gatctgtgaa cctccactaa atcttttggt tttactagaa tcgatctaag
ttgaccgatc 780agttagctcg attatagcta ccagaatttg
gcttgacctt gatggagaga tccatgttca 840tgttacctgg gaaatgattt
gtatatgtga attgaaatct gaactgttga agttagattg 900aatctgaaca
ctgtcaatgt tagattgaat ctgaacactg tttaagttag atgaagtttg
960tgtatagatt cttcgaaact ttaggatttg tagtgtcgta cgttgaacag
aaagctattt 1020ctgattcaat cagggtttat ttgactgtat tgaactcttt
ttgtgtgttt gcagctcat 1079171943DNANicotiana tabacum 17aagcttttta
tttagctttt tcctccctat ttcaatatat aatggctcaa tttttgtcag 60atagcaataa
aaccatacaa gaaaataaaa caaatcacaa aatacaaaaa gaggttatat
120ctccatgtat gcaatttcat tatatgcata taagcatctt acgtataaaa
aaaaagaggg 180aatcatggac gtgtctttct aatccaagta gggtcaactt
tatagggtcg gtgtatgtgt 240agtttaatcg aaaaagaatt ccatcattag
gtaatttaca attagatcct taaattatac 300aaatatataa gggtataaaa
gttgatcaat atttcaggga tattttagtc gttcaacatt 360tagtataaat
tattcgtact tttataataa taaatagata gataaacata gatatagata
420taaatataga tagataaatg ggggatttgc atctataccc actttttggg
tcacgtttta 480atttgtgccc gctttgcaaa aaaaattgca agcgtacaca
ctttttcgcg taacttcagc 540atacggggct aaagtagcaa agacagtcac
gcaaaacttc agcatacttc agtctttgct 600acttcagccc cgtatgctga
agttatgcga aaagcgggta tgcttgtaat ttttttgcaa 660agcgagcata
agttaaaacg tgacacaaaa agcgggtata gatacaaatg gccctttttt
720ttctagccaa attttattca tttttttgga atactttttc actttatttt
aaaattagtg 780tttggttata aatttttaaa tacaacttgg agttggactc
caaagtcttt acatacttat 840ttttagtttt attaccctat tttttttaac
atgagatatt tacttttaca gatctaaaaa 900tgatattttc ttagttttaa
cactataaat agccatgaag gcccatttcc tccctttgca 960aaaagtatac
ccaaacgcaa ctccgtcttc acctccaact ccaacttcat aatttcaatt
1020aaagtgaaaa ttattttaag agaccatttg gacatgataa ttttttcact
ttttccgaac 1080ttttttttac tttttttcaa atcagtgttt ggccataaaa
ttttcatttt tcacttgaag 1140ttgaattttt gaatttttcg agaattcgaa
aaaccccaga aagctgtttt tcaaaatttt 1200cactcggatc ctcacaaaac
ttccaaaata acccaaaatt atattcatgt ccaacacaac 1260tctaattttc
aaataccatt ttcacttgaa aaagaaattc accttttttt tttttttgaa
1320ctttacaatt cttatgtcca aacgccccct tcgaatctac ggccaacgtt
tattaagtaa 1380ggaaagaaaa atggctataa taattatatc ccttttgaag
taaatataat tctaccaaat 1440taattaatat gcttaaaaac aataaaaata
atcaaaattg ctagagagga caaccaatta 1500gccgaagcat tgtcaagatt
gagcagggcg cagaatgaag aaagtagttt tttatctttt 1560gatgccctac
gccttttgta ttaaaatact atatacaaga tttgaaaaag acgagttcca
1620ttcaaaacag ttcccttgtc ccgaaatgtt cattgatgaa gtaatatgca
cttttaaaat 1680tatttttttc cagtttatcc taaaaaaaat attattttta
taatcacata gaaataatat 1740atatcaaata acaaagggaa aaagaaagta
gggaaagaaa ataataattg aagtgggctg 1800ggctttgaca tggaaaggaa
tggcttagta ataattgaag ttagcatcgg atctatttga 1860agtgccactc
atccctcaga aaaacagtgt tagtattttc tctcacaaat tgattctgtg
1920gtccgaattg gagttcctaa atc 194318690DNAArabidopsis thaliana
18ttcgttgaaa aatcatcgaa attttcgacg gattccaatg atcaaaaatt cgtcaataat
60ttccaacgat attctgacta aactaaatct gatgaaatat ttttgacggc tttccaacca
120aaatatttcg ttgtgacttg tcaaaaatcc gttagaatac taagcaactt
ttcgacagat 180tttcagcaaa aatattcggt aatataacgt gttaaaaata
tgataaaaaa aaaaacttga 240tgaatctact aaaactaaat tttcaatcat
atatatctat tattcatata tttcattcat 300tttattattt ttctcttaac
aattatttag ttattctggt atcgtgtaat tatattcata 360tgatttattc
tgatattgat tcggttagca tccggataaa tctgggttgg gctttttaac
420ttggtttttc taagaaaaat tctaatatga tttggttagc atccggatta
gtctagtttg 480gtaggcctgc ctttgtgatt cttaactcgg tcttttgtat
gggtttgaac aattactaca 540ccatttagat tcttctgacc catatcaaat
aaagatccac ttaggcccat tagggttaga 600acaaacatga ggttgcagaa
taaaaagggt tcattttcct cactctcaag ttggatctca 660aaaccctaat
atctgaactt cgccgtcgag 69019995DNAArabidopsis thaliana 19ttctgttcgt
atatttgtaa ctattatgtg tatttttatt ttgttagtat tactaattca 60agtggtttaa
gttgttgaga ctctttaaaa tctaagcatt ttataaacaa taatatataa
120ttattgttta ggctaaattt gtcactaatt aaggtttgga tacatagtgt
ctaaactaag 180ctaataatat cacttaacgt ttacttgtaa cgctaggtga
tgatgtcgtc aagtcaattg 240gtacaaggaa taaacgagtg gtcatatgac
attatgacca tatgaattca aactccagta 300atccaatggt aattggattc
aatgatcaag acttgaacca cgtaatccac ccttatcctt 360agaagctcat
aaatatcact aaagggacag gcaacactta accagtagtt gtccaataat
420ttagttttcc aaaatgaaaa attattgttg tcatctattt taggtgtttt
agttcaatgt 480ggattcctcg tcctaacaaa tacttgacga atatatctag
actataaaat tggttatgag 540ttctactttt ttttgtttgt gaaattatca
aaatttgtta tatttattta tttattctca 600ttaatttgag tactaatttt
taaattattt atactaaaaa caattactaa gatacaaaaa 660tggataagag
catggtgtat agatatttaa tgggatagaa tatttcccat aattgtatgt
720gtgtgagagg ttttgttttc gtaaggaaag aaacaaaaac catttgacca
aagaaaagca 780aaagaaggca aggaatcaaa caacaaatgt tgcaaggcag
aaataatgga cgttatgtta 840atgtagtgtc gtcacacgtg acttaaaaga
gacgagtctg cgtgtcaaac taaaaatgta 900tgcaactata aaaatgggat
ttgattatct ttttagtacc gaagcctacc aaccacatgc 960acactaattc
tactcgccaa ataaagtgaa aagag 995201017DNAArabidopsis thaliana
20aagtaacttt tagaattgat tcaatctttt tagaatagat tttttttttt tttttttttg
60gatttcgctg aggttttacc attttgttac tcagcatatt ttaacgatgt tgcatttgtg
120tcccatatac gttattgtta gtgaaaaata taatgtaaga ataatttata
taactatcct 180actagcaaag ctaacgcaaa ttttgaactc gaactttagt
taccgtgaat gaaaataaca 240gacttgaact ttataatact cgtagtatac
gtaatttttg ctttttgcag atatgcttgc 300cactaataaa gtcataaatt
ttatattttc ataaactata gttatacact tttgactaaa 360caaacaaaat
cggtttagca aaagaaaaag ttacttttct gatgaactag gataaggaat
420tcggaactga attttgctac gttctctctg gaccacacac actgaacacc
cttttaagat 480tttctccttc tctttttcaa cgtaatttat cttttgatca
gaaacgacaa aaaagaagtc 540taacaatatc aaacaatttt tttatagata
tttttagata tttttcctgc taattttatc 600tagtgtagac aaacccaaat
atacgattat tataaaaaca cgaaatacca agtggacgac 660tgaggttaat
agatctagcc gtagaataaa gatctgcatg aaaggcggtg agaatctaaa
720cggtgataag accataacac acggaacatc ggtacgctct cgaacgtaca
agaatcgacg 780acacacaaac actccacaat tatttgaaca ctggacaatt
attgaaccga cgtacgagaa 840tcaatgcgct gagggtaaag acgtaaatga
agaactagtt ttggagataa gagcggagaa 900agattgcgac acatgtatgg
tcaatattaa tctcatttag cttataaatt tgggagcttc 960ctctatcatt
aattttcatt cataaatttt tcttcaattt gaattttctc gagaaaa
101721273DNAArabidopsis thaliana 21gcaagtgtgt tgcctttgtg tggaaatgaa
gaggtacttg cgaggacttt gcgtttatca 60gtttatgtgt ttgtatatct atttgatcca
gttattatgg attatatacg cttgaaactc 120attttaagcc attgttattg
aacgtttatc aaatacttta ttatgccaag caagtcaaac 180acatgcttgt
tgattgaaat caagctatag aaatctcttc ttcacataca gcagtttaga
240ttcacaatac aacaagcgaa acgataaagt ttc 273221131DNAArabidopsis
thaliana 22gtccgtcgct tctcttccat ttcttctcat tttcgatttt gattcttatt
tctttccagt 60agctcctgct ctgtgaattt ctccgctcac gatagatctg cttatactcc
ttacattcaa 120ccttagatct ggtctcgatt ctctgtttct ctgttttttt
cttttggtcg agaatctgat 180gtttgtttat gttctgtcac cattaataat
aatgaactct ctcattcata caatgattag 240tttctctcgt ctacaaaacg
atatgttgca ttttcacttt tcttcttttt ttctaagatg 300atttgctttg
accaatttgt ttagatcttt attttatttt attttctggt gggttggtgg
360aaattgaaaa aaaaaaaaac agcataaatt gttatttgtt aatgtattca
ttttttggct 420atttgttctg ggtaaaaatc tgcttctact attgaatctt
tcctggattt tttactccta 480ttgggttttt atagtaaaaa tacataataa
aaggaaaaca aaagttttat agattctctt 540aaacccctta cgataaaagt
tggaatcaaa ataattcagg atcagatgct ctttgattga 600ttcagatgcg
attacagttg catggcaaat tttctagatc cgtcgtcaca ttttattttc
660tgtttaaata tctaaatctg atatatgatg tcgacaaatt ctggtggctt
atacatcact 720tcaactgttt tcttttggct ttgtttgtca acttggtttt
caatacgatt tgtgatttcg 780atcgctgaat ttttaataca agcaaactga
tgttaaccac aagcaagaga tgtgacctgc 840cttattaaca tcgtattact
tactactagt cgtattctca acgcaatcgt ttttgtattt 900ctcacattat
gccgcttctc tactctttat tccttttggt ccacgcattt tctatttgtg
960gcaatccctt tcacaacctg atttcccact ttggatcatt tgtctgaaga
ctctcttgaa 1020tcgttaccac ttgtttcttg tgcatgctct gttttttaga
attaatgata aaactattcc 1080atagtcttga gttttcagct tgttgattct
tttgcttttg gttttctgca g 1131235688DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
23ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat cctttctttt
60cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat cctattttgt
120ttctttactt atttatttgc ttctatgtct ttgtacaaag atttaaactc
tatggcacat 180attttaaagt tgttagaaaa taaattcttt caagattgat
gaaagaactt tttaattgta 240gatatttcgt agattttatt ctcttactac
caatataacg cttgaattga cgaaaatttg 300tgtccaaata tctagcaaaa
aggtatccaa tgaaaatata tcatatgtga tcttcaaatc 360ttgtgtctta
tgcaagattg atactttgtt caatggaaga gattgtgtgc atatttttaa
420aatttttatt agtaataaag attctatata gctgttatag agggataatt
ttacaaagaa 480cactataaat atgattgttg ttgttagggt gtcaatggtt
cggttcgact ggttatttta 540taaaatttgt accataccat ttttttcgat
attctatttt gtataaccaa aattagactt 600ttcgaaatcg tcccaatcat
gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660catttttttt
taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt
720cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg
ataatgaatt 780aaaaaacatg aaagatggct agagtataga tacacaacta
ttcgacagca acgtaaaaga 840aaccaagtaa aagcaaagaa aatataaatc
acacgagtgg aaagatatta accaagttgg 900gattcaagaa taaagtctat
attaaatatt caaaaagata aatttaaata atatgaaagg 960aaacatattc
aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc
1020taataaagat acttgaaata gcttagttta aatataaata gcataataga
ttttaggaat 1080tagtattttg agtttaatta cttattgact tgtaacagtt
tttataattc caaggcccat 1140gaaaaattta atgctttatt agttttaaac
ttactatata aatttttcat atgtaaaatt 1200taatcggtat agttcgatat
tttttcaatt tatttttata aaataaaaaa cttaccctaa 1260ttatcggtac
agttatagat ttatataaaa atctacggtt cttcagaaga aacctaaaaa
1320tcggttcggt gcggacggtt cgatcggttt agtcgatttt caaatattca
ttgacactcc 1380tagttgttgt tataggtaaa aagcagttac agagaggtaa
aatataactt aaaaaatcag 1440ttctaaggaa aaattgactt ttatagtaaa
tgactgttat ataaggatgt tgttacagag 1500aggtatgagt gtagttggta
aattatgttc ttgacggtgt atgtcacata ttatttatta 1560aaactagaaa
aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa atcggctgaa
1620tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg
actgttgatg 1680atataaacaa agggcacatt ggtcaataac cataaaaaat
tatatgacag ctacagttgg 1740tagcatgtgc tcagctattg aacaaatcta
aagaaggtac atctgtaacc ggaacaccac 1800ttaaatgact aaattaccct
catcagaaag cagatggagt gctacaaata acacactatt 1860caacaaccat
aaataaaacg tgttcagcta ctaaaacaaa tataaataaa tctatgtttg
1920taagcactcc agccatgtta atggagtgct attgcctgtt aactctcact
tataaaatag 1980tagtagaaaa aatatgaacc aaaacacaac aacatctcaa
aatatttgaa gtaacacaga 2040attttacata caccaaactt ataaatcaag
tattttcatt gtaacaaatt ccatgaaaca 2100tgaaaacaaa gctataatga
aattaccaac tcaagcaata aggttggaaa agagccatct 2160gagatattcc
agcaatttac atctttttgt ttgattacac agtgaaggat cttttgtttg
2220acaactagta aaatgattct tatttgcacc tttcagctat tcagctgctt
ttactccaac 2280cctatagcag aagtaatggc gctcatgctc gttttgtacg
ccttccaact tcaagggcga 2340gctctgtatc gatcttcagg gaaatgtcaa
gtgctttcac ggaatgcgtc agggcaccac 2400tagaaatgta ggtaacacca
gtttgtccaa tcttgtgtac tgtttcaagg gtaacatttc 2460ctgaagcctc
cgtatcaaac ctcccattga tcaattctac agcctcctta agcatggata
2520catcaatatc tccgttagat aatggaacaa ccatattgtc cagcattatc
ctagtcaacg 2580aagtctttgt ttgagatgca tagtctagaa cctcacgtac
ttcttcaatt gtcctggttt 2640caacctcaac ccctatttga agtttatttt
gctccaaata ctgatccaca gattttagag 2700ctttgccgac acctccagca
gcagatatgt gattgtcttt tatcattacc atatcaaata 2760agcccattct
gtgattcttc cccccaccga tcaataccgc ccatttatcc accaaacgta
2820atccaggagc agttttccta gtctccaaga tgtaagcagg gtgtgcagca
tctgccattt 2880ccttagttag tgtagctatt ccactcattc tttgcataaa
attgagaaca accctctcag 2940ctataacaat gttgtaagcg tttccttgta
ctttgccaaa tttcaagcct ttatgaactt 3000tatcgccatc atttacatac
cactccacct ttaatgaagg atcaacttcc gcgaatatca 3060tctcagcaag
tgcaattcct gctatgatcc cgtcttcctt tgctagaaaa tgagcatcgg
3120attccatatc aagaggaatt gtcgccttac aagtcacatc tcctaaattc
ccagcatctt 3180cagagagtgc aagtttcata acttccttta aatcataagt
tgggtgtgct ggtggtttca 3240cctctaatga ctccactctt gtattcttgg
tggctattgc tgacattttc accaccaacc 3300ttggagctgt aattgcataa
ggatgcactg tagcagtgaa aggaatagct ctaaacatgg 3360tttttttttg
ggggggttgt gaaatgaatt ttgtggaaaa tagtttttgg ggcacatcaa
3420tcctgcggtg acattcggaa tgtttctaac aagaaagata tcgttggtcc
gagccttgct 3480ctacatcata gctcagtgca taggggccct gtgcgggtgc
gccttagtca agacattgca 3540gcgagatcat tacaaccact atggcggtgg
cgctaaccag ctcgttgatg gttatagccg 3600aggcactggc cttgctgttg
agattatggg cacctttatt cttctgtata ctgtcttctc 3660cgccactgat
cccaaacgca atgctagaga ttcccatgtt cctgtcttgg ctccactccc
3720cattggcttt gctgtcttca ttgttcacct cgccaccatt cccgtcaccg
gcactggcat 3780caacccagcg agcaaaaact attttccaca aaattcattt
cacaaccccc ccaaaaaaaa 3840accatgttta gagctattcc tttcactgct
acagtgcatc cttatgcaat tacagctcca 3900aggttggtgg tgaaaatgtc
agcaatagcc accaagaata caagagtgga gtcattagag 3960gtgaaaccac
cagcacaccc aacttatgat ttaaaggaag ttatgaaact tgcactctct
4020gaagatgctg ggaatttagg agatgtgact tgtaaggcga caattcctct
tgatatggaa 4080tccgatgctc attttctagc aaaggaagac gggatcatag
caggaattgc acttgctgag 4140atgatattcg cggaagttga tccttcatta
aaggtggagt ggtatgtaaa tgatggcgat 4200aaagttcata aaggcttgaa
atttggcaaa gtacaaggaa acgcttacaa cattgttata 4260gctgagaggg
ttgttctcaa ttttatgcaa agaatgagtg gaatagctac actaactaag
4320gaaatggcag atgctgcaca ccctgcttac atcttggaga ctaggaaaac
tgctcctgga 4380ttacgtttgg tggataaatg ggcggtattg atcggtgggg
ggaagaatca cagaatgggc 4440ttatttgata tggtaatgat aaaagacaat
cacatatctg ctgctggagg tgtcggcaaa 4500gctctaaaat ctgtggatca
gtatttggag caaaataaac ttcaaatagg ggttgaggtt 4560gaaaccagga
caattgaaga agtacgtgag gttctagact atgcatctca aacaaagact
4620tcgttgacta ggataatgct ggacaatatg gttgttccat tatctaacgg
agatattgat 4680gtatccatgc ttaaggaggc tgtagaattg atcaatggga
ggtttgatac ggaggcttca 4740ggaaatgtta cccttgaaac agtacacaag
attggacaaa ctggtgttac ctacatttct 4800agtggtgccc tgacgcattc
cgtgaaagca cttgacattt ccctgaagat cgatacagag 4860ctcgcccttg
aagttggaag gcgtacaaaa cgagcatgag cgccattact tctgctatag
4920ggttggagta aaagcagctg aatagctgaa aggtgcaaat aagaatcatt
ttactagttg 4980tcaaacaaaa gatccttcac tgtgtaatca aacaaaaaga
tgtaaattgc tggaatatct 5040cagatggctc ttttccaacc ttattgcttg
agttggtaat ttcattatag ctttgttttc 5100atgtttcatg gaatttgtta
caatgaaaat acttgattta taagtttggt gtatgtaaaa 5160ttctgtgtta
cttcaaatat tttgagatgt tgagctcgtg aaatggcctc tttagttttt
5220gattgaatca taggggtatt agttttctat ggccgggagt ggtcttcttg
cttaattgta 5280atggaataac cagagaggaa ctactgtgtt atctttgagg
aatgttgggc ttttttcgtt 5340tgaattatca tgaatgaaat tttacttttt
cccaatacaa gtttgttttc gtttcttggt 5400ttttgttatc ccttggttta
tgtcttggtt tggcttaaat gattgaagat tacactacct 5460atgtttctgc
tattcctgtt gaagatcaca tttgataata atgcatcgaa tgcattaaag
5520tttcttattg gctctgtcaa aagtattgaa ggtggatttt tctaattggc
aagagaaagt 5580attaaagagg tgatttatta gtacttatat ttttctcagc
atctctcttt cagtgttgga 5640gcttcataaa attagcactt cagagtttca
gtcgggagct gaattcga 5688244134DNAArtificial SequenceArtificially
created chimeric nucleic acid sequence 24ctcgaggatc taaattgtga
gttcaatctc ttccctattg gattgattat cctttctttt 60cttccaattt gtgtttcttt
ttgcctaatt tattgtgtta tcccctttat cctattttgt 120ttctttactt
atttatttgc ttctatgtct ttgtacaaag atttaaactc tatggcacat
180attttaaagt tgttagaaaa taaattcttt caagattgat gaaagaactt
tttaattgta 240gatatttcgt agattttatt ctcttactac caatataacg
cttgaattga cgaaaatttg 300tgtccaaata tctagcaaaa aggtatccaa
tgaaaatata tcatatgtga tcttcaaatc 360ttgtgtctta tgcaagattg
atactttgtt caatggaaga gattgtgtgc atatttttaa 420aatttttatt
agtaataaag attctatata gctgttatag agggataatt ttacaaagaa
480cactataaat atgattgttg ttgttagggt gtcaatggtt cggttcgact
ggttatttta 540taaaatttgt accataccat ttttttcgat attctatttt
gtataaccaa aattagactt 600ttcgaaatcg tcccaatcat gtcggtttca
cttcggtatc ggtaccgttc ggttaatttt 660catttttttt taaatgtcat
taaaattcac tagtaaaaat agaatgcaat aacatacgtt 720cttttatagg
acttagcaaa agctctctag acatttttac tgtttaaagg ataatgaatt
780aaaaaacatg aaagatggct agagtataga tacacaacta ttcgacagca
acgtaaaaga 840aaccaagtaa aagcaaagaa aatataaatc acacgagtgg
aaagatatta accaagttgg 900gattcaagaa taaagtctat attaaatatt
caaaaagata aatttaaata atatgaaagg 960aaacatattc aatacattgt
agtttgctac tcataatcgc tagaatactt tgtgccttgc 1020taataaagat
acttgaaata gcttagttta aatataaata gcataataga ttttaggaat
1080tagtattttg agtttaatta cttattgact tgtaacagtt tttataattc
caaggcccat 1140gaaaaattta atgctttatt agttttaaac ttactatata
aatttttcat atgtaaaatt 1200taatcggtat agttcgatat tttttcaatt
tatttttata aaataaaaaa cttaccctaa 1260ttatcggtac agttatagat
ttatataaaa atctacggtt cttcagaaga aacctaaaaa 1320tcggttcggt
gcggacggtt cgatcggttt agtcgatttt caaatattca ttgacactcc
1380tagttgttgt tataggtaaa aagcagttac agagaggtaa aatataactt
aaaaaatcag 1440ttctaaggaa aaattgactt ttatagtaaa tgactgttat
ataaggatgt tgttacagag 1500aggtatgagt gtagttggta aattatgttc
ttgacggtgt atgtcacata ttatttatta 1560aaactagaaa aaacagcgtc
aaaactagca aaaatccaac ggacaaaaaa atcggctgaa 1620tttgatttgg
ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg actgttgatg
1680atataaacaa agggcacatt ggtcaataac cataaaaaat tatatgacag
ctacagttgg 1740tagcatgtgc tcagctattg aacaaatcta aagaaggtac
atctgtaacc ggaacaccac 1800ttaaatgact aaattaccct catcagaaag
cagatggagt gctacaaata acacactatt 1860caacaaccat aaataaaacg
tgttcagcta ctaaaacaaa tataaataaa tctatgtttg 1920taagcactcc
agccatgtta atggagtgct attgcctgtt aactctcact tataaaatag
1980tagtagaaaa aatatgaacc aaaacacaac tttatcgcca tcatttacat
accactccac 2040ctttaatgaa ggatcaactt ccgcgaatat catctcagca
agtgcaattc ctgctatgat 2100cccgtcttcc tttgctagaa aatgagcatc
ggattccata tcaagaggaa ttgtcgcctt 2160acaagtcaca tctcctaaat
tcccagcatc ttcagagagt gcaagtttca taacttcctt 2220taaatcataa
gttgggtgtg ctggtggttt cacctctaat gactccactc ttgtattctt
2280ggtggctatt gctgacattt tcaccaccaa ccttggagct gtaattgcat
aaggatgcac 2340tgtagcagtg aaaggaatag ctctaaacat gtccgtcgct
tctcttccat ttcttctcat 2400tttcgatttt gattcttatt tctttccagt
agctcctgct ctgtgaattt ctccgctcac 2460gatagatctg cttatactcc
ttacattcaa ccttagatct ggtctcgatt ctctgtttct 2520ctgttttttt
cttttggtcg agaatctgat gtttgtttat
gttctgtcac cattaataat 2580aatgaactct ctcattcata caatgattag
tttctctcgt ctacaaaacg atatgttgca 2640ttttcacttt tcttcttttt
ttctaagatg atttgctttg accaatttgt ttagatcttt 2700attttatttt
attttctggt gggttggtgg aaattgaaaa aaaaaaaaac agcataaatt
2760gttatttgtt aatgtattca ttttttggct atttgttctg ggtaaaaatc
tgcttctact 2820attgaatctt tcctggattt tttactccta ttgggttttt
atagtaaaaa tacataataa 2880aaggaaaaca aaagttttat agattctctt
aaacccctta cgataaaagt tggaatcaaa 2940ataattcagg atcagatgct
ctttgattga ttcagatgcg attacagttg catggcaaat 3000tttctagatc
cgtcgtcaca ttttattttc tgtttaaata tctaaatctg atatatgatg
3060tcgacaaatt ctggtggctt atacatcact tcaactgttt tcttttggct
ttgtttgtca 3120acttggtttt caatacgatt tgtgatttcg atcgctgaat
ttttaataca agcaaactga 3180tgttaaccac aagcaagaga tgtgacctgc
cttattaaca tcgtattact tactactagt 3240cgtattctca acgcaatcgt
ttttgtattt ctcacattat gccgcttctc tactctttat 3300tccttttggt
ccacgcattt tctatttgtg gcaatccctt tcacaacctg atttcccact
3360ttggatcatt tgtctgaaga ctctcttgaa tcgttaccac ttgtttcttg
tgcatgctct 3420gttttttaga attaatgata aaactattcc atagtcttga
gttttcagct tgttgattct 3480tttgcttttg gttttctgca gatgtttaga
gctattcctt tcactgctac agtgcatcct 3540tatgcaatta cagctccaag
gttggtggtg aaaatgtcag caatagccac caagaataca 3600agagtggagt
cattagaggt gaaaccacca gcacacccaa cttatgattt aaaggaagtt
3660atgaaacttg cactctctga agatgctggg aatttaggag atgtgacttg
taaggcgaca 3720attcctcttg atatggaatc cgatgctcat tttctagcaa
aggaagacgg gatcatagca 3780ggaattgcac ttgctgagat gatattcgcg
gaagttgatc cttcattaaa ggtggagtgg 3840tatgtaaatg atggcgataa
agcaagtgtg ttgcctttgt gtggaaatga agaggtactt 3900gcgaggactt
tgcgtttatc agtttatgtg tttgtatatc tatttgatcc agttattatg
3960gattatatac gcttgaaact cattttaagc cattgttatt gaacgtttat
caaatacttt 4020attatgccaa gcaagtcaaa cacatgcttg ttgattgaaa
tcaagctata gaaatctctt 4080cttcacatac agcagtttag attcacaata
caacaagcga aacgataaag tttc 4134253896DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
25ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat cctttctttt
60cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat cctattttgt
120ttctttactt atttatttgc ttctatgtct ttgtacaaag atttaaactc
tatggcacat 180attttaaagt tgttagaaaa taaattcttt caagattgat
gaaagaactt tttaattgta 240gatatttcgt agattttatt ctcttactac
caatataacg cttgaattga cgaaaatttg 300tgtccaaata tctagcaaaa
aggtatccaa tgaaaatata tcatatgtga tcttcaaatc 360ttgtgtctta
tgcaagattg atactttgtt caatggaaga gattgtgtgc atatttttaa
420aatttttatt agtaataaag attctatata gctgttatag agggataatt
ttacaaagaa 480cactataaat atgattgttg ttgttagggt gtcaatggtt
cggttcgact ggttatttta 540taaaatttgt accataccat ttttttcgat
attctatttt gtataaccaa aattagactt 600ttcgaaatcg tcccaatcat
gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660catttttttt
taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt
720cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg
ataatgaatt 780aaaaaacatg aaagatggct agagtataga tacacaacta
ttcgacagca acgtaaaaga 840aaccaagtaa aagcaaagaa aatataaatc
acacgagtgg aaagatatta accaagttgg 900gattcaagaa taaagtctat
attaaatatt caaaaagata aatttaaata atatgaaagg 960aaacatattc
aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc
1020taataaagat acttgaaata gcttagttta aatataaata gcataataga
ttttaggaat 1080tagtattttg agtttaatta cttattgact tgtaacagtt
tttataattc caaggcccat 1140gaaaaattta atgctttatt agttttaaac
ttactatata aatttttcat atgtaaaatt 1200taatcggtat agttcgatat
tttttcaatt tatttttata aaataaaaaa cttaccctaa 1260ttatcggtac
agttatagat ttatataaaa atctacggtt cttcagaaga aacctaaaaa
1320tcggttcggt gcggacggtt cgatcggttt agtcgatttt caaatattca
ttgacactcc 1380tagttgttgt tataggtaaa aagcagttac agagaggtaa
aatataactt aaaaaatcag 1440ttctaaggaa aaattgactt ttatagtaaa
tgactgttat ataaggatgt tgttacagag 1500aggtatgagt gtagttggta
aattatgttc ttgacggtgt atgtcacata ttatttatta 1560aaactagaaa
aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa atcggctgaa
1620tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg
actgttgatg 1680atataaacaa agggcacatt ggtcaataac cataaaaaat
tatatgacag ctacagttgg 1740tagcatgtgc tcagctattg aacaaatcta
aagaaggtac atctgtaacc ggaacaccac 1800ttaaatgact aaattaccct
catcagaaag cagatggagt gctacaaata acacactatt 1860caacaaccat
aaataaaacg tgttcagcta ctaaaacaaa tataaataaa tctatgtttg
1920taagcactcc agccatgtta atggagtgct attgcctgtt aactctcact
tataaaatag 1980tagtagaaaa aatatgaacc aaaacacaac cgattcaaaa
cttcagcagg aaaatttata 2040ttaaagtacg aaacaccaca gagaaagaat
tttacaatga ttgtgaattg gaaattatga 2100taattactgc agaattctcc
tacaatacgt cgcatttcac ttatttattc acttgattaa 2160gactcgatct
tggtccaatg attgtactac cagtgttcat tattcacttg attaagactc
2220gatcatactt ctggcgaaag atggtaaaat ggtccgtcgc ttctcttcca
tttcttctca 2280ttttcgattt tgattcttat ttctttccag tagctcctgc
tctgtgaatt tctccgctca 2340cgatagatct gcttatactc cttacattca
accttagatc tggtctcgat tctctgtttc 2400tctgtttttt tcttttggtc
gagaatctga tgtttgttta tgttctgtca ccattaataa 2460taatgaactc
tctcattcat acaatgatta gtttctctcg tctacaaaac gatatgttgc
2520attttcactt ttcttctttt tttctaagat gatttgcttt gaccaatttg
tttagatctt 2580tattttattt tattttctgg tgggttggtg gaaattgaaa
aaaaaaaaaa cagcataaat 2640tgttatttgt taatgtattc attttttggc
tatttgttct gggtaaaaat ctgcttctac 2700tattgaatct ttcctggatt
ttttactcct attgggtttt tatagtaaaa atacataata 2760aaaggaaaac
aaaagtttta tagattctct taaacccctt acgataaaag ttggaatcaa
2820aataattcag gatcagatgc tctttgattg attcagatgc gattacagtt
gcatggcaaa 2880ttttctagat ccgtcgtcac attttatttt ctgtttaaat
atctaaatct gatatatgat 2940gtcgacaaat tctggtggct tatacatcac
ttcaactgtt ttcttttggc tttgtttgtc 3000aacttggttt tcaatacgat
ttgtgatttc gatcgctgaa tttttaatac aagcaaactg 3060atgttaacca
caagcaagag atgtgacctg ccttattaac atcgtattac ttactactag
3120tcgtattctc aacgcaatcg tttttgtatt tctcacatta tgccgcttct
ctactcttta 3180ttccttttgg tccacgcatt ttctatttgt ggcaatccct
ttcacaacct gatttcccac 3240tttggatcat ttgtctgaag actctcttga
atcgttacca cttgtttctt gtgcatgctc 3300tgttttttag aattaatgat
aaaactattc catagtcttg agttttcagc ttgttgattc 3360ttttgctttt
ggttttctgc agcattttac catctttcgc cagaagtatg atcgagtctt
3420aatcaagtga ataatgaaca ctggtagtac aatcattgga ccaagatcga
gtcttaatca 3480agtgaataaa taagtgaaat gcgacgtatt gtaggagaat
tctgcagtaa ttatcataat 3540ttccaattca caatcattgt aaaattcttt
ctctgtggtg tttcgtactt taatataaat 3600tttcctgctg aagttttgaa
tcggcaagtg tgttgccttt gtgtggaaat gaagaggtac 3660ttgcgaggac
tttgcgttta tcagtttatg tgtttgtata tctatttgat ccagttatta
3720tggattatat acgcttgaaa ctcattttaa gccattgtta ttgaacgttt
atcaaatact 3780ttattatgcc aagcaagtca aacacatgct tgttgattga
aatcaagcta tagaaatctc 3840ttcttcacat acagcagttt agattcacaa
tacaacaagc gaaacgataa agtttc 3896264670DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
26ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat cctttctttt
60cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat cctattttgt
120ttctttactt atttatttgc ttctatgtct ttgtacaaag atttaaactc
tatggcacat 180attttaaagt tgttagaaaa taaattcttt caagattgat
gaaagaactt tttaattgta 240gatatttcgt agattttatt ctcttactac
caatataacg cttgaattga cgaaaatttg 300tgtccaaata tctagcaaaa
aggtatccaa tgaaaatata tcatatgtga tcttcaaatc 360ttgtgtctta
tgcaagattg atactttgtt caatggaaga gattgtgtgc atatttttaa
420aatttttatt agtaataaag attctatata gctgttatag agggataatt
ttacaaagaa 480cactataaat atgattgttg ttgttagggt gtcaatggtt
cggttcgact ggttatttta 540taaaatttgt accataccat ttttttcgat
attctatttt gtataaccaa aattagactt 600ttcgaaatcg tcccaatcat
gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660catttttttt
taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt
720cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg
ataatgaatt 780aaaaaacatg aaagatggct agagtataga tacacaacta
ttcgacagca acgtaaaaga 840aaccaagtaa aagcaaagaa aatataaatc
acacgagtgg aaagatatta accaagttgg 900gattcaagaa taaagtctat
attaaatatt caaaaagata aatttaaata atatgaaagg 960aaacatattc
aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc
1020taataaagat acttgaaata gcttagttta aatataaata gcataataga
ttttaggaat 1080tagtattttg agtttaatta cttattgact tgtaacagtt
tttataattc caaggcccat 1140gaaaaattta atgctttatt agttttaaac
ttactatata aatttttcat atgtaaaatt 1200taatcggtat agttcgatat
tttttcaatt tatttttata aaataaaaaa cttaccctaa 1260ttatcggtac
agttatagat ttatataaaa atctacggtt cttcagaaga aacctaaaaa
1320tcggttcggt gcggacggtt cgatcggttt agtcgatttt caaatattca
ttgacactcc 1380tagttgttgt tataggtaaa aagcagttac agagaggtaa
aatataactt aaaaaatcag 1440ttctaaggaa aaattgactt ttatagtaaa
tgactgttat ataaggatgt tgttacagag 1500aggtatgagt gtagttggta
aattatgttc ttgacggtgt atgtcacata ttatttatta 1560aaactagaaa
aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa atcggctgaa
1620tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg
actgttgatg 1680atataaacaa agggcacatt ggtcaataac cataaaaaat
tatatgacag ctacagttgg 1740tagcatgtgc tcagctattg aacaaatcta
aagaaggtac atctgtaacc ggaacaccac 1800ttaaatgact aaattaccct
catcagaaag cagatggagt gctacaaata acacactatt 1860caacaaccat
aaataaaacg tgttcagcta ctaaaacaaa tataaataaa tctatgtttg
1920taagcactcc agccatgtta atggagtgct attgcctgtt aactctcact
tataaaatag 1980tagtagaaaa aatatgaacc aaaacacaac ggttgtgtat
ttcacttttg gatatagctc 2040agtggcttcg acacctgtag gaggctgaac
ctcaaagttt gcagaatctc cattaacaaa 2100aactgaatgg catatggcca
aattagtcct taatggcaga ggtccctctt gtacaatctg 2160gagaatatct
tcctctgata gatataactt ctcgagggtc ttcccaattt tgtcctccca
2220caaggacact atctcgttga aggatagaat attggcaggt ggtctcatgt
gaagagtctt 2280attcaatgtc cgtggatcat ctactgcttc gatagtgtat
gtcgctatgt cttcttcctt 2340cacatatatt gctttgggat ttccatcgcc
aaaaatgaca actttgtctc taggaggggt 2400tttggcctct aactgcccca
agttgggcaa gaagaaatct gcaaaccaat tgcagattac 2460atatgtgtat
ggaattcctt ctgcctctat catcctcctg attcttacct ttagagcgaa
2520gagtgatgca gctggttcaa ttgcacgagc atgatccaca tcaaatccaa
attctgaagg 2580aagaaatctc ttgatatttc cagcttcttt aattgctttg
atgatgttca cttgatcagt 2640ccgtcgcttc tcttccattt cttctcattt
tcgattttga ttcttatttc tttccagtag 2700ctcctgctct gtgaatttct
ccgctcacga tagatctgct tatactcctt acattcaacc 2760ttagatctgg
tctcgattct ctgtttctct gtttttttct tttggtcgag aatctgatgt
2820ttgtttatgt tctgtcacca ttaataataa tgaactctct cattcataca
atgattagtt 2880tctctcgtct acaaaacgat atgttgcatt ttcacttttc
ttcttttttt ctaagatgat 2940ttgctttgac caatttgttt agatctttat
tttattttat tttctggtgg gttggtggaa 3000attgaaaaaa aaaaaaacag
cataaattgt tatttgttaa tgtattcatt ttttggctat 3060ttgttctggg
taaaaatctg cttctactat tgaatctttc ctggattttt tactcctatt
3120gggtttttat agtaaaaata cataataaaa ggaaaacaaa agttttatag
attctcttaa 3180accccttacg ataaaagttg gaatcaaaat aattcaggat
cagatgctct ttgattgatt 3240cagatgcgat tacagttgca tggcaaattt
tctagatccg tcgtcacatt ttattttctg 3300tttaaatatc taaatctgat
atatgatgtc gacaaattct ggtggcttat acatcacttc 3360aactgttttc
ttttggcttt gtttgtcaac ttggttttca atacgatttg tgatttcgat
3420cgctgaattt ttaatacaag caaactgatg ttaaccacaa gcaagagatg
tgacctgcct 3480tattaacatc gtattactta ctactagtcg tattctcaac
gcaatcgttt ttgtatttct 3540cacattatgc cgcttctcta ctctttattc
cttttggtcc acgcattttc tatttgtggc 3600aatccctttc acaacctgat
ttcccacttt ggatcatttg tctgaagact ctcttgaatc 3660gttaccactt
gtttcttgtg catgctctgt tttttagaat taatgataaa actattccat
3720agtcttgagt tttcagcttg ttgattcttt tgcttttggt tttctgcagt
gatcaagtga 3780acatcatcaa agcaattaaa gaagctggaa atatcaagag
atttcttcct tcagaatttg 3840gatttgatgt ggatcatgct cgtgcaattg
aaccagctgc atcactcttc gctctaaagg 3900taagaatcag gaggatgata
gaggcagaag gaattccata cacatatgta atctgcaatt 3960ggtttgcaga
tttcttcttg cccaacttgg ggcagttaga ggccaaaacc cctcctagag
4020acaaagttgt catttttggc gatggaaatc ccaaagcaat atatgtgaag
gaagaagaca 4080tagcgacata cactatcgaa gcagtagatg atccacggac
attgaataag actcttcaca 4140tgagaccacc tgccaatatt ctatccttca
acgagatagt gtccttgtgg gaggacaaaa 4200ttgggaagac cctcgagaag
ttatatctat cagaggaaga tattctccag attgtacaag 4260agggacctct
gccattaagg actaatttgg ccatatgcca ttcagttttt gttaatggag
4320attctgcaaa ctttgaggtt cagcctccta caggtgtcga agccactgag
ctatatccaa 4380aagtgaaata cacaaccgca agtgtgttgc ctttgtgtgg
aaatgaagag gtacttgcga 4440ggactttgcg tttatcagtt tatgtgtttg
tatatctatt tgatccagtt attatggatt 4500atatacgctt gaaactcatt
ttaagccatt gttattgaac gtttatcaaa tactttatta 4560tgccaagcaa
gtcaaacaca tgcttgttga ttgaaatcaa gctatagaaa tctcttcttc
4620acatacagca gtttagattc acaatacaac aagcgaaacg ataaagtttc
4670275390DNAArtificial SequenceArtificially created chimeric
nucleic acid sequence 27ctcgaggatc taaattgtga gttcaatctc ttccctattg
gattgattat cctttctttt 60cttccaattt gtgtttcttt ttgcctaatt tattgtgtta
tcccctttat cctattttgt 120ttctttactt atttatttgc ttctatgtct
ttgtacaaag atttaaactc tatggcacat 180attttaaagt tgttagaaaa
taaattcttt caagattgat gaaagaactt tttaattgta 240gatatttcgt
agattttatt ctcttactac caatataacg cttgaattga cgaaaatttg
300tgtccaaata tctagcaaaa aggtatccaa tgaaaatata tcatatgtga
tcttcaaatc 360ttgtgtctta tgcaagattg atactttgtt caatggaaga
gattgtgtgc atatttttaa 420aatttttatt agtaataaag attctatata
gctgttatag agggataatt ttacaaagaa 480cactataaat atgattgttg
ttgttagggt gtcaatggtt cggttcgact ggttatttta 540taaaatttgt
accataccat ttttttcgat attctatttt gtataaccaa aattagactt
600ttcgaaatcg tcccaatcat gtcggtttca cttcggtatc ggtaccgttc
ggttaatttt 660catttttttt taaatgtcat taaaattcac tagtaaaaat
agaatgcaat aacatacgtt 720cttttatagg acttagcaaa agctctctag
acatttttac tgtttaaagg ataatgaatt 780aaaaaacatg aaagatggct
agagtataga tacacaacta ttcgacagca acgtaaaaga 840aaccaagtaa
aagcaaagaa aatataaatc acacgagtgg aaagatatta accaagttgg
900gattcaagaa taaagtctat attaaatatt caaaaagata aatttaaata
atatgaaagg 960aaacatattc aatacattgt agtttgctac tcataatcgc
tagaatactt tgtgccttgc 1020taataaagat acttgaaata gcttagttta
aatataaata gcataataga ttttaggaat 1080tagtattttg agtttaatta
cttattgact tgtaacagtt tttataattc caaggcccat 1140gaaaaattta
atgctttatt agttttaaac ttactatata aatttttcat atgtaaaatt
1200taatcggtat agttcgatat tttttcaatt tatttttata aaataaaaaa
cttaccctaa 1260ttatcggtac agttatagat ttatataaaa atctacggtt
cttcagaaga aacctaaaaa 1320tcggttcggt gcggacggtt cgatcggttt
agtcgatttt caaatattca ttgacactcc 1380tagttgttgt tataggtaaa
aagcagttac agagaggtaa aatataactt aaaaaatcag 1440ttctaaggaa
aaattgactt ttatagtaaa tgactgttat ataaggatgt tgttacagag
1500aggtatgagt gtagttggta aattatgttc ttgacggtgt atgtcacata
ttatttatta 1560aaactagaaa aaacagcgtc aaaactagca aaaatccaac
ggacaaaaaa atcggctgaa 1620tttgatttgg ttccaacatt taaaaaagtt
tcagtgagaa agaatcggtg actgttgatg 1680atataaacaa agggcacatt
ggtcaataac cataaaaaat tatatgacag ctacagttgg 1740tagcatgtgc
tcagctattg aacaaatcta aagaaggtac atctgtaacc ggaacaccac
1800ttaaatgact aaattaccct catcagaaag cagatggagt gctacaaata
acacactatt 1860caacaaccat aaataaaacg tgttcagcta ctaaaacaaa
tataaataaa tctatgtttg 1920taagcactcc agccatgtta atggagtgct
attgcctgtt aactctcact tataaaatag 1980tagtagaaaa aatatgaacc
aaaacacaac tttatcgcca tcatttacat accactccac 2040ctttaatgaa
ggatcaactt ccgcgaatat catctcagca agtgcaattc ctgctatgat
2100cccgtcttcc tttgctagaa aatgagcatc ggattccata tcaagaggaa
ttgtcgcctt 2160acaagtcaca tctcctaaat tcccagcatc ttcagagagt
gcaagtttca taacttcctt 2220taaatcataa gttgggtgtg ctggtggttt
cacctctaat gactccactc ttgtattctt 2280ggtggctatt gctgacattt
tcaccaccaa ccttggagct gtaattgcat aaggatgcac 2340tgtagcagtg
aaaggaatag ctctaaacat ggttgtgtat ttcacttttg gatatagctc
2400agtggcttcg acacctgtag gaggctgaac ctcaaagttt gcagaatctc
cattaacaaa 2460aactgaatgg catatggcca aattagtcct taatggcaga
ggtccctctt gtacaatctg 2520gagaatatct tcctctgata gatataactt
ctcgagggtc ttcccaattt tgtcctccca 2580caaggacact atctcgttga
aggatagaat attggcaggt ggtctcatgt gaagagtctt 2640attcaatgtc
cgtggatcat ctactgcttc gatagtgtat gtcgctatgt cttcttcctt
2700cacatatatt gctttgggat ttccatcgcc aaaaatgaca actttgtctc
taggaggggt 2760tttggcctct aactgcccca agttgggcaa gaagaaatct
gcaaaccaat tgcagattac 2820atatgtgtat ggaattcctt ctgcctctat
catcctcctg attcttacct ttagagcgaa 2880gagtgatgca gctggttcaa
ttgcacgagc atgatccaca tcaaatccaa attctgaagg 2940aagaaatctc
ttgatatttc cagcttcttt aattgctttg atgatgttca cttgatcagt
3000ccgtcgcttc tcttccattt cttctcattt tcgattttga ttcttatttc
tttccagtag 3060ctcctgctct gtgaatttct ccgctcacga tagatctgct
tatactcctt acattcaacc 3120ttagatctgg tctcgattct ctgtttctct
gtttttttct tttggtcgag aatctgatgt 3180ttgtttatgt tctgtcacca
ttaataataa tgaactctct cattcataca atgattagtt 3240tctctcgtct
acaaaacgat atgttgcatt ttcacttttc ttcttttttt ctaagatgat
3300ttgctttgac caatttgttt agatctttat tttattttat tttctggtgg
gttggtggaa 3360attgaaaaaa aaaaaaacag cataaattgt tatttgttaa
tgtattcatt ttttggctat 3420ttgttctggg taaaaatctg cttctactat
tgaatctttc ctggattttt tactcctatt 3480gggtttttat agtaaaaata
cataataaaa ggaaaacaaa agttttatag attctcttaa 3540accccttacg
ataaaagttg gaatcaaaat aattcaggat cagatgctct ttgattgatt
3600cagatgcgat tacagttgca tggcaaattt tctagatccg tcgtcacatt
ttattttctg 3660tttaaatatc taaatctgat atatgatgtc gacaaattct
ggtggcttat acatcacttc 3720aactgttttc ttttggcttt gtttgtcaac
ttggttttca atacgatttg tgatttcgat 3780cgctgaattt ttaatacaag
caaactgatg ttaaccacaa gcaagagatg tgacctgcct 3840tattaacatc
gtattactta ctactagtcg tattctcaac gcaatcgttt ttgtatttct
3900cacattatgc cgcttctcta ctctttattc cttttggtcc acgcattttc
tatttgtggc 3960aatccctttc acaacctgat ttcccacttt ggatcatttg
tctgaagact ctcttgaatc 4020gttaccactt gtttcttgtg catgctctgt
tttttagaat taatgataaa actattccat 4080agtcttgagt tttcagcttg
ttgattcttt tgcttttggt tttctgcagt gatcaagtga 4140acatcatcaa
agcaattaaa gaagctggaa atatcaagag atttcttcct tcagaatttg
4200gatttgatgt ggatcatgct cgtgcaattg aaccagctgc atcactcttc
gctctaaagg 4260taagaatcag gaggatgata gaggcagaag gaattccata
cacatatgta atctgcaatt 4320ggtttgcaga tttcttcttg cccaacttgg
ggcagttaga ggccaaaacc cctcctagag 4380acaaagttgt catttttggc
gatggaaatc ccaaagcaat atatgtgaag gaagaagaca 4440tagcgacata
cactatcgaa gcagtagatg atccacggac attgaataag actcttcaca
4500tgagaccacc tgccaatatt ctatccttca acgagatagt gtccttgtgg
gaggacaaaa 4560ttgggaagac cctcgagaag ttatatctat cagaggaaga
tattctccag attgtacaag 4620agggacctct gccattaagg actaatttgg
ccatatgcca
ttcagttttt gttaatggag 4680attctgcaaa ctttgaggtt cagcctccta
caggtgtcga agccactgag ctatatccaa 4740aagtgaaata cacaaccatg
tttagagcta ttcctttcac tgctacagtg catccttatg 4800caattacagc
tccaaggttg gtggtgaaaa tgtcagcaat agccaccaag aatacaagag
4860tggagtcatt agaggtgaaa ccaccagcac acccaactta tgatttaaag
gaagttatga 4920aacttgcact ctctgaagat gctgggaatt taggagatgt
gacttgtaag gcgacaattc 4980ctcttgatat ggaatccgat gctcattttc
tagcaaagga agacgggatc atagcaggaa 5040ttgcacttgc tgagatgata
ttcgcggaag ttgatccttc attaaaggtg gagtggtatg 5100taaatgatgg
cgataaagca agtgtgttgc ctttgtgtgg aaatgaagag gtacttgcga
5160ggactttgcg tttatcagtt tatgtgtttg tatatctatt tgatccagtt
attatggatt 5220atatacgctt gaaactcatt ttaagccatt gttattgaac
gtttatcaaa tactttatta 5280tgccaagcaa gtcaaacaca tgcttgttga
ttgaaatcaa gctatagaaa tctcttcttc 5340acatacagca gtttagattc
acaatacaac aagcgaaacg ataaagtttc 5390283773DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
28gacggtccga tgtgagactt ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca
60ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa
120atgccatcat tgcgataaag gaaaggccat cgttgaagat gcctctgccg
acagtggtcc 180caaagatgga cccccaccca cgaggagcat cgtggaaaaa
gaagacgttc caaccacgtc 240ttcaaagcaa gtggattgat gtgatggtcc
gatgtgagac ttttcaacaa agggtaatat 300ccggaaacct cctcggattc
cattgcccag ctatctgtca ctttattgtg aagatagtgg 360aaaaggaagg
tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag
420atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc
atcgtggaaa 480aagaagacgt tccaaccacg tcttcaaagc aagtggattg
atgtgatatc tccactgacg 540taagggatga cgcacaatcc cactatcctt
cgcaagaccc ttcctctata taaggaagtt 600catttcattt ggagaggaat
catactcttt tccttccctg gttttaacag tgaaatcact 660atgcgaacta
cgtagaagca ttatcagtgg aggatggagt ctcagggctt cctttatgca
720tctatagagg acttccatct cggaaaggat gtcatgatcg accttattcc
catgtttctt 780catcagattc ttctgttcat ctacgacggc agacatgtac
ttgttgttgc agagaaggta 840tgcccctgcc caagtggagg tgatggaact
ggtgtgttgc ccagcgaaaa gagcagcaat 900cagaagacct gtgatctcag
actctgtcgt tgcccgccca tctttgtact tggagtcaat 960gaagcattgt
aacatatcgc tctccgcctt gcctgtacgt tttctagaat ctatgatgtt
1020tgcaaagatc tccgcgagct tcttgcgggc attgtcacga cggcgatggg
ctggaatggg 1080aaggtaggga aagattacac tgataggaag catcccattg
tccaggtcat ggaagagagc 1140agagacatcc tcaaagagtt tattgcgaac
ctcttctccc aacagacatc tactagctgt 1200cagtatgata agatgctcca
gttcatactt caagtccact tcaccactat caccccattt 1260tgagaagtac
tcctcagctt ccatgaccat ctgatccaca tatcccttca atttatttac
1320cctcaaagat tcagtaaaga acctaaattg ctcttgtctg atagtataat
caacgtcaaa 1380aaccacacca gggccaaaag taggcacatt gaactgataa
acctcttgtt gactgagatc 1440ggtttctggg gccttaaaga aatgggccga
cacttctggg ccaacgaaga acgtgatatt 1500cttgtccgtc gcttctcttc
catttcttct cattttcgat tttgattctt atttctttcc 1560agtagctcct
gctctgtgaa tttctccgct cacgatagat ctgcttatac tccttacatt
1620caaccttaga tctggtctcg attctctgtt tctctgtttt tttcttttgg
tcgagaatct 1680gatgtttgtt tatgttctgt caccattaat aataatgaac
tctctcattc atacaatgat 1740tagtttctct cgtctacaaa acgatatgtt
gcattttcac ttttcttctt tttttctaag 1800atgatttgct ttgaccaatt
tgtttagatc tttattttat tttattttct ggtgggttgg 1860tggaaattga
aaaaaaaaaa aacagcataa attgttattt gttaatgtat tcattttttg
1920gctatttgtt ctgggtaaaa atctgcttct actattgaat ctttcctgga
ttttttactc 1980ctattgggtt tttatagtaa aaatacataa taaaaggaaa
acaaaagttt tatagattct 2040cttaaacccc ttacgataaa agttggaatc
aaaataattc aggatcagat gctctttgat 2100tgattcagat gcgattacag
ttgcatggca aattttctag atccgtcgtc acattttatt 2160ttctgtttaa
atatctaaat ctgatatatg atgtcgacaa attctggtgg cttatacatc
2220acttcaactg ttttcttttg gctttgtttg tcaacttggt tttcaatacg
atttgtgatt 2280tcgatcgctg aatttttaat acaagcaaac tgatgttaac
cacaagcaag agatgtgacc 2340tgccttatta acatcgtatt acttactact
agtcgtattc tcaacgcaat cgtttttgta 2400tttctcacat tatgccgctt
ctctactctt tattcctttt ggtccacgca ttttctattt 2460gtggcaatcc
ctttcacaac ctgatttccc actttggatc atttgtctga agactctctt
2520gaatcgttac cacttgtttc ttgtgcatgc tctgtttttt agaattaatg
ataaaactat 2580tccatagtct tgagttttca gcttgttgat tcttttgctt
ttggttttct gcagaagaat 2640atcacgttct tcgttggccc agaagtgtcg
gcccatttct ttaaggcccc agaaaccgat 2700ctcagtcaac aagaggttta
tcagttcaat gtgcctactt ttggccctgg tgtggttttt 2760gacgttgatt
atactatcag acaagagcaa tttaggttct ttactgaatc tttgagggta
2820aataaattga agggatatgt ggatcagatg gtcatggaag ctgaggagta
cttctcaaaa 2880tggggtgata gtggtgaagt ggacttgaag tatgaactgg
agcatcttat catactgaca 2940gctagtagat gtctgttggg agaagaggtt
cgcaataaac tctttgagga tgtctctgct 3000ctcttccatg acctggacaa
tgggatgctt cctatcagtg taatctttcc ctaccttccc 3060attccagccc
atcgccgtcg tgacaatgcc cgcaagaagc tcgcggagat ctttgcaaac
3120atcatagatt ctagaaaacg tacaggcaag gcggagagcg atatgttaca
atgcttcatt 3180gactccaagt acaaagatgg gcgggcaacg acagagtctg
agatcacagg tcttctgatt 3240gctgctcttt tcgctgggca acacaccagt
tccatcacct ccacttgggc aggggcatac 3300cttctctgca acaacaagta
catgtctgcc gtcgtagatg aacagaagaa tctgatgaag 3360aaacatggga
ataaggtcga tcatgacatc ctttccgaga tggaagtcct ctatagatgc
3420ataaaggaag ccctgagact ccatcctcca ctgataatgc ttctacgtag
ttcgcatagt 3480gatttcactg ttaaaaccag ggaaggaaaa gagtatgatg
atcgttcaaa catttggcaa 3540taaagtttct taagattgaa tcctgttgcc
ggtcttgcga tgattatcat ataatttctg 3600ttgaattacg ttaagcatgt
aataattaac atgtaatgca tgacgttatt tatgagatgg 3660gtttttatga
ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag
3720cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcg
3773293563DNAArtificial SequenceArtificially created chimeric
nucleic acid sequence 29gacggtccga tgtgagactt ttcaacaaag ggtaatatcc
ggaaacctcc tcggattcca 60ttgcccagct atctgtcact ttattgtgaa gatagtggaa
aaggaaggtg gctcctacaa 120atgccatcat tgcgataaag gaaaggccat
cgttgaagat gcctctgccg acagtggtcc 180caaagatgga cccccaccca
cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 240ttcaaagcaa
gtggattgat gtgatggtcc gatgtgagac ttttcaacaa agggtaatat
300ccggaaacct cctcggattc cattgcccag ctatctgtca ctttattgtg
aagatagtgg 360aaaaggaagg tggctcctac aaatgccatc attgcgataa
aggaaaggcc atcgttgaag 420atgcctctgc cgacagtggt cccaaagatg
gacccccacc cacgaggagc atcgtggaaa 480aagaagacgt tccaaccacg
tcttcaaagc aagtggattg atgtgatatc tccactgacg 540taagggatga
cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt
600catttcattt ggagaggacc atatattagc aaatgcccac ctttatgagc
ttgtcaatag 660gtatatatct tagaacaagg acatcaatgg caaaaatagc
aagacatgaa atcaaattgt 720gccagacaag caacacagaa aaagaaaccc
tccacccaca acgccctcca aaaactgtag 780tcaccttaat tagggcggtc
atattcaatg tgtaaagttc tgtgcgaaga atcttacaga 840tttgctagct
aaagcaaaaa gctaagtgac taaactccat attactgaga gtctgaaatg
900ggcttgcgaa ccacgaagaa gtacattggt gtgaaaatcc ctttcttggc
accaccgaca 960agaccttctg cagctttctc taagaaagct tgaacccttt
gactaccttt aggagcaagt 1020cccacgtatt caagcgccga aaccagattt
ctggtgaaaa gtctgccaac tgctgttagg 1080cggaagctac tgagcgagaa
gtgactcgta tccaaaggca agtaccatgg aacaggtgag 1140tcatcagcca
gatccttgtc ccatacaact tcaaaaccag cttgtttggc tgcttcgagg
1200cactgtgttg tcaatctaac ctcagggagg ccatttccga gctcaatttc
ggccttgatc 1260ctgttgtgct cttcgttatt ggggttgtaa gaatcggtca
tgcaccactc atacacagcg 1320aaacattgac caggcttcag cacccggtaa
atctctttat agcatcccaa tggatctggt 1380gcatggcagg tagcttctgt
ccgtcgcttc tcttccattt cttctcattt tcgattttga 1440ttcttatttc
tttccagtag ctcctgctct gtgaatttct ccgctcacga tagatctgct
1500tatactcctt acattcaacc ttagatctgg tctcgattct ctgtttctct
gtttttttct 1560tttggtcgag aatctgatgt ttgtttatgt tctgtcacca
ttaataataa tgaactctct 1620cattcataca atgattagtt tctctcgtct
acaaaacgat atgttgcatt ttcacttttc 1680ttcttttttt ctaagatgat
ttgctttgac caatttgttt agatctttat tttattttat 1740tttctggtgg
gttggtggaa attgaaaaaa aaaaaaacag cataaattgt tatttgttaa
1800tgtattcatt ttttggctat ttgttctggg taaaaatctg cttctactat
tgaatctttc 1860ctggattttt tactcctatt gggtttttat agtaaaaata
cataataaaa ggaaaacaaa 1920agttttatag attctcttaa accccttacg
ataaaagttg gaatcaaaat aattcaggat 1980cagatgctct ttgattgatt
cagatgcgat tacagttgca tggcaaattt tctagatccg 2040tcgtcacatt
ttattttctg tttaaatatc taaatctgat atatgatgtc gacaaattct
2100ggtggcttat acatcacttc aactgttttc ttttggcttt gtttgtcaac
ttggttttca 2160atacgatttg tgatttcgat cgctgaattt ttaatacaag
caaactgatg ttaaccacaa 2220gcaagagatg tgacctgcct tattaacatc
gtattactta ctactagtcg tattctcaac 2280gcaatcgttt ttgtatttct
cacattatgc cgcttctcta ctctttattc cttttggtcc 2340acgcattttc
tatttgtggc aatccctttc acaacctgat ttcccacttt ggatcatttg
2400tctgaagact ctcttgaatc gttaccactt gtttcttgtg catgctctgt
tttttagaat 2460taatgataaa actattccat agtcttgagt tttcagcttg
ttgattcttt tgcttttggt 2520tttctgcaga gaagctacct gccatgcacc
agatccattg ggatgctata aagagattta 2580ccgggtgctg aagcctggtc
aatgtttcgc tgtgtatgag tggtgcatga ccgattctta 2640caaccccaat
aacgaagagc acaacaggat caaggccgaa attgagctcg gaaatggcct
2700ccctgaggtt agattgacaa cacagtgcct cgaagcagcc aaacaagctg
gttttgaagt 2760tgtatgggac aaggatctgg ctgatgactc acctgttcca
tggtacttgc ctttggatac 2820gagtcacttc tcgctcagta gcttccgcct
aacagcagtt ggcagacttt tcaccagaaa 2880tctggtttcg gcgcttgaat
acgtgggact tgctcctaaa ggtagtcaaa gggttcaagc 2940tttcttagag
aaagctgcag aaggtcttgt cggtggtgcc aagaaaggga ttttcacacc
3000aatgtacttc ttcgtggttc gcaagcccat ttcagactct cagtaatatg
gagtttagtc 3060acttagcttt ttgctttagc tagcaaatct gtaagattct
tcgcacagaa ctttacacat 3120tgaatatgac cgccctaatt aaggtgacta
cagtttttgg agggcgttgt gggtggaggg 3180tttctttttc tgtgttgctt
gtctggcaca atttgatttc atgtcttgct atttttgcca 3240ttgatgtcct
tgttctaaga tatataccta ttgacaagct cataaaggtg ggcatttgct
3300aatatatggg atcgttcaaa catttggcaa taaagtttct taagattgaa
tcctgttgcc 3360ggtcttgcga tgattatcat ataatttctg ttgaattacg
ttaagcatgt aataattaac 3420atgtaatgca tgacgttatt tatgagatgg
gtttttatga ttagagtccc gcaattatac 3480atttaatacg cgatagaaaa
caaaatatag cgcgcaaact aggataaatt atcgcgcgcg 3540gtgtcatcta
tgttactaga tcg 3563302881DNAArtificial SequenceArtificially created
chimeric nucleic acid sequence 30gacggtccga tgtgagactt ttcaacaaag
ggtaatatcc ggaaacctcc tcggattcca 60ttgcccagct atctgtcact ttattgtgaa
gatagtggaa aaggaaggtg gctcctacaa 120atgccatcat tgcgataaag
gaaaggccat cgttgaagat gcctctgccg acagtggtcc 180caaagatgga
cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc
240ttcaaagcaa gtggattgat gtgatggtcc gatgtgagac ttttcaacaa
agggtaatat 300ccggaaacct cctcggattc cattgcccag ctatctgtca
ctttattgtg aagatagtgg 360aaaaggaagg tggctcctac aaatgccatc
attgcgataa aggaaaggcc atcgttgaag 420atgcctctgc cgacagtggt
cccaaagatg gacccccacc cacgaggagc atcgtggaaa 480aagaagacgt
tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg
540taagggatga cgcacaatcc cactatcctt cgcaagaccc ttcctctata
taaggaagtt 600catttcattt ggagaggaac aatcctagcc caacaagccc
agctacatag tgacaatatt 660cgtcataatc atcagttgtt tccacctcct
tgcatatgaa ttttgccatt cctgcaccca 720tcctcatggt aatatcctca
attgcctgct gataatgttt cctaagctcc agaaaagcag 780ttgaaacatg
atggaactgg tccatgagaa ccttgtactc ttttgtacca catgaaaaat
840gccattcacg atcataaaca tgctgatgaa aagagatcag aataggtact
ttaacatcgg 900tgggaatgct ggtatcatcc tcaacagtgt caagtgctcg
aagaaccaaa tagaaaatgc 960acacggcgtc acgaagctcg acgggaagtt
gttgaatgac gagagcaaag ctacgagaaa 1020ccttatgaag cattgagtaa
cagaagcccc aatgtgggtc cgtcgcttct cttccatttc 1080ttctcatttt
cgattttgat tcttatttct ttccagtagc tcctgctctg tgaatttctc
1140cgctcacgat agatctgctt atactcctta cattcaacct tagatctggt
ctcgattctc 1200tgtttctctg tttttttctt ttggtcgaga atctgatgtt
tgtttatgtt ctgtcaccat 1260taataataat gaactctctc attcatacaa
tgattagttt ctctcgtcta caaaacgata 1320tgttgcattt tcacttttct
tctttttttc taagatgatt tgctttgacc aatttgttta 1380gatctttatt
ttattttatt ttctggtggg ttggtggaaa ttgaaaaaaa aaaaaacagc
1440ataaattgtt atttgttaat gtattcattt tttggctatt tgttctgggt
aaaaatctgc 1500ttctactatt gaatctttcc tggatttttt actcctattg
ggtttttata gtaaaaatac 1560ataataaaag gaaaacaaaa gttttataga
ttctcttaaa ccccttacga taaaagttgg 1620aatcaaaata attcaggatc
agatgctctt tgattgattc agatgcgatt acagttgcat 1680ggcaaatttt
ctagatccgt cgtcacattt tattttctgt ttaaatatct aaatctgata
1740tatgatgtcg acaaattctg gtggcttata catcacttca actgttttct
tttggctttg 1800tttgtcaact tggttttcaa tacgatttgt gatttcgatc
gctgaatttt taatacaagc 1860aaactgatgt taaccacaag caagagatgt
gacctgcctt attaacatcg tattacttac 1920tactagtcgt attctcaacg
caatcgtttt tgtatttctc acattatgcc gcttctctac 1980tctttattcc
ttttggtcca cgcattttct atttgtggca atccctttca caacctgatt
2040tcccactttg gatcatttgt ctgaagactc tcttgaatcg ttaccacttg
tttcttgtgc 2100atgctctgtt ttttagaatt aatgataaaa ctattccata
gtcttgagtt ttcagcttgt 2160tgattctttt gcttttggtt ttctgcagcc
acattggggc ttctgttact caatgcttca 2220taaggtttct cgtagctttg
ctctcgtcat tcaacaactt cccgtcgagc ttcgtgacgc 2280cgtgtgcatt
ttctatttgg ttcttcgagc acttgacact gttgaggatg ataccagcat
2340tcccaccgat gttaaagtac ctattctgat ctcttttcat cagcatgttt
atgatcgtga 2400atggcatttt tcatgtggta caaaagagta caaggttctc
atggaccagt tccatcatgt 2460ttcaactgct tttctggagc ttaggaaaca
ttatcagcag gcaattgagg atattaccat 2520gaggatgggt gcaggaatgg
caaaattcat atgcaaggag gtggaaacaa ctgatgatta 2580tgacgaatat
tgtcactatg tagctgggct tgttgggcta ggattgtgat cgttcaaaca
2640tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg
attatcatat 2700aatttctgtt gaattacgtt aagcatgtaa taattaacat
gtaatgcatg acgttattta 2760tgagatgggt ttttatgatt agagtcccgc
aattatacat ttaatacgcg atagaaaaca 2820aaatatagcg cgcaaactag
gataaattat cgcgcgcggt gtcatctatg ttactagatc 2880g
2881313703DNAArtificial SequenceArtificially created chimeric
nucleic acid sequence 31gacggtccga tgtgagactt ttcaacaaag ggtaatatcc
ggaaacctcc tcggattcca 60ttgcccagct atctgtcact ttattgtgaa gatagtggaa
aaggaaggtg gctcctacaa 120atgccatcat tgcgataaag gaaaggccat
cgttgaagat gcctctgccg acagtggtcc 180caaagatgga cccccaccca
cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc 240ttcaaagcaa
gtggattgat gtgatggtcc gatgtgagac ttttcaacaa agggtaatat
300ccggaaacct cctcggattc cattgcccag ctatctgtca ctttattgtg
aagatagtgg 360aaaaggaagg tggctcctac aaatgccatc attgcgataa
aggaaaggcc atcgttgaag 420atgcctctgc cgacagtggt cccaaagatg
gacccccacc cacgaggagc atcgtggaaa 480aagaagacgt tccaaccacg
tcttcaaagc aagtggattg atgtgatatc tccactgacg 540taagggatga
cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt
600catttcattt ggagaggaca tatacaaaag caaactttct gagcaaacat
aaagagtttg 660agatgccatt tctctctaaa ttacaatcta gtcacaatta
acaaacaaac gaaagacagt 720acaacagaaa agattattta aaaaaaaagg
ggttatcttc cttggcgcat gaatgaaatt 780aaccaaatgt tgaattacaa
gttaacccca ctggtcacca tcccacctaa cacctttcgt 840agatcttcac
caaatatttg caggtttttt tacacgaact cagaaaaaaa tacgacccta
900cctcctcaca tgccttcata gaaagaatac aacactacat acagatccac
gtccaccctt 960gatttgcttg tctttttctt cttgatcttt ctccacatga
ctaatgcctt acactttttc 1020aactttttgg tctacccttt acttattgct
ctccctaatt ggaaaatttt attcctactt 1080ttattgtaat ccatttcttt
aataatgatg gtccataaag gatggtgatg tacacgatgt 1140tgggataata
aatttgtctt tttccttact aagaggaaat cttagtaaca tctttgctag
1200atctattgta tttcatgtga ctcttaacca gctgccctgc tgagatagca
gacatgagag 1260ataactcacc agcaagaaca gaacctgcta ctattgtggc
caagagcctt gcatttgacc 1320ctgctgcctc cctgtttgca cctttcactc
ctaataagtt caagcaagct gactgtgatg 1380caagttgagt tccaccacca
actgtgccaa cctcaataga aggcatagtt actgaaatat 1440ggaggtcttt
gccatcattt acagcctcgt ccgtcgcttc tcttccattt cttctcattt
1500tcgattttga ttcttatttc tttccagtag ctcctgctct gtgaatttct
ccgctcacga 1560tagatctgct tatactcctt acattcaacc ttagatctgg
tctcgattct ctgtttctct 1620gtttttttct tttggtcgag aatctgatgt
ttgtttatgt tctgtcacca ttaataataa 1680tgaactctct cattcataca
atgattagtt tctctcgtct acaaaacgat atgttgcatt 1740ttcacttttc
ttcttttttt ctaagatgat ttgctttgac caatttgttt agatctttat
1800tttattttat tttctggtgg gttggtggaa attgaaaaaa aaaaaaacag
cataaattgt 1860tatttgttaa tgtattcatt ttttggctat ttgttctggg
taaaaatctg cttctactat 1920tgaatctttc ctggattttt tactcctatt
gggtttttat agtaaaaata cataataaaa 1980ggaaaacaaa agttttatag
attctcttaa accccttacg ataaaagttg gaatcaaaat 2040aattcaggat
cagatgctct ttgattgatt cagatgcgat tacagttgca tggcaaattt
2100tctagatccg tcgtcacatt ttattttctg tttaaatatc taaatctgat
atatgatgtc 2160gacaaattct ggtggcttat acatcacttc aactgttttc
ttttggcttt gtttgtcaac 2220ttggttttca atacgatttg tgatttcgat
cgctgaattt ttaatacaag caaactgatg 2280ttaaccacaa gcaagagatg
tgacctgcct tattaacatc gtattactta ctactagtcg 2340tattctcaac
gcaatcgttt ttgtatttct cacattatgc cgcttctcta ctctttattc
2400cttttggtcc acgcattttc tatttgtggc aatccctttc acaacctgat
ttcccacttt 2460ggatcatttg tctgaagact ctcttgaatc gttaccactt
gtttcttgtg catgctctgt 2520tttttagaat taatgataaa actattccat
agtcttgagt tttcagcttg ttgattcttt 2580tgcttttggt tttctgcagg
aggctgtaaa tgatggcaaa gacctccata tttcagtaac 2640tatgccttct
attgaggttg gcacagttgg tggtggaact caacttgcat cacagtcagc
2700ttgcttgaac ttattaggag tgaaaggtgc aaacagggag gcagcagggt
caaatgcaag 2760gctcttggcc acaatagtag caggttctgt tcttgctggt
gagttatctc tcatgtctgc 2820tatctcagca gggcagctgg ttaagagtca
catgaaatac aatagatcta gcaaagatgt 2880tactaagatt tcctcttagt
aaggaaaaag acaaatttat tatcccaaca tcgtgtacat 2940caccatcctt
tatggaccat cattattaaa gaaatggatt acaataaaag taggaataaa
3000attttccaat tagggagagc aataagtaaa gggtagacca aaaagttgaa
aaagtgtaag 3060gcattagtca tgtggagaaa gatcaagaag aaaaagacaa
gcaaatcaag ggtggacgtg 3120gatctgtatg tagtgttgta ttctttctat
gaaggcatgt gaggaggtag ggtcgtattt 3180ttttctgagt tcgtgtaaaa
aaacctgcaa atatttggtg aagatctacg aaaggtgtta 3240ggtgggatgg
tgaccagtgg ggttaacttg taattcaaca tttggttaat ttcattcatg
3300cgccaaggaa gataacccct ttttttttaa ataatctttt ctgttgtact
gtctttcgtt 3360tgtttgttaa ttgtgactag attgtaattt agagagaaat
ggcatctcaa actctttatg 3420tttgctcaga aagtttgctt ttgtatatgg
atcgttcaaa catttggcaa taaagtttct 3480taagattgaa tcctgttgcc
ggtcttgcga tgattatcat ataatttctg ttgaattacg 3540ttaagcatgt
aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga
3600ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag
cgcgcaaact 3660aggataaatt atcgcgcgcg
gtgtcatcta tgttactaga tcg 3703324286DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
32ttctgttcgt atatttgtaa ctattatgtg tatttttatt ttgttagtat tactaattca
60agtggtttaa gttgttgaga ctctttaaaa tctaagcatt ttataaacaa taatatataa
120ttattgttta ggctaaattt gtcactaatt aaggtttgga tacatagtgt
ctaaactaag 180ctaataatat cacttaacgt ttacttgtaa cgctaggtga
tgatgtcgtc aagtcaattg 240gtacaaggaa taaacgagtg gtcatatgac
attatgacca tatgaattca aactccagta 300atccaatggt aattggattc
aatgatcaag acttgaacca cgtaatccac ccttatcctt 360agaagctcat
aaatatcact aaagggacag gcaacactta accagtagtt gtccaataat
420ttagttttcc aaaatgaaaa attattgttg tcatctattt taggtgtttt
agttcaatgt 480ggattcctcg tcctaacaaa tacttgacga atatatctag
actataaaat tggttatgag 540ttctactttt ttttgtttgt gaaattatca
aaatttgtta tatttattta tttattctca 600ttaatttgag tactaatttt
taaattattt atactaaaaa caattactaa gatacaaaaa 660tggataagag
catggtgtat agatatttaa tgggatagaa tatttcccat aattgtatgt
720gtgtgagagg ttttgttttc gtaaggaaag aaacaaaaac catttgacca
aagaaaagca 780aaagaaggca aggaatcaaa caacaaatgt tgcaaggcag
aaataatgga cgttatgtta 840atgtagtgtc gtcacacgtg acttaaaaga
gacgagtctg cgtgtcaaac taaaaatgta 900tgcaactata aaaatgggat
ttgattatct ttttagtacc gaagcctacc aaccacatgc 960acactaattc
tactcgccaa ataaagtgaa aagagccata tattagcaaa tgcccacctt
1020tatgagcttg tcaataggta tatatcttag aacaaggaca tcaatggcaa
aaatagcaag 1080acatgaaatc aaattgtgcc agacaagcaa cacagaaaaa
gaaaccctcc acccacaacg 1140ccctccaaaa actgtagtca ccttaattag
ggcggtcata ttcaatgtgt aaagttctgt 1200gcgaagaatc ttacagattt
gctagctaaa gcaaaaagct aagtgactaa actccatatt 1260actgagagtc
tgaaatgggc ttgcgaacca cgaagaagta cattggtgtg aaaatccctt
1320tcttggcacc accgacaaga ccttctgcag ctttctctaa gaaagcttga
accctttgac 1380tacctttagg agcaagtccc acgtattcaa gcgccgaaac
cagatttctg gtgaaaagtc 1440tgccaactgc tgttaggcgg aagctactga
gcgagaagtg actcgtatcc aaaggcaagt 1500accatggaac aggtgagtca
tcagccagat ccttgtccca tacaacttca aaaccagctt 1560gtttggctgc
ttcgaggcac tgtgttgtca atctaacctc agggaggcca tttccgagct
1620caatttcggc cttgatcctg ttgtgctctt cgttattggg gttgtaagaa
tcggtcatgc 1680accactcata cacagcgaaa cattgaccag gcttcagcac
ccggtaaatc tctttatagc 1740atcccaatgg atctggtgca tggcaggtag
cttctgtaag ttcctgtttt cacctgcacc 1800atgaaaaata tactattact
attatttttc atttatttgt gtggtccata ttgctatgtg 1860tgaaatgaaa
aaatattttt tttctcaaac tacaatattg tcagaaagaa aggaattaat
1920attccgaatt tataccaaaa aattaatttc ttttttctct ttggtaagct
ggattctgtt 1980attctttggt aaaacggaga ataattttgt ttatcaactt
ctgttgattt tatgaacaat 2040tctcaattaa ttgaaggggt agtttaaggc
tgatgaatct tttggatgag ttacttgagc 2100agtatggatt gactcacatg
actaactgct tcactagctt ccaatatttt ttagttatta 2160catgttgtgt
atgttgatta ttgtgctcta agcaatcgga ttctcttgtt aaataaaaac
2220tatcatagtt tatttattca ataatcgagt ttgagctaac actcctgtct
atctggaata 2280caaaaggaaa gataataaaa gtttttggta ccttgaaaac
tagaagtatc aggaagggga 2340gccttgaaca aaggtcaagt tgtctccgtt
tgacctacat gtcatgttcg agccattgat 2400gcttgcatca ggatagactg
cctacatcac cccctcttgc ggtacggccc ttccccggac 2460ctgcgtgaac
gcgggatact ttgtgcaccg gaaaactaca agtatcccta acacatatca
2520ggattttagt gatatccctt cactgccgtg ttcgataaag gttacataaa
gttttaaatt 2580tatgggtgct aaatatcaca gctaaatata cacattaaag
atattactgc atccatatat 2640gttgccatga ccatacatca agtatacatc
cacccctaat ttttgagtgt ttttgagatg 2700cagcaaagtt gaaggagatt
ataatagttt gatgtggaga gactaatttt ttttttaaca 2760tcactttcta
agggtgctat cttttcacca ccatcactgg tggcttgttg atttgtagct
2820aatcattatc ttttgatgaa aacaaggaca ttctttagtg cactaagatt
gttaaacgtt 2880cgtgcttcat tgtaaatgta atatactcgc gcttgttggc
atgaacactt ggaattgttt 2940actggaacac tgcagagaag ctacctgcca
tgcaccagat ccattgggat gctataaaga 3000gatttaccgg gtgctgaagc
ctggtcaatg tttcgctgtg tatgagtggt gcatgaccga 3060ttcttacaac
cccaataacg aagagcacaa caggatcaag gccgaaattg agctcggaaa
3120tggcctccct gaggttagat tgacaacaca gtgcctcgaa gcagccaaac
aagctggttt 3180tgaagttgta tgggacaagg atctggctga tgactcacct
gttccatggt acttgccttt 3240ggatacgagt cacttctcgc tcagtagctt
ccgcctaaca gcagttggca gacttttcac 3300cagaaatctg gtttcggcgc
ttgaatacgt gggacttgct cctaaaggta gtcaaagggt 3360tcaagctttc
ttagagaaag ctgcagaagg tcttgtcggt ggtgccaaga aagggatttt
3420cacaccaatg tacttcttcg tggttcgcaa gcccatttca gactctcagt
aatatggagt 3480ttagtcactt agctttttgc tttagctagc aaatctgtaa
gattcttcgc acagaacttt 3540acacattgaa tatgaccgcc ctaattaagg
tgactacagt ttttggaggg cgttgtgggt 3600ggagggtttc tttttctgtg
ttgcttgtct ggcacaattt gatttcatgt cttgctattt 3660ttgccattga
tgtccttgtt ctaagatata tacctattga caagctcata aaggtgggca
3720tttgctaata tatggtttcc ctttgctttt gtgtaaacct caaaacttta
tcccccatct 3780ttgattttat cccttgtttt tctgcttttt tcttctttct
tgggttttaa tttccggact 3840taacgtttgt tttccggttt gcgagacata
ttctatcgga ttctcaactg tctgatgaaa 3900taaatatgta atgttctata
agtctttcaa tttgatatgc atatcaacaa aaagaaaata 3960ggacaatgcg
gctacaaata tgaaatttac aagtttaaga accatgagtc gctaaagaaa
4020tcattaagaa aattagtttc acattcaatt cttgtcacat gattaacgag
cttgagaggt 4080ttagagtaac aatatcttga agcaaaagat gacccacttg
aaatctagtg atggatacat 4140aagtggacgt gccttgttta ggataggatt
ctggataaga gtctcgaata ttcattttta 4200ccaagtatat tcaaggatct
tgtggatcat atatttcctc aatcaaaggg acttgaccca 4260aattcacata
aagatatttt ggagtc 4286338956DNAArtificial SequenceArtificially
created chimeric nucleic acid sequence 33ctcgaggatc taaattgtga
gttcaatctc ttccctattg gattgattat cctttctttt 60cttccaattt gtgtttcttt
ttgcctaatt tattgtgtta tcccctttat cctattttgt 120ttctttactt
atttatttgc ttctatgtct ttgtacaaag atttaaactc tatggcacat
180attttaaagt tgttagaaaa taaattcttt caagattgat gaaagaactt
tttaattgta 240gatatttcgt agattttatt ctcttactac caatataacg
cttgaattga cgaaaatttg 300tgtccaaata tctagcaaaa aggtatccaa
tgaaaatata tcatatgtga tcttcaaatc 360ttgtgtctta tgcaagattg
atactttgtt caatggaaga gattgtgtgc atatttttaa 420aatttttatt
agtaataaag attctatata gctgttatag agggataatt ttacaaagaa
480cactataaat atgattgttg ttgttagggt gtcaatggtt cggttcgact
ggttatttta 540taaaatttgt accataccat ttttttcgat attctatttt
gtataaccaa aattagactt 600ttcgaaatcg tcccaatcat gtcggtttca
cttcggtatc ggtaccgttc ggttaatttt 660catttttttt taaatgtcat
taaaattcac tagtaaaaat agaatgcaat aacatacgtt 720cttttatagg
acttagcaaa agctctctag acatttttac tgtttaaagg ataatgaatt
780aaaaaacatg aaagatggct agagtataga tacacaacta ttcgacagca
acgtaaaaga 840aaccaagtaa aagcaaagaa aatataaatc acacgagtgg
aaagatatta accaagttgg 900gattcaagaa taaagtctat attaaatatt
caaaaagata aatttaaata atatgaaagg 960aaacatattc aatacattgt
agtttgctac tcataatcgc tagaatactt tgtgccttgc 1020taataaagat
acttgaaata gcttagttta aatataaata gcataataga ttttaggaat
1080tagtattttg agtttaatta cttattgact tgtaacagtt tttataattc
caaggcccat 1140gaaaaattta atgctttatt agttttaaac ttactatata
aatttttcat atgtaaaatt 1200taatcggtat agttcgatat tttttcaatt
tatttttata aaataaaaaa cttaccctaa 1260ttatcggtac agttatagat
ttatataaaa atctacggtt cttcagaaga aacctaaaaa 1320tcggttcggt
gcggacggtt cgatcggttt agtcgatttt caaatattca ttgacactcc
1380tagttgttgt tataggtaaa aagcagttac agagaggtaa aatataactt
aaaaaatcag 1440ttctaaggaa aaattgactt ttatagtaaa tgactgttat
ataaggatgt tgttacagag 1500aggtatgagt gtagttggta aattatgttc
ttgacggtgt atgtcacata ttatttatta 1560aaactagaaa aaacagcgtc
aaaactagca aaaatccaac ggacaaaaaa atcggctgaa 1620tttgatttgg
ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg actgttgatg
1680atataaacaa agggcacatt ggtcaataac cataaaaaat tatatgacag
ctacagttgg 1740tagcatgtgc tcagctattg aacaaatcta aagaaggtac
atctgtaacc ggaacaccac 1800ttaaatgact aaattaccct catcagaaag
cagatggagt gctacaaata acacactatt 1860caacaaccat aaataaaacg
tgttcagcta ctaaaacaaa tataaataaa tctatgtttg 1920taagcactcc
agccatgtta atggagtgct attgcctgtt aactctcact tataaaatag
1980tagtagaaaa aatatgaacc aaaacacaac ggttgtgtat ttcacttttg
gatatagctc 2040agtggcttcg acacctgtag gaggctgaac ctcaaagttt
gcagaatctc cattaacaaa 2100aactgaatgg catatggcca aattagtcct
taatggcaga ggtccctctt gtacaatctg 2160gagaatatct tcctctgata
gatataactt ctcgagggtc ttcccaattt tgtcctccca 2220caaggacact
atctcgttga aggatagaat attggcaggt ggtctcatgt gaagagtctt
2280attcaatgtc cgtggatcat ctactgcttc gatagtgtat gtcgctatgt
cttcttcctt 2340cacatatatt gctttgggat ttccatcgcc aaaaatgaca
actttgtctc taggaggggt 2400tttggcctct aactgcccca agttgggcaa
gaagaaatct gcaaaccaat tgcagattac 2460atatgtgtat ggaattcctt
ctgcctctat catcctcctg attcttacct ttagagcgaa 2520gagtgatgca
gctggttcaa ttgcacgagc atgatccaca tcaaatccaa attctgaagg
2580aagaaatctc ttgatatttc cagcttcttt aattgctttg atgatgttca
cttgatcagt 2640ccgtcgcttc tcttccattt cttctcattt tcgattttga
ttcttatttc tttccagtag 2700ctcctgctct gtgaatttct ccgctcacga
tagatctgct tatactcctt acattcaacc 2760ttagatctgg tctcgattct
ctgtttctct gtttttttct tttggtcgag aatctgatgt 2820ttgtttatgt
tctgtcacca ttaataataa tgaactctct cattcataca atgattagtt
2880tctctcgtct acaaaacgat atgttgcatt ttcacttttc ttcttttttt
ctaagatgat 2940ttgctttgac caatttgttt agatctttat tttattttat
tttctggtgg gttggtggaa 3000attgaaaaaa aaaaaaacag cataaattgt
tatttgttaa tgtattcatt ttttggctat 3060ttgttctggg taaaaatctg
cttctactat tgaatctttc ctggattttt tactcctatt 3120gggtttttat
agtaaaaata cataataaaa ggaaaacaaa agttttatag attctcttaa
3180accccttacg ataaaagttg gaatcaaaat aattcaggat cagatgctct
ttgattgatt 3240cagatgcgat tacagttgca tggcaaattt tctagatccg
tcgtcacatt ttattttctg 3300tttaaatatc taaatctgat atatgatgtc
gacaaattct ggtggcttat acatcacttc 3360aactgttttc ttttggcttt
gtttgtcaac ttggttttca atacgatttg tgatttcgat 3420cgctgaattt
ttaatacaag caaactgatg ttaaccacaa gcaagagatg tgacctgcct
3480tattaacatc gtattactta ctactagtcg tattctcaac gcaatcgttt
ttgtatttct 3540cacattatgc cgcttctcta ctctttattc cttttggtcc
acgcattttc tatttgtggc 3600aatccctttc acaacctgat ttcccacttt
ggatcatttg tctgaagact ctcttgaatc 3660gttaccactt gtttcttgtg
catgctctgt tttttagaat taatgataaa actattccat 3720agtcttgagt
tttcagcttg ttgattcttt tgcttttggt tttctgcagt gatcaagtga
3780acatcatcaa agcaattaaa gaagctggaa atatcaagag atttcttcct
tcagaatttg 3840gatttgatgt ggatcatgct cgtgcaattg aaccagctgc
atcactcttc gctctaaagg 3900taagaatcag gaggatgata gaggcagaag
gaattccata cacatatgta atctgcaatt 3960ggtttgcaga tttcttcttg
cccaacttgg ggcagttaga ggccaaaacc cctcctagag 4020acaaagttgt
catttttggc gatggaaatc ccaaagcaat atatgtgaag gaagaagaca
4080tagcgacata cactatcgaa gcagtagatg atccacggac attgaataag
actcttcaca 4140tgagaccacc tgccaatatt ctatccttca acgagatagt
gtccttgtgg gaggacaaaa 4200ttgggaagac cctcgagaag ttatatctat
cagaggaaga tattctccag attgtacaag 4260agggacctct gccattaagg
actaatttgg ccatatgcca ttcagttttt gttaatggag 4320attctgcaaa
ctttgaggtt cagcctccta caggtgtcga agccactgag ctatatccaa
4380aagtgaaata cacaaccgca agtgtgttgc ctttgtgtgg aaatgaagag
gtacttgcga 4440ggactttgcg tttatcagtt tatgtgtttg tatatctatt
tgatccagtt attatggatt 4500atatacgctt gaaactcatt ttaagccatt
gttattgaac gtttatcaaa tactttatta 4560tgccaagcaa gtcaaacaca
tgcttgttga ttgaaatcaa gctatagaaa tctcttcttc 4620acatacagca
gtttagattc acaatacaac aagcgaaacg ataaagtttc ttctgttcgt
4680atatttgtaa ctattatgtg tatttttatt ttgttagtat tactaattca
agtggtttaa 4740gttgttgaga ctctttaaaa tctaagcatt ttataaacaa
taatatataa ttattgttta 4800ggctaaattt gtcactaatt aaggtttgga
tacatagtgt ctaaactaag ctaataatat 4860cacttaacgt ttacttgtaa
cgctaggtga tgatgtcgtc aagtcaattg gtacaaggaa 4920taaacgagtg
gtcatatgac attatgacca tatgaattca aactccagta atccaatggt
4980aattggattc aatgatcaag acttgaacca cgtaatccac ccttatcctt
agaagctcat 5040aaatatcact aaagggacag gcaacactta accagtagtt
gtccaataat ttagttttcc 5100aaaatgaaaa attattgttg tcatctattt
taggtgtttt agttcaatgt ggattcctcg 5160tcctaacaaa tacttgacga
atatatctag actataaaat tggttatgag ttctactttt 5220ttttgtttgt
gaaattatca aaatttgtta tatttattta tttattctca ttaatttgag
5280tactaatttt taaattattt atactaaaaa caattactaa gatacaaaaa
tggataagag 5340catggtgtat agatatttaa tgggatagaa tatttcccat
aattgtatgt gtgtgagagg 5400ttttgttttc gtaaggaaag aaacaaaaac
catttgacca aagaaaagca aaagaaggca 5460aggaatcaaa caacaaatgt
tgcaaggcag aaataatgga cgttatgtta atgtagtgtc 5520gtcacacgtg
acttaaaaga gacgagtctg cgtgtcaaac taaaaatgta tgcaactata
5580aaaatgggat ttgattatct ttttagtacc gaagcctacc aaccacatgc
acactaattc 5640tactcgccaa ataaagtgaa aagagccata tattagcaaa
tgcccacctt tatgagcttg 5700tcaataggta tatatcttag aacaaggaca
tcaatggcaa aaatagcaag acatgaaatc 5760aaattgtgcc agacaagcaa
cacagaaaaa gaaaccctcc acccacaacg ccctccaaaa 5820actgtagtca
ccttaattag ggcggtcata ttcaatgtgt aaagttctgt gcgaagaatc
5880ttacagattt gctagctaaa gcaaaaagct aagtgactaa actccatatt
actgagagtc 5940tgaaatgggc ttgcgaacca cgaagaagta cattggtgtg
aaaatccctt tcttggcacc 6000accgacaaga ccttctgcag ctttctctaa
gaaagcttga accctttgac tacctttagg 6060agcaagtccc acgtattcaa
gcgccgaaac cagatttctg gtgaaaagtc tgccaactgc 6120tgttaggcgg
aagctactga gcgagaagtg actcgtatcc aaaggcaagt accatggaac
6180aggtgagtca tcagccagat ccttgtccca tacaacttca aaaccagctt
gtttggctgc 6240ttcgaggcac tgtgttgtca atctaacctc agggaggcca
tttccgagct caatttcggc 6300cttgatcctg ttgtgctctt cgttattggg
gttgtaagaa tcggtcatgc accactcata 6360cacagcgaaa cattgaccag
gcttcagcac ccggtaaatc tctttatagc atcccaatgg 6420atctggtgca
tggcaggtag cttctgtaag ttcctgtttt cacctgcacc atgaaaaata
6480tactattact attatttttc atttatttgt gtggtccata ttgctatgtg
tgaaatgaaa 6540aaatattttt tttctcaaac tacaatattg tcagaaagaa
aggaattaat attccgaatt 6600tataccaaaa aattaatttc ttttttctct
ttggtaagct ggattctgtt attctttggt 6660aaaacggaga ataattttgt
ttatcaactt ctgttgattt tatgaacaat tctcaattaa 6720ttgaaggggt
agtttaaggc tgatgaatct tttggatgag ttacttgagc agtatggatt
6780gactcacatg actaactgct tcactagctt ccaatatttt ttagttatta
catgttgtgt 6840atgttgatta ttgtgctcta agcaatcgga ttctcttgtt
aaataaaaac tatcatagtt 6900tatttattca ataatcgagt ttgagctaac
actcctgtct atctggaata caaaaggaaa 6960gataataaaa gtttttggta
ccttgaaaac tagaagtatc aggaagggga gccttgaaca 7020aaggtcaagt
tgtctccgtt tgacctacat gtcatgttcg agccattgat gcttgcatca
7080ggatagactg cctacatcac cccctcttgc ggtacggccc ttccccggac
ctgcgtgaac 7140gcgggatact ttgtgcaccg gaaaactaca agtatcccta
acacatatca ggattttagt 7200gatatccctt cactgccgtg ttcgataaag
gttacataaa gttttaaatt tatgggtgct 7260aaatatcaca gctaaatata
cacattaaag atattactgc atccatatat gttgccatga 7320ccatacatca
agtatacatc cacccctaat ttttgagtgt ttttgagatg cagcaaagtt
7380gaaggagatt ataatagttt gatgtggaga gactaatttt ttttttaaca
tcactttcta 7440agggtgctat cttttcacca ccatcactgg tggcttgttg
atttgtagct aatcattatc 7500ttttgatgaa aacaaggaca ttctttagtg
cactaagatt gttaaacgtt cgtgcttcat 7560tgtaaatgta atatactcgc
gcttgttggc atgaacactt ggaattgttt actggaacac 7620tgcagagaag
ctacctgcca tgcaccagat ccattgggat gctataaaga gatttaccgg
7680gtgctgaagc ctggtcaatg tttcgctgtg tatgagtggt gcatgaccga
ttcttacaac 7740cccaataacg aagagcacaa caggatcaag gccgaaattg
agctcggaaa tggcctccct 7800gaggttagat tgacaacaca gtgcctcgaa
gcagccaaac aagctggttt tgaagttgta 7860tgggacaagg atctggctga
tgactcacct gttccatggt acttgccttt ggatacgagt 7920cacttctcgc
tcagtagctt ccgcctaaca gcagttggca gacttttcac cagaaatctg
7980gtttcggcgc ttgaatacgt gggacttgct cctaaaggta gtcaaagggt
tcaagctttc 8040ttagagaaag ctgcagaagg tcttgtcggt ggtgccaaga
aagggatttt cacaccaatg 8100tacttcttcg tggttcgcaa gcccatttca
gactctcagt aatatggagt ttagtcactt 8160agctttttgc tttagctagc
aaatctgtaa gattcttcgc acagaacttt acacattgaa 8220tatgaccgcc
ctaattaagg tgactacagt ttttggaggg cgttgtgggt ggagggtttc
8280tttttctgtg ttgcttgtct ggcacaattt gatttcatgt cttgctattt
ttgccattga 8340tgtccttgtt ctaagatata tacctattga caagctcata
aaggtgggca tttgctaata 8400tatggtttcc ctttgctttt gtgtaaacct
caaaacttta tcccccatct ttgattttat 8460cccttgtttt tctgcttttt
tcttctttct tgggttttaa tttccggact taacgtttgt 8520tttccggttt
gcgagacata ttctatcgga ttctcaactg tctgatgaaa taaatatgta
8580atgttctata agtctttcaa tttgatatgc atatcaacaa aaagaaaata
ggacaatgcg 8640gctacaaata tgaaatttac aagtttaaga accatgagtc
gctaaagaaa tcattaagaa 8700aattagtttc acattcaatt cttgtcacat
gattaacgag cttgagaggt ttagagtaac 8760aatatcttga agcaaaagat
gacccacttg aaatctagtg atggatacat aagtggacgt 8820gccttgttta
ggataggatt ctggataaga gtctcgaata ttcattttta ccaagtatat
8880tcaaggatct tgtggatcat atatttcctc aatcaaaggg acttgaccca
aattcacata 8940aagatatttt ggagtc 8956343600DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
34tctagaatgt tcgtgcgtca aatggataaa caaaaaaata gcataagtta gttttgttac
60tcgagagtta tgtattataa ggtataggga aatgactcaa acataccact gaacttaacg
120aaacgacgca tatatatact acttaactta acgaaaaagg ggtgagagtg
gatgggtgct 180ggtaaataat gaagggttta tataacgtca cgtgtcaaaa
ttcgatagta gtagtttcgt 240tagttgtaat agcatatatg gcccaaagtt
ataatataga taatatgttt atgtccaact 300attaacgagt gacatagaca
gttcattttg tgaagttcaa tgacatattt gagccctttc 360ccttttatta
tctcctttta tttgttctaa taaaagaatg gcatttatta tgtacataga
420caaataacta ttttctttgg aatataattt gtttatatat tttaaaatca
tgtctcaatt 480tagtttgttt tgtgcatatt tcaactattc aattttgtcc
atatatttat taccttcccc 540catttacaag cattgaaccg ctttgctcac
caaaacttat gcacattgca aaaatatcat 600gtaaaggttt tatgtatgct
gtaattaagg tctgaactca tcgtgatttt atttttaggc 660ttcattgacc
actaccaaac tctttgatgc tacattttct aattatattg gagttcgatt
720atatccgaat tcgcgttgcg tagggcccat tcgagggaaa acactcccta
tcaaggattt 780tttcataccc agagctcgaa ctcaagacat ctggttaagg
gaagaacagt ctcatccact 840gcaccatatc cttttgtggt caacaagtaa
attttatgta gaaccaaaaa ctatactcga 900attgataaaa taaatggtgt
aaaatattgt tttctttctt acattttgga cagtaaatat 960gtaggacaat
aataattagc gtggggtctt aagaaaatta gcatagattt ccagaaattc
1020caaatcaacc ggcagttcca ggtttgaaaa ctacaactca ttccgacggt
tcaaacttca 1080aaccatgctt gctgactcgg cttcttcttt ctttttcacc
aagacagagc agtagtcacg 1140tgacacccct cacgtgcctc ccccctttat
atttcagact gcaaccctac actttcgcta 1200cattcactac catattcttt
tcactaagca attttctctc ctacttttct ttaaacccct 1260tttttctccc
ctaagccatg gcatctagat catgttacgt cctgtagaaa ccccaacccg
1320tgaaatcaaa aaactcgacg gcctgtgggc attcagtctg gatcgcgaaa
actgtggaat 1380tgatcagcgt tggtgggaaa gcgcgttaca agaaagccgg
gcaattgctg tgccaggcag 1440ttttaacgat cagttcgccg
atgcagatat tcgtaattat gcgggcaacg tctggtatca 1500gcgcgaagtc
tttataccga aaggttgggc aggccagcgt atcgtgctgc gtttcgatgc
1560ggtcactcat tacggcaaag tgtgggtcaa taatcaggaa gtgatggagc
atcagggcgg 1620ctatacgcca tttgaagccg atgtcacgcc gtatgttatt
gccgggaaaa gtgtacgtat 1680caccgtttgt gtgaacaacg aactgaactg
gcagactatc ccgccgggaa tggtgattac 1740cgacgaaaac ggcaagaaaa
agcagtctta cttccatgat ttctttaact atgccggaat 1800ccatcgcagc
gtaatgctct acaccacgcc gaacacctgg gtggacgata tcaccgtggt
1860gacgcatgtc gcgcaagact gtaaccacgc gtctgttgac tggcaggtgg
tggccaatgg 1920tgatgtcagc gttgaactgc gtgatgcgga tcaacaggtg
gttgcaactg gacaaggcac 1980tagcgggact ttgcaagtgg tgaatccgca
cctctggcaa ccgggtgaag gttatctcta 2040tgaactgtgc gtcacagcca
aaagccagac agagtgtgat atctacccgc ttcgcgtcgg 2100catccggtca
gtggcagtga agggcgaaca gttcctgatt aaccacaaac cgttctactt
2160tactggcttt ggtcgtcatg aagatgcgga cttgcgtggc aaaggattcg
ataacgtgct 2220gatggtgcac gaccacgcat taatggactg gattggggcc
aactcctacc gtacctcgca 2280ttacccttac gctgaagaga tgctcgactg
ggcagatgaa catggcatcg tggtgattga 2340tgaaactgct gctgtcggct
ttaacctctc tttaggcatt ggtttcgaag cgggcaacaa 2400gccgaaagaa
ctgtacagcg aagaggcagt caacggggaa actcagcaag cgcacttaca
2460ggcgattaaa gagctgatag cgcgtgacaa aaaccaccca agcgtggtga
tgtggagtat 2520tgccaacgaa ccggataccc gtccgcaagg tgcacgggaa
tatttcgcgc cactggcgga 2580agcaacgcgt aaactcgacc cgacgcgtcc
gatcacctgc gtcaatgtaa tgttctgcga 2640cgctcacacc gataccatca
gcgatctctt tgatgtgctg tgcctgaacc gttattacgg 2700atggtatgtc
caaagcggcg atttggaaac ggcagagaag gtactggaaa aagaacttct
2760ggcctggcag gagaaactgc atcagccgat tatcatcacc gaatacggcg
tggatacgtt 2820agccgggctg cactcaatgt acaccgacat gtggagtgaa
gagtatcagt gtgcatggct 2880ggatatgtat caccgcgtct ttgatcgcgt
cagcgccgtc gtcggtgaac aggtatggaa 2940tttcgccgat tttgcgacct
cgcaaggcat attgcgcgtt ggcggtaaca agaaagggat 3000cttcactcgc
gaccgcaaac cgaagtcggc ggcttttctg ctgcaaaaac gctggactgg
3060catgaacttc ggtgaaaaac cgcagcaggg aggcaaacaa tgagagctcg
tgaaatggcc 3120tctttagttt ttgattgaat cataggggta ttagttttct
atggccggga gtggtcttct 3180tgcttaattg taatggaata accagagagg
aactactgtg ttatctttga ggaatgttgg 3240gcttttttcg tttgaattat
catgaatgaa attttacttt ttcccaatac aagtttgttt 3300tcgtttcttg
gtttttgtta tcccttggtt tatgtcttgg tttggcttaa atgattgaag
3360attacactac ctatgtttct gctattcctg ttgaagatca catttgataa
taatgcatcg 3420aatgcattaa agtttcttat tggctctgtc aaaagtattg
aaggtggatt tttctaattg 3480gcaagagaaa gtattaaaga ggtgatttat
tagtacttat atttttctca gcatctctct 3540ttcagtgttg gagcttcata
aaattagcac ttcagagttt cagtcgggag ctgaattcga 3600353387DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
35cgttttgacg agttcggatg tagtagtagc cattatttaa tgtacatact aatcgtgaat
60agtgaatatg atgaaacatt gtatcttatt gtataaatat ccataaacac atcatgaaag
120acactttctt tcacggtctg aattaattat gatacaattc taatagaaaa
cgaattaaat 180tacgttgaat tgtatgaaat ctaattgaac aagccaacca
cgacgacgac taacgttgcc 240tggattgact cggtttaagt taaccactaa
aaaaacggag ctgtcatgta acacgcggat 300cgagcaggtc acagtcatga
agccatcaaa gcaaaagaac taatccaagg gctgagatga 360ttaattagtt
taaaaattag ttaacacgag ggaaaaggct gtctgacagc caggtcacgt
420tatctttacc tgtggtcgaa atgattcgtg tctgtcgatt ttaattattt
ttttgaaagg 480ccgaaaataa agttgtaaga gataaacccg cctatataaa
ttcatatatt ttctctccgc 540tttgaattgt ctcgttgtcc tcctcacttt
catcggccgt ttttgaatct ccggcgactt 600gacagagaag aacaaggaag
aagactaaga gagaaagtaa gagataatcc aggagattca 660ttctccgttt
tgaatcttcc tcaatctcat cttcttccgc tctttctttc caaggtaata
720ggaactttct ggatctactt tatttgctgg atctcgatct tgttttctca
atttccttga 780gatctggaat tcgtttaatt tggatctgtg aacctccact
aaatcttttg gttttactag 840aatcgatcta agttgaccga tcagttagct
cgattatagc taccagaatt tggcttgacc 900ttgatggaga gatccatgtt
catgttacct gggaaatgat ttgtatatgt gaattgaaat 960ctgaactgtt
gaagttagat tgaatctgaa cactgtcaat gttagattga atctgaacac
1020tgtttaagtt agatgaagtt tgtgtataga ttcttcgaaa ctttaggatt
tgtagtgtcg 1080tacgttgaac agaaagctat ttctgattca atcagggttt
atttgactgt attgaactct 1140ttttgtgtgt ttgcagctca tatggttgtg
tttgggaatg tttctgcggc gaatttgcct 1200tatcaaaacg ggtttttgga
ggcactttca tctggaggtt gtgaactaat gggacatagc 1260tttagggttc
ccacttctca agcgcttaag acaagaacaa ggaggaggag tactgctggt
1320cctttgcagg tagtttgtgt ggatattcca aggccagagc tagagaacac
tgtcaatttc 1380ttggaagctg ctagtttatc tgcatccttc cgtagtgctc
ctcgtcctgc taagcctttg 1440aaagttgtaa ttgctggtgc tggattggct
ggattgtcaa ctgcaaagta cctggctgat 1500gcaggccaca aacctctgtt
gcttgaagca agagatgttc ttggtggaaa gatagctgca 1560tggaaggatg
aagatgggga ctggtatgag actggtttac atattttctt cggtgcttat
1620ccgaatgtgc agaatttatt tggagaactt gggatcaatg atcggttgca
gtggaaggaa 1680cactccatga tttttgctat gccaagtaaa cctggagaat
ttagtagatt tgacttccca 1740gatgtcctac cagcaccctt aaatggtatt
tgggctattt tgcggaacaa cgagatgctg 1800acatggccag agaaaataaa
gtttgctatt ggacttttgc cagccatggt cggcggtcag 1860gcttatgttg
aggcccaaga tggtttatca gtcaaagaat ggatggaaaa gcagggagta
1920cctgagcgcg tgaccgacga ggtgtttatt gccatgtcaa aggcgctaaa
ctttataaac 1980cctgatgaac tgtcaatgca atgcattttg atagctttga
accggtttct tcaggaaaaa 2040catggttcca agatggcatt cttggatggt
aatcctccgg aaaggctttg tatgccagta 2100gtggatcata ttcgatcact
aggtggggaa gtgcaactta attctaggat aaagaaaatt 2160gagctcaatg
acgatggcac ggttaagagt ttcttactca ctaatggaag cactgtcgaa
2220ggagacgctt atgtgtttgc cgctccagtc gatatcctga agctcctttt
accagatccc 2280tggaaagaaa taccgtactt caagaaattg gataaattag
ttggagtacc agttattaat 2340gttcatatat ggtttgatcg aaaactgaag
aacacatatg atcacctact ctttagcaga 2400agtaaccttc tgagcgtgta
tgccgacatg tccttaactt gtaaggaata ttacgatcct 2460aaccggtcaa
tgctggagct agtatttgca ccagcagagg aatggatatc acggactgat
2520tctgacatca tagatgcaac aatgaaagaa cttgagaaac tcttccctga
tgaaatctca 2580gctgaccaaa gcaaagctaa aattctgaag taccatgtcg
ttaagactcc aagatctggg 2640tacaagacca tcccaaactg tgaaccatgt
cgtcctctac aaagatcacc tattgaagga 2700ttctacttag ctggagatta
cacaaaacag aagtacttag cttccatgga aggcgctgtc 2760ctctctggca
aattctgctc tcagtctatt gttcaggatt acgagctact ggctgcgtct
2820ggaccaagaa agttgtcgga ggcaacagta tcatcatcat gagaaaaggg
cgaattcgtt 2880aaccgcagac gagctcgtga aatggcctct ttagtttttg
attgaatcat aggggtatta 2940gttttctatg gccgggagtg gtcttcttgc
ttaattgtaa tggaataacc agagaggaac 3000tactgtgtta tctttgagga
atgttgggct tttttcgttt gaattatcat gaatgaaatt 3060ttactttttc
ccaatacaag tttgttttcg tttcttggtt tttgttatcc cttggtttat
3120gtcttggttt ggcttaaatg attgaagatt acactaccta tgtttctgct
attcctgttg 3180aagatcacat ttgataataa tgcatcgaat gcattaaagt
ttcttattgg ctctgtcaaa 3240agtattgaag gtggattttt ctaattggca
agagaaagta ttaaagaggt gatttattag 3300tacttatatt tttctcagca
tctctctttc agtgttggag cttcataaaa ttagcacttc 3360agagtttcag
tcgggagctg aattcga 3387361701DNAArabidopsis thaliana 36atggttgtgt
ttgggaatgt ttctgcggcg aatttgcctt atcaaaacgg gtttttggag 60gcactttcat
ctggaggttg tgaactaatg ggacatagct ttagggttcc cacttctcaa
120gcgcttaaga caagaacaag gaggaggagt actgctggtc ctttgcaggt
agtttgtgtg 180gatattccaa ggccagagct agagaacact gtcaatttct
tggaagctgc tagtttatct 240gcatccttcc gtagtgctcc tcgtcctgct
aagcctttga aagttgtaat tgctggtgct 300ggattggctg gattgtcaac
tgcaaagtac ctggctgatg caggccacaa acctctgttg 360cttgaagcaa
gagatgttct tggtggaaag atagctgcat ggaaggatga agatggggac
420tggtatgaga ctggtttaca tattttcttc ggtgcttatc cgaatgtgca
gaatttattt 480ggagaacttg ggatcaatga tcggttgcag tggaaggaac
actccatgat ttttgctatg 540ccaagtaaac ctggagaatt tagtagattt
gacttcccag atgtcctacc agcaccctta 600aatggtattt gggctatttt
gcggaacaac gagatgctga catggccaga gaaaataaag 660tttgctattg
gacttttgcc agccatggtc ggcggtcagg cttatgttga ggcccaagat
720ggtttatcag tcaaagaatg gatggaaaag cagggagtac ctgagcgcgt
gaccgacgag 780gtgtttattg ccatgtcaaa ggcgctaaac tttataaacc
ctgatgaact gtcaatgcaa 840tgcattttga tagctttgaa ccggtttctt
caggaaaaac atggttccaa gatggcattc 900ttggatggta atcctccgga
aaggctttgt atgccagtag tggatcatat tcgatcacta 960ggtggggaag
tgcaacttaa ttctaggata aagaaaattg agctcaatga cgatggcacg
1020gttaagagtt tcttactcac taatggaagc actgtcgaag gagacgctta
tgtgtttgcc 1080gctccagtcg atatcctgaa gctcctttta ccagatccct
ggaaagaaat accgtacttc 1140aagaaattgg ataaattagt tggagtacca
gttattaatg ttcatatatg gtttgatcga 1200aaactgaaga acacatatga
tcacctactc tttagcagaa gtaaccttct gagcgtgtat 1260gccgacatgt
ccttaacttg taaggaatat tacgatccta accggtcaat gctggagcta
1320gtatttgcac cagcagagga atggatatca cggactgatt ctgacatcat
agatgcaaca 1380atgaaagaac ttgagaaact cttccctgat gaaatctcag
ctgaccaaag caaagctaaa 1440attctgaagt accatgtcgt taagactcca
agatctgtgt acaagaccat cccaaactgt 1500gaaccatgtc gtcctctaca
aagatcacct attgaaggat tctacttagc tggagattac 1560acaaaacaga
agtacttagc ttccatggaa ggcgctgtcc tctctggcaa attctgctct
1620cagtctattg ttcaggatta cgagctactg gctgcgtctg gaccaagaaa
gttgtcggag 1680gcaacagtat catcatcatg a 1701372010DNANicotiana
tabacum 37ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat
cctttctttt 60cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat
cctattttgt 120ttctttactt atttatttgc ttctatgtct ttgtacaaag
atttaaactc tatggcacat 180attttaaagt tgttagaaaa taaattcttt
caagattgat gaaagaactt tttaattgta 240gatatttcgt agattttatt
ctcttactac caatataacg cttgaattga cgaaaatttg 300tgtccaaata
tctagcaaaa aggtatccaa tgaaaatata tcatatgtga tcttcaaatc
360ttgtgtctta tgcaagattg atactttgtt caatggaaga gattgtgtgc
atatttttaa 420aatttttatt agtaataaag attctatata gctgttatag
agggataatt ttacaaagaa 480cactataaat atgattgttg ttgttagggt
gtcaatggtt cggttcgact ggttatttta 540taaaatttgt accataccat
ttttttcgat attctatttt gtataaccaa aattagactt 600ttcgaaatcg
tcccaatcat gtcggtttca cttcggtatc ggtaccgttc ggttaatttt
660catttttttt taaatgtcat taaaattcac tagtaaaaat agaatgcaat
aacatacgtt 720cttttatagg acttagcaaa agctctctag acatttttac
tgtttaaagg ataatgaatt 780aaaaaacatg aaagatggct agagtataga
tacacaacta ttcgacagca acgtaaaaga 840aaccaagtaa aagcaaagaa
aatataaatc acacgagtgg aaagatatta accaagttgg 900gattcaagaa
taaagtctat attaaatatt caaaaagata aatttaaata atatgaaagg
960aaacatattc aatacattgt agtttgctac tcataatcgc tagaatactt
tgtgccttgc 1020taataaagat acttgaaata gcttagttta aatataaata
gcataataga ttttaggaat 1080tagtattttg agtttaatta cttattgact
tgtaacagtt tttataattc caaggcccat 1140gaaaaattta atgctttatt
agttttaaac ttactatata aatttttcat atgtaaaatt 1200taatcggtat
agttcgatat tttttcaatt tatttttata aaataaaaaa cttaccctaa
1260ttatcggtac agttatagat ttatataaaa atctacggtt cttcagaaga
aacctaaaaa 1320tcggttcggt gcggacggtt cgatcggttt agtcgatttt
caaatattca ttgacactcc 1380tagttgttgt tataggtaaa aagcagttac
agagaggtaa aatataactt aaaaaatcag 1440ttctaaggaa aaattgactt
ttatagtaaa tgactgttat ataaggatgt tgttacagag 1500aggtatgagt
gtagttggta aattatgttc ttgacggtgt atgtcacata ttatttatta
1560aaactagaaa aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa
atcggctgaa 1620tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa
agaatcggtg actgttgatg 1680atataaacaa agggcacatt ggtcaataac
cataaaaaat tatatgacag ctacagttgg 1740tagcatgtgc tcagctattg
aacaaatcta aagaaggtac atctgtaacc ggaacaccac 1800ttaaatgact
aaattaccct catcagaaag cagatggagt gctacaaata acacactatt
1860caacaaccat aaataaaacg tgttcagcta ctaaaacaaa tataaataaa
tctatgtttg 1920taagcactcc agccatgtta atggagtgct attgcctgtt
aactctcact tataaaatag 1980tagtagaaaa aatatgaacc aaaacacaac
201038254DNAAgrobacterium tumefaciens 38gatcgttcaa acatttggca
ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60atgattatca tataatttct
gttgaattac gttaagcatg taataattaa catgtaatgc 120atgacgttat
ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac
180gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc
ggtgtcatct 240atgttactag atcg 25439202DNANicotiana tabacum
39tatgggaagg ttctgacttt ggatggagca attcaacata cagagaatgg tggatttcca
60tacactgaaa tgattgttca tctaccactt ggttccatcc caaacccaaa aaaggttttg
120atcatcggcg gaggaattgg ttttacatta ttcgaaatgc ttcgttatcc
ttcaatcgaa 180aaaattgaca ttgttgagat cg 20240344DNANicotiana tabacum
40ccagcaaaag atttgtttga gaggccattc tttgaggcag tagccaaagc ccttaggcca
60ggaggagttg tatgcacaca ggctgaaagc atttggcttc atatgcatat tattaagcaa
120atcattgcta actgtcgtca agtctttaag ggttctgtca actatgcttg
gacaaccgtt 180ccaacatatc ccaccggtgt gatcggttat atgctctgct
ctactgaagg gccagaagtt 240gacttcaaga atccagtaaa tccaattgac
aaagagacaa ctcaagtcaa gtccaaatta 300ggacctctca agttctacaa
ctctgatatt cacaaagcag catt 34441155DNAArabidopsis thaliana
41gtaataagat cttcaacacc tacaccattt ttttaatcac tactacccat tgcattgaac
60aaacttccaa gttcttctta gcttcagatt aagaaagtac cctttcttgg ctttgttgat
120gtggtaccat tgtccattgt cttgtgtgtt tccag 155424115DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
42ctcgaggatc taaattgtga gttcaatctc ttccctattg gattgattat cctttctttt
60cttccaattt gtgtttcttt ttgcctaatt tattgtgtta tcccctttat cctattttgt
120ttctttactt atttatttgc ttctatgtct ttgtacaaag atttaaactc
tatggcacat 180attttaaagt tgttagaaaa taaattcttt caagattgat
gaaagaactt tttaattgta 240gatatttcgt agattttatt ctcttactac
caatataacg cttgaattga cgaaaatttg 300tgtccaaata tctagcaaaa
aggtatccaa tgaaaatata tcatatgtga tcttcaaatc 360ttgtgtctta
tgcaagattg atactttgtt caatggaaga gattgtgtgc atatttttaa
420aatttttatt agtaataaag attctatata gctgttatag agggataatt
ttacaaagaa 480cactataaat atgattgttg ttgttagggt gtcaatggtt
cggttcgact ggttatttta 540taaaatttgt accataccat ttttttcgat
attctatttt gtataaccaa aattagactt 600ttcgaaatcg tcccaatcat
gtcggtttca cttcggtatc ggtaccgttc ggttaatttt 660catttttttt
taaatgtcat taaaattcac tagtaaaaat agaatgcaat aacatacgtt
720cttttatagg acttagcaaa agctctctag acatttttac tgtttaaagg
ataatgaatt 780aaaaaacatg aaagatggct agagtataga tacacaacta
ttcgacagca acgtaaaaga 840aaccaagtaa aagcaaagaa aatataaatc
acacgagtgg aaagatatta accaagttgg 900gattcaagaa taaagtctat
attaaatatt caaaaagata aatttaaata atatgaaagg 960aaacatattc
aatacattgt agtttgctac tcataatcgc tagaatactt tgtgccttgc
1020taataaagat acttgaaata gcttagttta aatataaata gcataataga
ttttaggaat 1080tagtattttg agtttaatta cttattgact tgtaacagtt
tttataattc caaggcccat 1140gaaaaattta atgctttatt agttttaaac
ttactatata aatttttcat atgtaaaatt 1200taatcggtat agttcgatat
tttttcaatt tatttttata aaataaaaaa cttaccctaa 1260ttatcggtac
agttatagat ttatataaaa atctacggtt cttcagaaga aacctaaaaa
1320tcggttcggt gcggacggtt cgatcggttt agtcgatttt caaatattca
ttgacactcc 1380tagttgttgt tataggtaaa aagcagttac agagaggtaa
aatataactt aaaaaatcag 1440ttctaaggaa aaattgactt ttatagtaaa
tgactgttat ataaggatgt tgttacagag 1500aggtatgagt gtagttggta
aattatgttc ttgacggtgt atgtcacata ttatttatta 1560aaactagaaa
aaacagcgtc aaaactagca aaaatccaac ggacaaaaaa atcggctgaa
1620tttgatttgg ttccaacatt taaaaaagtt tcagtgagaa agaatcggtg
actgttgatg 1680atataaacaa agggcacatt ggtcaataac cataaaaaat
tatatgacag ctacagttgg 1740tagcatgtgc tcagctattg aacaaatcta
aagaaggtac atctgtaacc ggaacaccac 1800ttaaatgact aaattaccct
catcagaaag cagatggagt gctacaaata acacactatt 1860caacaaccat
aaataaaacg tgttcagcta ctaaaacaaa tataaataaa tctatgtttg
1920taagcactcc agccatgtta atggagtgct attgcctgtt aactctcact
tataaaatag 1980tagtagaaaa aatatgaacc aaaacacaac tttatcgcca
tcatttacat accactccac 2040ctttaatgaa ggatcaactt ccgcgaatat
catctcagca agtgcaattc ctgctatgat 2100cccgtcttcc tttgctagaa
aatgagcatc ggattccata tcaagaggaa ttgtcgcctt 2160acaagtcaca
tctcctaaat tcccagcatc ttcagagagt gcaagtttca taacttcctt
2220taaatcataa gttgggtgtg ctggtggttt cacctctaat gactccactc
ttgtattctt 2280ggtggctatt gctgacattt tcaccaccaa ccttggagct
gtaattgcat aaggatgcac 2340tgtagcagtg aaaggaatag ctctaaacat
gtccgtcgct tctcttccat ttcttctcat 2400tttcgatttt gattcttatt
tctttccagt agctcctgct ctgtgaattt ctccgctcac 2460gatagatctg
cttatactcc ttacattcaa ccttagatct ggtctcgatt ctctgtttct
2520ctgttttttt cttttggtcg agaatctgat gtttgtttat gttctgtcac
cattaataat 2580aatgaactct ctcattcata caatgattag tttctctcgt
ctacaaaacg atatgttgca 2640ttttcacttt tcttcttttt ttctaagatg
atttgctttg accaatttgt ttagatcttt 2700attttatttt attttctggt
gggttggtgg aaattgaaaa aaaaaaaaac agcataaatt 2760gttatttgtt
aatgtattca ttttttggct atttgttctg ggtaaaaatc tgcttctact
2820attgaatctt tcctggattt tttactccta ttgggttttt atagtaaaaa
tacataataa 2880aaggaaaaca aaagttttat agattctctt aaacccctta
cgataaaagt tggaatcaaa 2940ataattcagg atcagatgct ctttgattga
ttcagatgcg attacagttg catggcaaat 3000tttctagatc cgtcgtcaca
ttttattttc tgtttaaata tctaaatctg atatatgatg 3060tcgacaaatt
ctggtggctt atacatcact tcaactgttt tcttttggct ttgtttgtca
3120acttggtttt caatacgatt tgtgatttcg atcgctgaat ttttaataca
agcaaactga 3180tgttaaccac aagcaagaga tgtgacctgc cttattaaca
tcgtattact tactactagt 3240cgtattctca acgcaatcgt ttttgtattt
ctcacattat gccgcttctc tactctttat 3300tccttttggt ccacgcattt
tctatttgtg gcaatccctt tcacaacctg atttcccact 3360ttggatcatt
tgtctgaaga ctctcttgaa tcgttaccac ttgtttcttg tgcatgctct
3420gttttttaga attaatgata aaactattcc atagtcttga gttttcagct
tgttgattct 3480tttgcttttg gttttctgca gatgtttaga gctattcctt
tcactgctac agtgcatcct 3540tatgcaatta cagctccaag gttggtggtg
aaaatgtcag caatagccac caagaataca 3600agagtggagt cattagaggt
gaaaccacca gcacacccaa cttatgattt aaaggaagtt 3660atgaaacttg
cactctctga agatgctggg aatttaggag atgtgacttg taaggcgaca
3720attcctcttg atatggaatc cgatgctcat tttctagcaa aggaagacgg
gatcatagca 3780ggaattgcac ttgctgagat gatattcgcg gaagttgatc
cttcattaaa ggtggagtgg 3840tatgtaaatg atggcgataa agatcgttca
aacatttggc aataaagttt cttaagattg 3900aatcctgttg ccggtcttgc
gatgattatc atataatttc tgttgaatta cgttaagcat 3960gtaataatta
acatgtaatg catgacgtta tttatgagat gggtttttat gattagagtc
4020ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa
ctaggataaa 4080ttatcgcgcg cggtgtcatc tatgttacta gatcg
4115432835DNAArtificial SequenceArtificially created chimeric
nucleic acid sequence 43gaattcaatg gagaaggaaa atatttccag tgtaaacaca
agtgaatgaa gagaagccaa 60aataatctct atcattcaag ccttaggtgg
agattaaaaa
aattatttac tttcttatca 120aagtaatagg tgatcaacag ctttcgtaaa
acgtcattag gagaatatta taatctcttt 180tatgctgaag aacccacata
aggaagatca taaaatacat gactttcaga tgacttcttg 240gagctttatt
tttaaagagt ggctagctgg tcagcaaaga ggtgctcgtc agatatcata
300aaattttact attatttgtt ttaagaggga gatggggcac acatgcttgt
gacaaaagta 360agaggaagaa aggagacaga agaggaaata gatttggggg
gggggggggg ggtttcacaa 420tcaaagaaaa tttttaaaat ggagagagaa
atgagcacac acatatacta acaaaatttt 480actaataatt gcaccgagac
aaacttatat tttagttcca aaatgtcagt ctaaccctgc 540acgttgtaat
gaatttttaa ctattatatt atatcgagtt gcgccctcca ctcctcggtg
600tccaaattgt atttaaatgc atagatgttt attgggagtg tacagcaagc
tttcggaaaa 660tacaaaccat aatactttct cttcttcaat ttgtttagtt
taattttgaa atttatcgcc 720atcatttaca taccactcca cctttaatga
aggatcaact tccgcgaata tcatctcagc 780aagtgcaatt cctgctatga
tcccgtcttc ctttgctaga aaatgagcat cggattccat 840atcaagagga
attgtcgcct tacaagtcac atctcctaaa ttcccagcat cttcagagag
900tgcaagtttc ataacttcct ttaaatcata agttgggtgt gctggtggtt
tcacctctaa 960tgactccact cttgtattct tggtggctat tgctgacatt
ttcaccacca accttggagc 1020tgtaattgca taaggatgca ctgtagcagt
gaaaggaata gctctaaaca tgtccgtcgc 1080ttctcttcca tttcttctca
ttttcgattt tgattcttat ttctttccag tagctcctgc 1140tctgtgaatt
tctccgctca cgatagatct gcttatactc cttacattca accttagatc
1200tggtctcgat tctctgtttc tctgtttttt tcttttggtc gagaatctga
tgtttgttta 1260tgttctgtca ccattaataa taatgaactc tctcattcat
acaatgatta gtttctctcg 1320tctacaaaac gatatgttgc attttcactt
ttcttctttt tttctaagat gatttgcttt 1380gaccaatttg tttagatctt
tattttattt tattttctgg tgggttggtg gaaattgaaa 1440aaaaaaaaaa
cagcataaat tgttatttgt taatgtattc attttttggc tatttgttct
1500gggtaaaaat ctgcttctac tattgaatct ttcctggatt ttttactcct
attgggtttt 1560tatagtaaaa atacataata aaaggaaaac aaaagtttta
tagattctct taaacccctt 1620acgataaaag ttggaatcaa aataattcag
gatcagatgc tctttgattg attcagatgc 1680gattacagtt gcatggcaaa
ttttctagat ccgtcgtcac attttatttt ctgtttaaat 1740atctaaatct
gatatatgat gtcgacaaat tctggtggct tatacatcac ttcaactgtt
1800ttcttttggc tttgtttgtc aacttggttt tcaatacgat ttgtgatttc
gatcgctgaa 1860tttttaatac aagcaaactg atgttaacca caagcaagag
atgtgacctg ccttattaac 1920atcgtattac ttactactag tcgtattctc
aacgcaatcg tttttgtatt tctcacatta 1980tgccgcttct ctactcttta
ttccttttgg tccacgcatt ttctatttgt ggcaatccct 2040ttcacaacct
gatttcccac tttggatcat ttgtctgaag actctcttga atcgttacca
2100cttgtttctt gtgcatgctc tgttttttag aattaatgat aaaactattc
catagtcttg 2160agttttcagc ttgttgattc ttttgctttt ggttttctgc
agatgtttag agctattcct 2220ttcactgcta cagtgcatcc ttatgcaatt
acagctccaa ggttggtggt gaaaatgtca 2280gcaatagcca ccaagaatac
aagagtggag tcattagagg tgaaaccacc agcacaccca 2340acttatgatt
taaaggaagt tatgaaactt gcactctctg aagatgctgg gaatttagga
2400gatgtgactt gtaaggcgac aattcctctt gatatggaat ccgatgctca
ttttctagca 2460aaggaagacg ggatcatagc aggaattgca cttgctgaga
tgatattcgc ggaagttgat 2520ccttcattaa aggtggagtg gtatgtaaat
gatggcgata aagcaagtgt gttgcctttg 2580tgtggaaatg aagaggtact
tgcgaggact ttgcgtttat cagtttatgt gtttgtatat 2640ctatttgatc
cagttattat ggattatata cgcttgaaac tcattttaag ccattgttat
2700tgaacgttta tcaaatactt tattatgcca agcaagtcaa acacatgctt
gttgattgaa 2760atcaagctat agaaatctct tcttcacata cagcagttta
gattcacaat acaacaagcg 2820aaacgataaa gtttc 2835441893DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
44aatatgaaag gaaacatatt caatacattg tagtttgcta ctcataatcg ctagaatact
60ttgtgccttg ctaataaaga tacttgaaat agcttagttt aaatataaat agcataatag
120attttaggaa ttagtatttt gagtttaatt acttattgac ttgtaacagt
ttttataatt 180ccaaggccca tgaaaaattt aatgctttat tagttttaaa
cttactatat aaatttttca 240tatgtaaaat ttaatcggta tagttcgata
ttttttcaat ttatttttat aaaataaaaa 300acttacccta attatcggta
cagttataga tttatataaa aatctacggt tcttcagaag 360aaacctaaaa
atcggttcgg tgcggacggt tcgatcggtt tagtcgattt tcaaatattc
420attgacactc ctagttgttg ttataggtaa aaagcagtta cagagaggta
aaatataact 480taaaaaatca gttctaagga aaaattgact tttatagtaa
atgactgtta tataaggatg 540ttgttacaga gaggtatgag tgtagttggt
aaattatgtt cttgacggtg tatgtcacat 600attatttatt aaaactagaa
aaaacagcgt caaaactagc aaaaatccaa cggacaaaaa 660aatcggctga
atttgatttg gttccaacat ttaaaaaagt ttcagtgaga aagaatcggt
720gactgttgat gatataaaca aagggcacat tggtcaataa ccataaaaaa
ttatatgaca 780gctacagttg gtagcatgtg ctcagctatt gaacaaatct
aaagaaggta catctgtaac 840cggaacacca cttaaatgac taaattaccc
tcatcagaaa gcagatggag tgctacaaat 900aacacactat tcaacaacca
taaataaaac gtgttcagct actaaaacaa atataaataa 960atctatgttt
gtaagcactc cagccatgtt aatggagtgc tattgcctgt taactctcac
1020ttataaaata gtagtagaaa aaatatgaac caaaacacaa ccgatctcaa
caatgtcaat 1080tttttcgatt gaaggataac gaagcatttc gaataatgta
aaaccaattc ctccgccgat 1140gatcaaaacc ttttttgggt ttgggatgga
accaagtggt agatgaacaa tcatttcagt 1200gtatggaaat ccaccattct
ctgtatgttg aattgctcca tccaaagtca gaaccttccc 1260atagtaataa
gatcttcaac acctacacca tttttttaat cactactacc cattgcattg
1320aacaaacttc caagttcttc ttagcttcag attaagaaag taccctttct
tggctttgtt 1380gatgtggtac cattgtccat tgtcttgtgt gtttccagta
tgggaaggtt ctgactttgg 1440atggagcaat tcaacataca gagaatggtg
gatttccata cactgaaatg attgttcatc 1500taccacttgg ttccatccca
aacccaaaaa aggttttgat catcggcgga ggaattggtt 1560ttacattatt
cgaaatgctt cgttatcctt caatcgaaaa aattgacatt gttgagatcg
1620gcaagtgtgt tgcctttgtg tggaaatgaa gaggtacttg cgaggacttt
gcgtttatca 1680gtttatgtgt ttgtatatct atttgatcca gttattatgg
attatatacg cttgaaactc 1740attttaagcc attgttattg aacgtttatc
aaatacttta ttatgccaag caagtcaaac 1800acatgcttgt tgattgaaat
caagctatag aaatctcttc ttcacataca gcagtttaga 1860ttcacaatac
aacaagcgaa acgataaagt ttc 1893454098DNAArtificial
SequenceArtificially created chimeric nucleic acid sequence
45gatctaaatt gtgagttcaa tctcttccct attggattga ttatcctttc ttttcttcca
60atttgtgttt ctttttgcct aatttattgt gttatcccct ttatcctatc ttgtttcttt
120acttatttat ttgcttctat gtctttgtac aaagatttaa actctatggc
acatatttta 180aagttgttag aaaataaatt ctttcaagat tgatgaaaga
actttttaat tgtagatatt 240tcgtagattt tattctctta ctaccaatat
aacgcttgaa ttgacgaaaa tttgtgtcca 300aatatttagc aaaaaggtat
ccaatgaaaa tatatcatat gtgatcttca aatcttgtgt 360cttatgcaag
attgatactt tgttcaatgg aagagattgt gtgcatattt tcaaaatttt
420tattagtaat aaagattcta tatagctgtt atagagggat aattttacaa
agaacactat 480aaatatgatt gttgttgtta ggggtgtcaa tggttcggtt
cgactggtta ttttataaaa 540tttgtaccat accatttttt cggatattct
attttgtata accaaaatta gacttttcga 600aatcgtccca atcatgtcgg
tttcacttcg gtatcggtac cgttcggtta attttcattt 660ttttttaaat
gtcattaaaa ttcactagta aaaatagaat gcaataacat acgttctttt
720ataggactta gcaaaactct ctagacattt ttactgttta aaggataatg
aattaaaaaa 780catgaaagat ggctagagta tagatacaca actattcgac
agcaacgtaa aagaaaccaa 840gtaaaagcaa agaaaatata aatcacacga
gtggaaagat attaaccaag ttgggattca 900agaataaagt ctatattaaa
tattcaaaaa gataaattta aataatatga aaggaaacat 960attcaataca
ttgtagtttg ctactcataa tcgctagaat actttgtgcc ttgctaataa
1020agatactaga aatagcttag tttaaatata aatagcataa tagattttag
gaattagtat 1080tttgagttta attacttatt gacttgtaac agtttttata
attccaaggc ccaatgaaaa 1140atttaatgct ttattagttt taaacttact
atataaattt ttcatatgta aaatttaatc 1200ggtatagttc gatatttttt
caatttattt ttataaaata aaaaacttac cctaattatc 1260ggtacagtta
tagatttata taaaaatcta cggttcttca gaagaaacct aaaaatcggt
1320tcggtgcggg acggttcgat cggtttagtc gattttcaaa tattcattga
cactcctagt 1380tgttgttata ggtaaaaagc agttacagag aggtaaaata
taacttaaaa aatcagttct 1440aaggaaaaat tgacttttat agtaaatgac
tgttatataa ggatgttgtt acagagaggt 1500atgagtgtag ttggtaaatt
atgttcttga cggtgtatgt cgcatattat ttattaaaac 1560tagaaaaaac
agcgtcaaaa ctagcaaaaa tccaaaggac aaaaaaatcg gctgaatttg
1620atttggttcc aacatttaaa aaagtttcag tgagaaagaa tacggtgact
gttgatgata 1680taaacaaagg gcacattggt caataaccat aaaaaattat
atgacagcta cagttggtag 1740catgtgctca gctattgaac aaatctaaag
aaggtacatc tgtaaccgga acagcactta 1800aatgactaaa ttaccctcat
cagaaagcag atggagtgct acaaataaca cactattcaa 1860caaccataaa
taaaacgtgt tcagctacta aaacaaatat aaataaatct atgtatgtaa
1920gcactccagc catgttaatg gagtgctatt gcctgttaac tctcactata
aaatagtagt 1980agaaaaaata tgaaccaaaa cacaacaatg ctgctttgtg
aatatcagag ttgtagaact 2040tgagaggtcc taatttggac ttgacttgag
ttgtctcttt gtcaattgga tttactggat 2100tcttgaagtc aacttctggc
ccttcagtag agcagagcat ataaccgatc acaccggtgg 2160gatatgttgg
aacggttgtc caagcatagt tgacagaacc cttaaagact tgacgacagt
2220tagcaatgat ttgcttaata atatgcatat gaagccaaat gctttcagcc
tgtgtgcata 2280caactcctcc tggcctaagg gctttggcta ctgcctcaaa
gaatggcctc tcaaacaaat 2340cttttgctgg gtccgtcgct tctcttccat
ttcttctcat tttcgatttt gattcttatt 2400tctttccagt agctcctgct
ctgtgaattt ctccgctcac gatagatctg cttatactcc 2460ttacattcaa
ccttagatct ggtctcgatt ctctgtttct ctgttttttt cttttggtcg
2520agaatctgat gtttgtttat gttctgtcac cattaataat aatgaactct
ctcattcata 2580caatgattag tttctctcgt ctacaaaacg atatgttgca
ttttcacttt tcttcttttt 2640ttctaagatg atttgctttg accaatttgt
ttagatcttt attttatttt attttctggt 2700gggttggtgg aaattgaaaa
aaaaaaaaac agcataaatt gttatttgtt aatgtattca 2760ttttttggct
atttgttctg ggtaaaaatc tgcttctact attgaatctt tcctggattt
2820tttactccta ttgggttttt atagtaaaaa tacataataa aaggaaaaca
aaagttttat 2880agattctctt aaacccctta cgataaaagt tggaatcaaa
ataattcagg atcagatgct 2940ctttgattga ttcagatgcg attacagttg
catggcaaat tttctagatc cgtcgtcaca 3000ttttattttc tgtttaaata
tctaaatctg atatatgatg tcgacaaatt ctggtggctt 3060atacatcact
tcaactgttt tcttttggct ttgtttgtca acttggtttt caatacgatt
3120tgtgatttcg atcgctgaat ttttaataca agcaaactga tgttaaccac
aagcaagaga 3180tgtgacctgc cttattaaca tcgtattact tactactagt
cgtattctca acgcaatcgt 3240ttttgtattt ctcacattat gccgcttctc
tactctttat tccttttggt ccacgcattt 3300tctatttgtg gcaatccctt
tcacaacctg atttcccact ttggatcatt tgtctgaaga 3360ctctcttgaa
tcgttaccac ttgtttcttg tgcatgctct gttttttaga attaatgata
3420aaactattcc atagtcttga gttttcagct tgttgattct tttgcttttg
gttttctgca 3480gccagcaaaa gatttgtttg agaggccatt ctttgaggca
gtagccaaag cccttaggcc 3540aggaggagtt gtatgcacac aggctgaaag
catttggctt catatgcata ttattaagca 3600aatcattgct aactgtcgtc
aagtctttaa gggttctgtc aactatgctt ggacaaccgt 3660tccaacatat
cccaccggtg tgatcggtta tatgctctgc tctactgaag ggccagaagt
3720tgacttcaag aatccagtaa atccaattga caaagagaca actcaagtca
agtccaaatt 3780aggacctctc aagttctaca actctgatat tcacaaagca
gcattgcaag tgtgttgcct 3840ttgtgtggaa atgaagaggt acttgcgagg
actttgcgtt tatcagttta tgtgtttgta 3900tatctatttg atccagttat
tatggattat atacgcttga aactcatttt aagccattgt 3960tattgaacgt
ttatcaaata ctttattatg ccaagcaagt caaacacatg cttgttgatt
4020gaaatcaagc tatagaaatc tcttcttcac atacagcagt ttagattcac
aatacaacaa 4080gcgaaacgat aaagtttc 409846307DNAAgrobacterium
tumefaciens 46gatcatgagc ggagaattaa gggagtcacg ttatgacccc
cgccgatgac gcgggacaag 60ccgttttacg tttggaactg acagaaccgc aacgttgaag
gagccactca gccgcgggtt 120tctggagttt aatgagctaa gcacatacgt
cagaaaccat tattgcgcgt tcaaaagtcg 180cctaaggtca ctatcagcta
gcaaatattt cttgtcaaaa atgctccact gacgttccat 240aaattcccct
cggtatccaa ttagagtctc atattcactc tcaatccaaa taatctgcac 300cggatct
30747795DNAEscherichia coli 47atgattgaac aagatggatt gcacgcaggt
tctccggccg cttgggtgga gaggctattc 60ggctatgact gggcacaaca gacaatcggc
tgctctgatg ccgccgtgtt ccggctgtca 120gcgcaggggc gcccggttct
ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180caggacgagg
cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
240ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
gccggggcag 300gatctcctgt catctcacct tgctcctgcc gagaaagtat
ccatcatggc tgatgcaatg 360cggcggctgc atacgcttga tccggctacc
tgcccattcg accaccaagc gaaacatcgc 420atcgagcgag cacgtactcg
gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480gagcatcagg
ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
540ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
ggtggaaaat 600ggccgctttt ctggattcat cgactgtggc cggctgggtg
tggcggaccg ctatcaggac 660atagcgttgg ctacccgtga tattgctgaa
gagcttggcg gcgaatgggc tgaccgcttc 720ctcgtgcttt acggtatcgc
cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780gacgagttct tctga
795481757DNAArtificial SequenceArtificially created chimeric
nucleic acid sequence 48gatcatgagc ggagaattaa gggagtcacg ttatgacccc
cgccgatgac gcgggacaag 60ccgttttacg tttggaactg acagaaccgc aacgttgaag
gagccactca gccgcgggtt 120tctggagttt aatgagctaa gcacatacgt
cagaaaccat tattgcgcgt tcaaaagtcg 180cctaaggtca ctatcagcta
gcaaatattt cttgtcaaaa atgctccact gacgttccat 240aaattcccct
cggtatccaa ttagagtctc atattcactc tcaatccaaa taatctgcac
300cggatctgga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
ctccggccgc 360ttgggtggag aggctattcg gctatgactg ggcacaacag
acaatcggct gctctgatgc 420cgccgtgttc cggctgtcag cgcaggggcg
cccggttctt tttgtcaaga ccgacctgtc 480cggtgccctg aatgaactgc
aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 540cgttccttgc
gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
600gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
agaaagtatc 660catcatggct gatgcaatgc ggcggctgca tacgcttgat
ccggctacct gcccattcga 720ccaccaagcg aaacatcgca tcgagcgagc
acgtactcgg atggaagccg gtcttgtcga 780tcaggatgat ctggacgaag
agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 840caaggcgcgc
atgcccgacg gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc
900gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
ggctgggtgt 960ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
attgctgaag agcttggcgg 1020cgaatgggct gaccgcttcc tcgtgcttta
cggtatcgcc gctcccgatt cgcagcgcat 1080cgccttctat cgccttcttg
acgagttctt ctgagcggga ctctggggtt cgaaatgacc 1140gaccaagcga
cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
1200aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
gcgcggggat 1260ctcatgctgg agttcttcgc ccacgggatc tctgcggaac
aggcggtcga aggtgccgat 1320atcattacga cagcaacggc cgacaagcac
aacgccacga tcctgagcga caatatgatc 1380gggcccggcg tccacatcaa
cggcgtcggc ggcgactgcc caggcaagac cgagatgcac 1440cgcgatatct
tgctgcgttc ggatattttc gtggagttcc cgccacagac ccggatgatc
1500cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt
tgccggtctt 1560gcgatgatta tcatataatt tctgttgaat tacgttaagc
atgtaataat taacatgtaa 1620tgcatgacgt tatttatgag atgggttttt
atgattagag tcccgcaatt atacatttaa 1680tacgcgatag aaaacaaaat
atagcgcgca aactaggata aattatcgcg cgcggtgtca 1740tctatgttac tagatcg
1757495872DNAArtificial SequenceArtificially created chimeric
nucleic acid sequence 49gatcatgagc ggagaattaa gggagtcacg ttatgacccc
cgccgatgac gcgggacaag 60ccgttttacg tttggaactg acagaaccgc aacgttgaag
gagccactca gccgcgggtt 120tctggagttt aatgagctaa gcacatacgt
cagaaaccat tattgcgcgt tcaaaagtcg 180cctaaggtca ctatcagcta
gcaaatattt cttgtcaaaa atgctccact gacgttccat 240aaattcccct
cggtatccaa ttagagtctc atattcactc tcaatccaaa taatctgcac
300cggatctgga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
ctccggccgc 360ttgggtggag aggctattcg gctatgactg ggcacaacag
acaatcggct gctctgatgc 420cgccgtgttc cggctgtcag cgcaggggcg
cccggttctt tttgtcaaga ccgacctgtc 480cggtgccctg aatgaactgc
aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 540cgttccttgc
gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
600gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
agaaagtatc 660catcatggct gatgcaatgc ggcggctgca tacgcttgat
ccggctacct gcccattcga 720ccaccaagcg aaacatcgca tcgagcgagc
acgtactcgg atggaagccg gtcttgtcga 780tcaggatgat ctggacgaag
agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 840caaggcgcgc
atgcccgacg gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc
900gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
ggctgggtgt 960ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
attgctgaag agcttggcgg 1020cgaatgggct gaccgcttcc tcgtgcttta
cggtatcgcc gctcccgatt cgcagcgcat 1080cgccttctat cgccttcttg
acgagttctt ctgagcggga ctctggggtt cgaaatgacc 1140gaccaagcga
cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
1200aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
gcgcggggat 1260ctcatgctgg agttcttcgc ccacgggatc tctgcggaac
aggcggtcga aggtgccgat 1320atcattacga cagcaacggc cgacaagcac
aacgccacga tcctgagcga caatatgatc 1380gggcccggcg tccacatcaa
cggcgtcggc ggcgactgcc caggcaagac cgagatgcac 1440cgcgatatct
tgctgcgttc ggatattttc gtggagttcc cgccacagac ccggatgatc
1500cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt
tgccggtctt 1560gcgatgatta tcatataatt tctgttgaat tacgttaagc
atgtaataat taacatgtaa 1620tgcatgacgt tatttatgag atgggttttt
atgattagag tcccgcaatt atacatttaa 1680tacgcgatag aaaacaaaat
atagcgcgca aactaggata aattatcgcg cgcggtgtca 1740tctatgttac
tagatcgctc gaggatctaa attgtgagtt caatctcttc cctattggat
1800tgattatcct ttcttttctt ccaatttgtg tttctttttg cctaatttat
tgtgttatcc 1860cctttatcct attttgtttc tttacttatt tatttgcttc
tatgtctttg tacaaagatt 1920taaactctat ggcacatatt ttaaagttgt
tagaaaataa attctttcaa gattgatgaa 1980agaacttttt aattgtagat
atttcgtaga ttttattctc ttactaccaa tataacgctt 2040gaattgacga
aaatttgtgt ccaaatatct agcaaaaagg tatccaatga aaatatatca
2100tatgtgatct tcaaatcttg tgtcttatgc aagattgata ctttgttcaa
tggaagagat 2160tgtgtgcata tttttaaaat ttttattagt aataaagatt
ctatatagct gttatagagg 2220gataatttta caaagaacac tataaatatg
attgttgttg ttagggtgtc aatggttcgg 2280ttcgactggt tattttataa
aatttgtacc ataccatttt tttcgatatt ctattttgta 2340taaccaaaat
tagacttttc gaaatcgtcc caatcatgtc ggtttcactt cggtatcggt
2400accgttcggt taattttcat ttttttttaa atgtcattaa aattcactag
taaaaataga 2460atgcaataac atacgttctt ttataggact tagcaaaagc
tctctagaca tttttactgt 2520ttaaaggata atgaattaaa aaacatgaaa
gatggctaga gtatagatac acaactattc 2580gacagcaacg taaaagaaac
caagtaaaag caaagaaaat ataaatcaca cgagtggaaa 2640gatattaacc
aagttgggat tcaagaataa agtctatatt aaatattcaa aaagataaat
2700ttaaataata tgaaaggaaa catattcaat acattgtagt ttgctactca
taatcgctag 2760aatactttgt gccttgctaa taaagatact tgaaatagct
tagtttaaat ataaatagca 2820taatagattt taggaattag tattttgagt
ttaattactt
attgacttgt aacagttttt 2880ataattccaa ggcccatgaa aaatttaatg
ctttattagt tttaaactta ctatataaat 2940ttttcatatg taaaatttaa
tcggtatagt tcgatatttt ttcaatttat ttttataaaa 3000taaaaaactt
accctaatta tcggtacagt tatagattta tataaaaatc tacggttctt
3060cagaagaaac ctaaaaatcg gttcggtgcg gacggttcga tcggtttagt
cgattttcaa 3120atattcattg acactcctag ttgttgttat aggtaaaaag
cagttacaga gaggtaaaat 3180ataacttaaa aaatcagttc taaggaaaaa
ttgactttta tagtaaatga ctgttatata 3240aggatgttgt tacagagagg
tatgagtgta gttggtaaat tatgttcttg acggtgtatg 3300tcacatatta
tttattaaaa ctagaaaaaa cagcgtcaaa actagcaaaa atccaacgga
3360caaaaaaatc ggctgaattt gatttggttc caacatttaa aaaagtttca
gtgagaaaga 3420atcggtgact gttgatgata taaacaaagg gcacattggt
caataaccat aaaaaattat 3480atgacagcta cagttggtag catgtgctca
gctattgaac aaatctaaag aaggtacatc 3540tgtaaccgga acaccactta
aatgactaaa ttaccctcat cagaaagcag atggagtgct 3600acaaataaca
cactattcaa caaccataaa taaaacgtgt tcagctacta aaacaaatat
3660aaataaatct atgtttgtaa gcactccagc catgttaatg gagtgctatt
gcctgttaac 3720tctcacttat aaaatagtag tagaaaaaat atgaaccaaa
acacaacttt atcgccatca 3780tttacatacc actccacctt taatgaagga
tcaacttccg cgaatatcat ctcagcaagt 3840gcaattcctg ctatgatccc
gtcttccttt gctagaaaat gagcatcgga ttccatatca 3900agaggaattg
tcgccttaca agtcacatct cctaaattcc cagcatcttc agagagtgca
3960agtttcataa cttcctttaa atcataagtt gggtgtgctg gtggtttcac
ctctaatgac 4020tccactcttg tattcttggt ggctattgct gacattttca
ccaccaacct tggagctgta 4080attgcataag gatgcactgt agcagtgaaa
ggaatagctc taaacatgtc cgtcgcttct 4140cttccatttc ttctcatttt
cgattttgat tcttatttct ttccagtagc tcctgctctg 4200tgaatttctc
cgctcacgat agatctgctt atactcctta cattcaacct tagatctggt
4260ctcgattctc tgtttctctg tttttttctt ttggtcgaga atctgatgtt
tgtttatgtt 4320ctgtcaccat taataataat gaactctctc attcatacaa
tgattagttt ctctcgtcta 4380caaaacgata tgttgcattt tcacttttct
tctttttttc taagatgatt tgctttgacc 4440aatttgttta gatctttatt
ttattttatt ttctggtggg ttggtggaaa ttgaaaaaaa 4500aaaaaacagc
ataaattgtt atttgttaat gtattcattt tttggctatt tgttctgggt
4560aaaaatctgc ttctactatt gaatctttcc tggatttttt actcctattg
ggtttttata 4620gtaaaaatac ataataaaag gaaaacaaaa gttttataga
ttctcttaaa ccccttacga 4680taaaagttgg aatcaaaata attcaggatc
agatgctctt tgattgattc agatgcgatt 4740acagttgcat ggcaaatttt
ctagatccgt cgtcacattt tattttctgt ttaaatatct 4800aaatctgata
tatgatgtcg acaaattctg gtggcttata catcacttca actgttttct
4860tttggctttg tttgtcaact tggttttcaa tacgatttgt gatttcgatc
gctgaatttt 4920taatacaagc aaactgatgt taaccacaag caagagatgt
gacctgcctt attaacatcg 4980tattacttac tactagtcgt attctcaacg
caatcgtttt tgtatttctc acattatgcc 5040gcttctctac tctttattcc
ttttggtcca cgcattttct atttgtggca atccctttca 5100caacctgatt
tcccactttg gatcatttgt ctgaagactc tcttgaatcg ttaccacttg
5160tttcttgtgc atgctctgtt ttttagaatt aatgataaaa ctattccata
gtcttgagtt 5220ttcagcttgt tgattctttt gcttttggtt ttctgcagat
gtttagagct attcctttca 5280ctgctacagt gcatccttat gcaattacag
ctccaaggtt ggtggtgaaa atgtcagcaa 5340tagccaccaa gaatacaaga
gtggagtcat tagaggtgaa accaccagca cacccaactt 5400atgatttaaa
ggaagttatg aaacttgcac tctctgaaga tgctgggaat ttaggagatg
5460tgacttgtaa ggcgacaatt cctcttgata tggaatccga tgctcatttt
ctagcaaagg 5520aagacgggat catagcagga attgcacttg ctgagatgat
attcgcggaa gttgatcctt 5580cattaaaggt ggagtggtat gtaaatgatg
gcgataaaga tcgttcaaac atttggcaat 5640aaagtttctt aagattgaat
cctgttgccg gtcttgcgat gattatcata taatttctgt 5700tgaattacgt
taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg
5760tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac
aaaatatagc 5820gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat
gttactagat cg 5872502006DNANicotiana tabacum 50gatctaaatt
gtgagttcaa tctcttccct attggattga ttatcctttc ttttcttcca 60atttgtgttt
ctttttgcct aatttattgt gttatcccct ttatcctatc ttgtttcttt
120acttatttat ttgcttctat gtctttgtac aaagatttaa actctatggc
acatatttta 180aagttgttag aaaataaatt ctttcaagat tgatgaaaga
actttttaat tgtagatatt 240tcgtagattt tattctctta ctaccaatat
aacgcttgaa ttgacgaaaa tttgtgtcca 300aatatttagc aaaaaggtat
ccaatgaaaa tatatcatat gtgatcttca aatcttgtgt 360cttatgcaag
attgatactt tgttcaatgg aagagattgt gtgcatattt tcaaaatttt
420tattagtaat aaagattcta tatagctgtt atagagggat aattttacaa
agaacactat 480aaatatgatt gttgttgtta ggggtgtcaa tggttcggtt
cgactggtta ttttataaaa 540tttgtaccat accatttttt cggatattct
attttgtata accaaaatta gacttttcga 600aatcgtccca atcatgtcgg
tttcacttcg gtatcggtac cgttcggtta attttcattt 660ttttttaaat
gtcattaaaa ttcactagta aaaatagaat gcaataacat acgttctttt
720ataggactta gcaaaactct ctagacattt ttactgttta aaggataatg
aattaaaaaa 780catgaaagat ggctagagta tagatacaca actattcgac
agcaacgtaa aagaaaccaa 840gtaaaagcaa agaaaatata aatcacacga
gtggaaagat attaaccaag ttgggattca 900agaataaagt ctatattaaa
tattcaaaaa gataaattta aataatatga aaggaaacat 960attcaataca
ttgtagtttg ctactcataa tcgctagaat actttgtgcc ttgctaataa
1020agatactaga aatagcttag tttaaatata aatagcataa tagattttag
gaattagtat 1080tttgagttta attacttatt gacttgtaac agtttttata
attccaaggc ccaatgaaaa 1140atttaatgct ttattagttt taaacttact
atataaattt ttcatatgta aaatttaatc 1200ggtatagttc gatatttttt
caatttattt ttataaaata aaaaacttac cctaattatc 1260ggtacagtta
tagatttata taaaaatcta cggttcttca gaagaaacct aaaaatcggt
1320tcggtgcggg acggttcgat cggtttagtc gattttcaaa tattcattga
cactcctagt 1380tgttgttata ggtaaaaagc agttacagag aggtaaaata
taacttaaaa aatcagttct 1440aaggaaaaat tgacttttat agtaaatgac
tgttatataa ggatgttgtt acagagaggt 1500atgagtgtag ttggtaaatt
atgttcttga cggtgtatgt cgcatattat ttattaaaac 1560tagaaaaaac
agcgtcaaaa ctagcaaaaa tccaaaggac aaaaaaatcg gctgaatttg
1620atttggttcc aacatttaaa aaagtttcag tgagaaagaa tacggtgact
gttgatgata 1680taaacaaagg gcacattggt caataaccat aaaaaattat
atgacagcta cagttggtag 1740catgtgctca gctattgaac aaatctaaag
aaggtacatc tgtaaccgga acagcactta 1800aatgactaaa ttaccctcat
cagaaagcag atggagtgct acaaataaca cactattcaa 1860caaccataaa
taaaacgtgt tcagctacta aaacaaatat aaataaatct atgtatgtaa
1920gcactccagc catgttaatg gagtgctatt gcctgttaac tctcactata
aaatagtagt 1980agaaaaaata tgaaccaaaa cacaac 20065175DNAArtificial
SequenceSynthetically prepared primer sequence 51ccttgcgctt
ctcagccacg caaactcaag aggatcgcat caccatcacc atcacagtga 60ccttgaccgg
tgcac 755270DNAArtificial SequenceSynthetically prepared primer
sequence 52ataatctaga tgatcatggc ttcctccaag ttactctccc tagccctctt
ccttgcgctt 60ctcagccacg 705347DNAArtificial SequenceSynthetically
prepared primer sequence 53attcgagctc ttaaagttca tcatgagcca
tagaaacagg cattact 475423PRTArtificial SequenceSynthetic amino acid
sequence 54Met Ile Met Ala Ser Ser Lys Leu Leu Ser Leu Ala Leu Phe
Leu Ala 1 5 10 15 Leu Leu Ser His Ala Asn Ser 20 559PRTArtificial
SequenceSynthetic amino acid sequence 55Arg Gly Ser His His His His
His His1 5 564PRTArtificial SequencePlant specific endoplasmic
reticulum retention signal sequence 56His Asp Glu Leu1
* * * * *