U.S. patent application number 09/950312 was filed with the patent office on 2003-10-30 for methods for identifying products employing gene expression.
Invention is credited to Keller, Lorraine Holowach, Palli, Subba Reddy, Weinstein, Barry.
Application Number | 20030203360 09/950312 |
Document ID | / |
Family ID | 24772266 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203360 |
Kind Code |
A1 |
Weinstein, Barry ; et
al. |
October 30, 2003 |
Methods for identifying products employing gene expression
Abstract
A method for identifying a product involves the steps of: (1)
associating with the product a marker ligand; and (2) detecting the
marker ligand in the product at a later point in time as a means of
identifying the product by contacting the product with a detector
composition. The detector composition comprises one or more first
nucleotide sequences encoding one or more natural or synthetic
ligand-dependent transcription factors, wherein said factors
comprise at least one ligand binding domain, at least one DNA
binding domain and at least one transactivation domain; and a
second nucleotide sequence encoding a reporter gene under the
regulatory control of a receptor response element or a modified or
synthetic response element, and a second promoter. The method may
also employ a corepressor or coactivator or a nucleotide sequence
encoding the corepressor or activator. Interaction between the
marker ligand and ligand binding domain is highly specific and
induces a change in the expression of the reporter gene, the change
producing a detectable signal identifying the presence of the
marker ligand in the product. The detector composition, a cell line
containing the first and second nucleotide sequences, kits using
them and products marked with specific marker ligands are useful in
this method.
Inventors: |
Weinstein, Barry; (Dresher,
PA) ; Keller, Lorraine Holowach; (Lansdale, PA)
; Palli, Subba Reddy; (Lansdale, PA) |
Correspondence
Address: |
New RheoGene I LLC
2650 Eishenhower Avenue
Norristown
PA
19403
US
|
Family ID: |
24772266 |
Appl. No.: |
09/950312 |
Filed: |
September 10, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09950312 |
Sep 10, 2001 |
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09690391 |
Oct 17, 2000 |
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6576422 |
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Current U.S.
Class: |
435/6.16 ;
435/7.2; 506/14 |
Current CPC
Class: |
C12Q 1/6897
20130101 |
Class at
Publication: |
435/6 ;
435/7.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567 |
Claims
What is claimed is:
1. A method for identifying a product, said method comprising the
steps of: (a) associating with said product a marker ligand; (b)
detecting said marker ligand in said product, a portion of said
product or an extract of said product at a later point in time as a
means of identifying the product by contacting said product with a
composition comprising (1) one or more first nucleotide sequences
encoding one or more natural or synthetic ligand-dependent
transcription factors, wherein said factors comprise at least one
ligand binding domain, at least one DNA binding domain and at least
one transactivation domain; and (2) a second nucleotide sequence
encoding a reporter gene under the regulatory control of a receptor
response element or a modified or synthetic response element, and a
second promoter; wherein the interaction between said marker ligand
and said at least one ligand binding domain is highly specific and
induces a change in the expression of said reporter gene, said
change producing a detectable signal identifying the presence of
said ligand in said product.
2. The method according to claim 1, wherein said transcription
factor is under the regulatory control of a first promoter.
3. The method according to claim 1, wherein said transactivation
domain is a carboxy terminal portion of a ligand binding domain
that enhances activation.
4. The method according to claim 1, wherein said transactivation
domain is a sequence independent of said ligand binding domain.
5. The method according to claim 1, wherein said composition
further comprises a third nucleotide sequence encoding a
coactivator or corepressor that interacts with the ligand-dependent
transcription factor to activate or repress expression of said
reporter gene.
6. The method according to claim 1, wherein said product is
contacted with a coactivator or repressor protein that interacts
with the ligand-dependent transcription factor to activate or
repress expression of said reporter gene.
7. The method according to claim 1, wherein said ligand dependent
transcription factor is a nuclear receptor superfamily protein or
functional fragment thereof.
8. The method according to claim 1, wherein said ligand dependent
transcription factor is a modified or synthetic protein having the
transcription activating properties of a nuclear receptor
superfamily protein.
9. The method according to claim 7 wherein said factor is a
modified insect nuclear receptor superfamily protein.
10. The method according to claim 7, wherein said factor is
selected from the group of nuclear receptor superfamily members
consisting of ecdysone, estrogen, retinoid X, progesterone,
glucocorticoid, vitamin D, retinoic acid, and peroxisome
proliferation receptor protein.
11. The method according to claim 1, wherein said factor is
selected from a tetracycline inducible lac operon and an IPTG
inducible receptor protein, a lactone receptor protein, and an
arabinose-inducible protein.
12. The method according to claim 1, wherein said composition is a
cell comprising said first nucleotide sequence and said second
nucleotide sequence.
13. The method according to claim 12, wherein said cell is a
eukaryotic cell.
14. The method according to claim 12, wherein said cell is a
prokaryotic cell.
15. The method according to claim 12, wherein said cell is
immobilized on a solid support.
16. The method according to claim 1, wherein said binding of said
marker ligand to said ligand binding domain triggers the binding of
the DNA binding domain to said response element, wherein said bound
response element activates or suppresses the expression of said
reporter gene.
17. The method according to claim 1, wherein said composition
contains multiple different ligand binding domains, which associate
to provide a single receptor.
18 The method according to claim 1, wherein said composition
contains a single ligand binding domain.
19. The method according to claim 1, wherein said ligand binding
domain is selected from the group consisting of the ligand binding
domain from a steroid/thyroid nuclear receptor superfamily member,
a synthetic or recombinantly modified domain thereof, a fragment of
said domain, and an analog thereof.
20. The method according to claim 1, wherein said DNA binding
domain mediates binding of said ligand-dependent transcription
factor to said response element of said second nucleotide
sequence.
21. The method according to claim 20, wherein said DNA binding
domain is heterologous to said ligand binding domain.
22. The method according to claim 20, wherein said DNA binding
domain is selected from the group consisting of a DNA binding
domain of GAL4, a DNA binding domain of LexA, a DNA binding domain
from a transcription factor; a DNA binding domain from a
steroid/thyroid nuclear receptor superfamily member, a DNA binding
domain from a bacterial LacZ, a DNA binding domain from a yeast
cell, a DNA binding domain from a plant cell, a DNA binding domain
from a virus, an artificial zinc finger region, a synthetic or
recombinant analog, combination or modification thereof.
23. The method according to claim 1, wherein said transactivation
domain amplifies a conformational change in the ligand dependent
transcription factor when the ligand binds to said ligand binding
domain.
24. The method according to claim 23, wherein said transactivation
domain is selected from the group consisting of a steroid/thyroid
hormone nuclear receptor activation domain, a synthetic or chimeric
activation domain, a polyglutamine activation domain, basic or
acidic amino acid activation domain, a viral activation domain, a
plant virus activation domain, or the VP16, GAL4, NF-kB, or BP64
activation domain, a modified activation domain, a fragment of any
of said activation domains and a modification thereof.
25. The method according to claim 24, wherein more than one
activation domain is employed to increase the strength of
activation.
26. The method according to claim 1, wherein said response element
is selected from the group consisting of a response element from
GAL4, a response element from a steroid/thyroid hormone nuclear
receptor, an artificial zinc finger, a LexA operon, a lac operon
response element, and a synthetic or recombinantly produced
response element that recognizes a DNA binding domain.
27. The method according to claim 1, wherein said first promoter
regulates the expression of said factor in a selected host cell or
virus.
28. The method according to claim 2, wherein said first promoter is
a constitutive promoter.
29. The method according to claim 1, wherein said second promoter
regulates the inducible expression of said reporter in the selected
host cell and initiates or suppresses transcription of said
reporter gene only in the presence of a complex formed by the
marker ligand, the ligand-dependent transcription factor, and said
response element.
30. The method according to claim 2, wherein said first and second
promoter are the same.
31. The method according to claim 30, wherein said second promoter
is an inducible promoter.
32. The method according to claim 1 wherein the signal produced by
said reporter gene is detectable by a method selected from the
group consisting of visual detection, microscopic detection,
ultraviolet light detection, electrical detection, change in
capacitance, hybridization, infrared detection, fluorescence
detection and nuclear magnetic resonance.
33. The method according to claim 32, wherein said reporter gene is
a gene encoding a fluorescent or luminescent protein.
34. The method according to claim 32, wherein said reporter gene
encodes a protein that interacts with a substrate to produce a
detectable signal.
35. The method according to claim 32, wherein said reporter gene
causes directly or indirectly a red or blue shift in the emission
spectrum of said detectable signal.
36. The method according to claim 34, wherein said protein is an
enzyme that can catalyze a detectable signal.
37. The method according to claim 1, wherein said marker ligand is
a molecule that specifically binds to said ligand binding
domain.
38. The method according to claim 37 wherein said marker ligand is
a small molecule that can specifically interact with a selected
ligand binding domain.
39. The method according to claim 35, wherein said ligand is a
synthetic chemical compound.
40. The method according to claim 38, wherein said marker ligand is
selected from the group consisting of ponasteroneA, muristerone A,
an alkylhydrazine, an N,N'-diacylhydrazine, an N-substituted-N,N'
diacylhydrazine, an N-substituted-N,N'-disubstituted hydrazine, a
dibenzoylalkyl cyanohydrazine, an N-alkyl-N,N'-diaroylhydrazine, an
N-acyl-N-alkyl-N'-aroylhydrazine, a
3,5-di-tert-butyl-r-hydroxy-N-isobuty- l-benzamide, and an
8-O-acetylharpagide.
41. The method according to claim 1, wherein said product is
selected from the group consisting of a liquid, a solid, a
dispersion, an emulsion, and a latex.
42. The method according to claim 41, wherein said marker ligand is
admixed directly into said product.
43. The method according to claim 41, wherein the product is a
solid and the marker ligand is applied to the surface of the
product, or to a tag or packaging associated with the product.
44. The method according to claim 41, wherein said ligand is
present in said liquid product at a concentration of between 0.1 to
10 parts per billion.
45. The method according to claim 41, wherein said ligand is
present in said solid product at a concentration of between 10 to
about 500 parts per billion.
46. A marked product comprising a liquid, a solid, a dispersion, an
emulsion, or a latex associated with a marker ligand selected from
the group consisting of ponasterone A, muristerone A, an
alkylhydrazine, an N,N'-diacylhydrazine, an N-substituted-N,N'
diacylhydrazine, an N-substituted-N,N'-disubstituted hydrazine, a
dibenzoylalkyl cyanohydrazine, an N-alkyl-N,N'-diaroylhydrazine, an
N-acyl-N-alkyl-N'-aroylhydrazine, a
3,5-di-tert-butyl-r-hydroxy-N-isobuty- l-benzamide, and an
8-O-acetylharpagide.
47. One or more cells comprising: (a) one or more first nucleotide
sequences encoding one or more natural or synthetic
ligand-dependent transcription factors, wherein said factors
comprise at least one ligand binding domain, at least one DNA
binding domain and at least one transactivation domain; and (b) a
second nucleotide sequence encoding a reporter gene under the
regulatory control of a receptor response element or a modified or
synthetic response element, and a second promoter; wherein
interaction between said marker ligand and said at least one ligand
binding domain is highly specific and induces a change in the
expression of said reporter gene, said change producing a
detectable signal identifying the presence of said marker
ligand.
48. A kit for identifying a marked product comprising: (a) a
detector composition comprising (1) one or more first nucleotide
sequences encoding one or more natural or synthetic
ligand-dependent transcription factors, wherein said factors
comprise at least one ligand binding domain, at least one DNA
binding domain and at least one transactivation domain; and (2) a
second nucleotide sequence encoding a reporter gene under the
regulatory control of a receptor response element or a modified or
synthetic response element, and a second promoter; wherein
interaction between said marker ligand and said at least one ligand
binding domain is highly specific and induces a change in the
expression of said reporter gene, said change producing a
detectable signal identifying the presence of said marker ligand;
and (b) means for detecting and measuring said signal.
49. The kit according to claim 48, wherein said composition (a) is
selected from the group consisting of one or more cells and one or
more lyophilized cells.
50. The kit according to claim 48, further comprising at least one
component selected from the group consisting of reagents necessary
to culture said one or more cells, reagents necessary to reactivate
said one or more lyophilized cells or cell lysates; instructions
for performing a detection assay, substrates to which the
composition has been pre-adsorbed in a lyophilized state, diluents
and buffers, indicator charts for signal comparisons, disposable
gloves, decontamination instructions, applicator sticks or
containers, and sample preparator cups.
51. A detector composition comprising: (a) a live cell comprising:
(1) one or more first nucleotide sequences encoding one or more
natural or synthetic ligand-dependent transcription factors,
wherein said factors comprise at least one ligand binding domain,
at least one DNA binding domain and at least one transactivation
domain; and (2) a second nucleotide sequence encoding a reporter
gene under the regulatory control of a receptor response element or
a modified or synthetic response element, and a second promoter;
wherein interaction between said marker ligand and said at least
one ligand binding domain is highly specific and induces a change
in the expression of said reporter gene, said change producing a
detectable signal identifying the presence of said marker ligand;
and (b) a solid support upon which said cell is immobilized.
52. The composition according to claim 51, wherein said support is
selected from the group consisting of a microcell, a microcapsule,
a microtiter plate, a bead, and a biochip.
53. The composition according to claim 51, wherein said one or more
cell is lyophilized.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
product identification; and more specifically, to the application
of biotechnological systems to mark products for
identification.
BACKGROUND OF THE INVENTION
[0002] The interaction between a variety of ligands and the
receptors with which they bind intracellularly has been exploited
in fields where the triggering of a receptor-induced promoter
enables the promoter-regulated expression of a gene encoding a
desired protein. Such inducible expression systems permit the
desired protein to be produced in the cell at an appropriate
timepoint. One such receptor system is the insect steroid hormone
receptor system disclosed in U.S. Pat. No. 5,514,578.
[0003] Such systems are used in drug or new compound screening. For
example, International patent application No. WO92/27356, published
Jun. 3, 1999, refers to methods for identifying modulators of
nuclear hormone receptor function by mixing the receptor, a peptide
sensor, and a test compound. The sensor provides direct binding to
the receptor, and an assay is performed to determine if the test
compound influenced the binding of the sensor protein to the
receptor. Additionally, Evans, U.S. Pat. No. 5,071,773 refers to
hormone receptor related bioassays as screens to determine whether
proteins are receptors that activate transcription or whether a
test agent is a ligand that activates a known receptor.
[0004] Such receptor systems have been proposed for
"fingerprinting" or as biosensors for marking products for
identification. For example, the interaction between certain
G-protein coupled cell surface receptors, tyrosine canasta
receptors, and ion channel receptors which have been mutated to
have altered binding to their natural ligands and various
non-natural ligands thereto, have been proposed for the generation
of a sample fingerprint. The fingerprint is proposed to enable the
authentification and monitoring of products for safety, security,
fraud and quality control. See, International Patent Application
No. WO99/51777, published Oct. 14, 1999; and International Patent
Application No. WO97/35985, published Oct. 2, 1997. International
Patent Application No. WO95/02823, published Jan. 26, 1995 refers
to a method of detecting a ligand by incubating cells transfected
with DNA coding for a receptor that can influence cell
amplification in response to the ligand, with a test substance that
is a potential agonist or antagonist of the receptor. A marker for
amplification in the cells is then used to assess the presence or
absence of amplification of cells.
[0005] Some disadvantages of these prior art detection systems
include a need for high concentration of ligand in the marked
product as well as a limitation on the number and character of the
ligands that can be used in the detection method.
[0006] There is a need in the art for additional uses of
receptor-ligand interactions for product identification which
interactions can generate simple and rapid signals at low
concentrations.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides an improved
method for identifying a product, which employs ligand-dependent
transcription factors. This method involves first associating a
marker ligand with the product, and then detecting the marker
ligand in the product or a portion or extract thereof at a later
point in time as a means of identifying the product. The
ligand-containing product is contacted with a detector composition
comprising one or more first nucleotide sequences encoding one or
more natural or synthetic ligand-dependent transcription factors.
The ligand-dependent transcription factor(s) comprise at least one
ligand binding domain, at least one DNA binding domain and at least
one transactivation domain. The factor(s) are preferably under the
regulatory control of a first promoter. The detector composition
also comprises a second nucleotide sequence encoding a reporter
gene under the regulatory control of a receptor response element or
a modified or synthetic response element, and a second promoter.
The interaction between the marker ligand and at least one of the
ligand binding domains is highly specific and induces a change in
the expression of the reporter gene. This change produces a
detectable signal identifying the presence of the ligand in the
product.
[0008] In another aspect, the invention provides an additional
method such as that described above, but wherein the detector
composition further comprises a third nucleotide sequence encoding
a coactivator or corepressor that interacts with the
ligand-dependent transcription factor to activate or repress
expression of said reporter gene. In still another embodiment of a
method of this invention, the product is contacted with the
detector composition and either a coactivator or repressor protein
that interacts with the ligand-dependent transcription factor to
activate or repress expression of said reporter gene.
[0009] In another aspect, the invention provides a marked product
comprising a liquid, a solid, a dispersion, an emulsion, or a latex
associated with a specified marker ligand described in detail
below.
[0010] In a further aspect, the invention provides one or more
cells or a stable cell line comprising the above-described first
nucleotide sequence(s) and the above-described second nucleotide
sequence, and optionally the coactivator or corepressor for use in
the claimed method.
[0011] In still another aspect, the invention provides a kit for
identifying a marked product comprising a detector composition
comprising the above-described first and second nucleotide
sequences, and means for detecting and measuring the signal.
[0012] In yet another aspect, the invention provides detector
compositions for such use.
[0013] Other aspects and advantages of the present invention are
described further in the following detailed description of the
preferred embodiments thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The present invention provides methods and compositions that
employ natural or synthetic ligand-dependent transcription factors
that participate in receptor-ligand interactions that enable rapid
and efficient product identification. The methods and compositions
of this invention generate simple and rapid signals at low
concentrations, and are thus safe and suitable for application to
many products and industries.
[0015] This invention provides a novel method for identifying a
product. According to this method, a product is first associated
with a marker ligand. The presence and/or quantity of marker ligand
in or on the product, or in or on an extract or portion of the
product, is detected at a later point in time as a means of
identifying the source of the product and/or validating the
authenticity of the product. Detection of the marker ligand is made
by using an "in vivo " step, i.e., by contacting the product with a
detector composition that comprises one or more first nucleotide
sequence(s) that encodes one or more natural or synthetic
ligand-dependent transcription factor(s), optionally under the
regulatory control of a first promoter. These transcription factors
contain three functional regions: a ligand binding domain, a DNA
binding domain and a transactivation domain. Another portion of
this detector composition is a second nucleotide sequence encoding
a reporter gene that is under the regulatory control of a receptor
response element or a modified or synthetic response element, and a
second promoter. Interaction between the marker ligand and at least
one of the ligand binding domains is highly specific and induces a
change in the expression of the reporter gene. That change in the
reporter gene thereby produces a detectable signal identifying the
presence of the ligand in the product. More specifically, the
binding of the marker ligand to the ligand binding domain in the
detector composition triggers the binding of the DNA binding domain
to the response element. When the response element becomes bound,
it activates or suppresses the expression of the reporter gene.
Optionally, a coactivator or corepressor protein or a nucleotide
sequence encoding same is added to the method or to the detector
composition.
[0016] One advantage of the method of this invention over those of
the above-mentioned references include high specificity for
artificial small molecule marker ligands, which are nonhazardous
when present in consumer products. Additionally the specificity
between the ligand binding domains and the ligands results in a
very low potential for interference in this method. The marker
ligands are also easy to synthesize and manufacture. Additionally,
unlike other marker systems, the present invention can use many
markers. Further, this method is highly sensitive, and can detect
very low concentrations of marker ligand in a product, e.g., at
parts per billion (ppb) to parts per trillion (ppt) levels. In
fact, concentration of marker ligands can be so low in this method
that the concentrations cannot be detected by conventional chemical
chromatographic or mass spectrometry methods. Finally, in contrast
to the known methods, cell proliferation in this biological method
of marker detection is independent of the presence of marker
ligand.
[0017] To understand this invention fully, the following
components, defined as follows, are used:
[0018] A. The Product
[0019] As used in the methods and compositions of this invention, a
"product" to which one or more marker ligands is added, may be any
type of product for which identifiable marking is desirable, so to
ascertain the source of the product. For example, such "marking"
may be necessary to police unlicensed or illegal duplication of the
product or for other security purposes, or to enable the
identification of an adulterated product for reasons of consumer
safety. The product may be in any physical form. For example, the
product may be a fluid or liquid, a solid, or some intermediate
therebetween, such as a dispersion, an emulsion, a latex, or a
semi-solid matrix.
[0020] Examples of typical solid products can include, without
limitation, pharmaceutical tablets, capsules and powders; solid
formulations of agrochemicals such as insecticides, herbicides,
fungicides, fertilizers, other agricultural chemicals, and seeds;
explosives; textiles such as clothing; recordings such as
gramophone records, tape cassettes, floppy discs and compact discs;
electrical goods such as television sets, computers and radios;
motor vehicle components and cameras; paper such as documents,
confidential papers, notes, securities, labels, and packaging;
chemical products such as inks, biocides, and rubbers; cosmetics
such as creams; food products; and construction materials, such as
asphalt additives, roof shingles, and concrete blocks, as well as
packaging materials.
[0021] Examples of fluid or liquid products include, without
limitation, oil-based products such as lubricating oils, hydraulic
oils, greases, gasoline, kerosene, crude petroleum, diesel fuel,
and liquified petroleum products, gasahol, biodiesel fuel, motor
oil transmission fluid; paints, paint additives, plastic additives,
adhesives, coatings, ceramics; oil-field chemicals, including
polymers; perfumes and other cosmetics; drinks such as bottled
water, milk, wine, whisky, sherry, gin and vodka and other
alcoholic and non-alcoholic beverages; liquid pharmaceutical
formulations such as syrups, emulsions and suspensions; water
treatment chemicals, such as polymers, scale inhibitors, and
chelating agents; liquid agrochemical formulations, such as
pesticides, insecticides, and herbicides; and industrial solvents.
The product is preferably liquid.
[0022] One of skill in the art may readily select the product to
which a marker ligand is to be introduced, according to this
invention without any unnecessary experimentation. It will be
appreciated that the marker ligand, described in detail below, may
be associated with the product in a wide variety of ways. Thus the
marker ligand may be present in or on all or part of the product,
or in or on all or part of a label, wrapper or container associated
with the product.
[0023] Preferably, the marker ligand is directly admixed into the
product, e.g., where the product is a fluid, or semi-solid, or even
a powder. Where the product is a solid, the marker ligand may be
present independently of the product, for example, the marker
ligand may be present in the product packaging, tags or labels.
Alternatively, where the product is a solid, the marker ligand may
be applied to the surface of the product and dried. Methods of
application of the marker ligand to a solid product may include
without limitation, roller transfer or paint coating, spray
coating, brush coating and dip coating. The application method must
be applied so that drying takes place on the product at a selected
temperature, e.g., room temperature or greater than room
temperature. General descriptions of these types of coating methods
may be found in conventional texts, such as Modern Coating and
Drying Techniques, (E. Cohen and E. Gutoff, eds; VCH Publishers)
New York (1992) and Web Processing and Converting Technology and
Equipment, (D. Satas, ed; Van Nostrand Reinhold) New York
(1984).
[0024] In one embodiment, the marker ligand is present in a product
at a concentration of between 1 parts by trillion (ppt) to about
500 parts per billion (w/w) of marker ligand. In another
embodiment, the marker ligand is present in the product at a
concentration of between 0.1 ppb to about 100 ppb of marker ligand.
In yet another embodiment, the marker ligand is present in a
product at a concentration of between about 0.1 to 10 ppb of
ligand.
[0025] According to the method of this invention, the presence of
marker ligand in or on the product may be detected by examining the
entire product, a portion (e.g., a small quantity) of the product,
or an extract of the product (e.g., such as where the product is a
solid).
[0026] The selection of the product and product form, the method of
incorporation of the marker ligand into or on the selected product,
and the amount of marker ligand to be associated with the product
are not limitations on the present invention, but are variables
that may be readily selected by the person of skill in the art in
view of the teachings provided herein.
[0027] B. Marker Ligands
[0028] The marker ligands useful in the present invention include
molecules that specifically bind to the ligand binding domain of a
natural or synthetic ligand-dependent transcription factor. The
marker ligand can specifically interact with at least one of the
selected ligand binding domains. Preferably, the marker ligand is a
synthetic chemical compound or composition which demonstrates
preferential specific binding to the ligand binding domains
described in detail below. The marker ligand is not normally
present in the product; for example, it is not a by-product of the
production process, normal impurity, or standard additive for that
product. Another advantage of the marker ligands useful in this
invention is that the marker ligands are inert in the sense that
they do not react with the product which they label.
[0029] Preferred marker ligands useful in this invention include
known compounds, or compounds readily synthesized by one of skill
in the art, as disclosed in U.S. Pat. Nos. 4,954,655; 4,985,461;
5,117,057; 5,530,028; 5,378,726; and 6,013,836. These patents are
incorporated by reference herein for the purpose of defining
certain chemical compounds useful as marker ligands in this
invention. Thus the marker ligand may include, without limitation,
ponasterone, ponasterone A, muristerone A, an alkylhydrazine, an
N,N'-diacylhydrazine, an N-substituted-N,N' diacylhydrazine, an
N-substituted-N,N'-disubstituted hydrazine, a dibenzoylalkyl
cyanohydrazine, an N-alkyl-N,N'-diaroylhydrazine, an
N-acyl-N-alkyl-N'-aroylhydrazine, a
3,5-di-tert-butyl-y-hydroxy-N-isobuty- l-benzamide, an
8-O-acetylharpagide. In embodiments in which the ligand binding
domain is derived, for example, from the ecdysone nuclear receptor,
a variety of alkylhydrazines, N-substituted-N,N'-diacylhydrazin-
es, and N-substituted-N,N'-disubstituted hydrazines may be employed
as the marker ligands [see, e.g., U.S. Pat. Nos. 4,954,655;
4,985,461; 5,530,028; and 6,013,836]. A particularly preferred
marker ligand which is used in the following examples is
N'-tert-butyl-N'-(3,5-dimethylbenzoy-
l)-3-methoxy-2-methylbenzohydrazide, which is informally referred
to as methoxyfenoxide.
[0030] Still other suitable compounds useful as marker ligands are
disclosed in pending U.S. patent application Ser. No. 09/315,451,
which is also incorporated by reference herein. One such marker
ligand is defined by the formula: 1
[0031] wherein:
[0032] E is a (C.sub.4-C.sub.6)alkyl containing a tertiary carbon
or a cyano(C.sub.3-C.sub.5)alkyl containing a tertiary carbon;
[0033] R.sup.1 is H, Me, Et, i-Pr, F, formyl, CF.sub.3, CHF.sub.2,
CHCl.sub.2, CH.sub.2F, CH.sub.2C.sub.1, CH.sub.2OH, CH.sub.2OMe,
CH.sub.2CN, CN, C.ident.CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe,
OEt, cyclopropyl, CF.sub.2CF.sub.3, CH.ident.CHCN, allyl, azido,
SCN, or SCHF.sub.2; R.sup.2 is H, Me, Et, n-Pr, i-Pr, formyl,
CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2OH,
CH.sub.2OMe, CH.sub.2CN, CN, C.ident.CH, 1-propynyl, 2-propynyl,
vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe.sub.2, NEt.sub.2,
SMe, SEt, SOCF.sub.3, OCF.sub.2CF.sub.2H, COEt, cyclopropyl,
CF.sub.2CF.sub.3, CH.ident.CHCN, allyl, azido, OCF.sub.3,
OCHF.sub.2, O-i-Pr, SCN, SCHF.sub.2, SOMe, NH--CN, or joined with
R.sup.3 and the phenyl carbons to which R.sup.2 and R.sup.3 are
attached to form an ethylenedioxy, a dihydrofuryl ring with the
oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the
oxygen adjacent to a phenyl carbon;
[0034] R.sup.3 is H, Et, or joined with R.sup.2 and the phenyl
carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a
phenyl carbon; R.sup.4, R.sup.5 and R.sup.6 are independently H,
Me, Et, F, Cl, Br, formyl, CF.sub.3, CHF.sub.2, CHCI.sub.2, CH2F,
CH.sub.2Cl, CH.sub.2OH, CN, C.ident.CH, 1-propynyl, 2-propynyl,
vinyl, OMe, OEt, SMe, or SEt;
[0035] provided that:
[0036] a) when R.sup.1 is Me and R.sup.2 is OMe; then R.sup.3 is H;
and the combination R.sup.4, R.sup.5, and R.sup.6 is 3,5-di-Me,
3,5-di-OMe-4-Me, 3,5-di-Cl, or 2,5-di-F;
[0037] b) when R.sup.1 is Me and R.sup.2 is OEt; then R.sup.3 is H
and the combination R.sup.4, R.sup.5, and R.sup.6 is 3,5-di-Me,
3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4-or 2,5-di-F, 2,4-or
2,5-di-Cl;
[0038] c) when R.sup.1 is Et and R.sup.2 is OMe or OEt; then
R.sup.3 is H and the combination R.sup.4, R.sup.5, and R.sup.6
is:
[0039] i) 3,5-di-OMe-4-Me, 3,5-di-Cl, 3,5-di-F, 2,4-or 2,5-di-F,
2,4-or 2,5-di-Cl, 3-OMe, 2-Cl-5-Me, 2-Br-5-Me, 2-Cl, 2-Br, or 3-Me;
or
[0040] ii) R.sup.6 is H, R.sup.4 is Me, and R.sup.5 is Et, F, Cl,
Br, formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl,
CH.sub.2OH, CN, C.ident.CH, 1-propynyl, 2-propynyl, vinyl, OMe,
OEt, SMe, or SEt;
[0041] d) when R.sup.1 is i-Pr; then R.sup.2 is OMe, or OEt;
R.sup.3 is H; and the combination R.sup.4, R.sup.5, and R.sup.6 is
3,5-di-Me;
[0042] e) when R.sup.3 is Et; then R.sup.2 is H, R.sup.1 is F or
Cl, and the combination R.sup.4, R.sup.5, and R.sup.6 is
3,5-di-Me;
[0043] f) when R.sup.2 and R.sup.3, together with the phenyl
carbons to which they are attached, form an ethylenedioxy ring;
then R.sup.1 is Me or Et and the combination R.sup.4, R.sup.5, and
R.sup.6 is 3,5-di-Me;
[0044] g) when R.sup.2 and R.sup.3, together with the phenyl
carbons to which they are attached, form a dihydrofuryl or
dihydropyryl ring; then R.sup.1 is Et and the combination R.sup.4,
R.sup.5, and R.sup.6 is 3,5-di-Me;
[0045] h) when R.sup.1 is formyl, CF.sub.3, CHF.sub.2, CHCl.sub.2,
CH.sub.2F, CH.sub.2Cl, CH.sub.2OH, CH.sub.2OMe, CH.sub.2CN, CN,
C.ident.CH, 1-propynyl, 2-propynyl, vinyl, OH, cyclopropyl,
CF.sub.2CF.sub.3, CH.ident.CHCN, allyl, azido, SCN, or SCHF.sub.2;
then R.sup.2 is OMe or OEt, R.sup.3 is H, and the combination
R.sup.4, R.sup.5, and R.sup.6 is 3,5-di-Me; and
[0046] i) when R.sup.2 is Me, Et, n-Pr, i-Pr, formyl, CF.sub.3,
CHF.sub.2, CHCl.sub.2, CH.sub.2F, CH.sub.2Cl, CH.sub.2OH,
CH.sub.2OMe, CH.sub.2CN, CN, C.ident.CH, 1-propynyl, 2-propynyl,
vinyl, Ac, F, Cl, OH, O-n-Pr, OAc, NMe.sub.2, NEt.sub.2, SMe, SEt,
SOCF.sub.3, OCF.sub.2CF.sub.2H, COEt, cyclopropyl,
CF.sub.2CF.sub.3, CH.ident.CHCN, allyl, azido, OCF.sub.3,
OCHF.sub.2, O-i-Pr, SCN, SCHF.sub.2, SOMe, or NH--CN; then R.sup.1
is Et, R.sup.3 is H, and the combination R.sup.4, R.sup.5, and
R.sup.6 is 3,5-di-Me.
[0047] One particularly desirable marker ligand has the above
specified formula in which E is t-butyl; R.sup.1 is Me, Et, i-Pr,
or F; R.sup.2 is OH, OMe, OEt, or joined with R.sup.3 and the
phenyl carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon; R.sup.3 is H, Et or joined with R.sup.2 and the
phenyl carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon; and R.sup.4, R.sup.5, and R.sup.6 are independently
Me, F, Cl, CH.sub.2OH, or OMe. Another desirable ligand has the
above-specified formula, in which E is t-butyl; R.sup.1 is Me, Et,
i-Pr, or F; R.sup.2 is OH, OMe, OEt, or joined with R.sup.3 and the
phenyl carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon; R.sup.3 is H, Et or joined with R.sup.2 and phenyl
carbons to which R.sup.2 and R.sup.3 are attached to form an
ethylenedioxy or dihydrofuryl ring with the oxygen adjacent to a
phenyl carbon; and R.sup.4, R.sup.5, and R.sup.6 are independently
Me, F, Cl, CH.sub.2OH, or OMe.
[0048] Still another marker ligand has the above-specified formula
in which E is t-butyl; R.sup.1 is Me, Et, i-Pr, or F; R is OH, OMe,
OEt, or joined with R.sup.3 and the phenyl carbons to which R.sup.2
and R.sup.3 are attached to form an ethylenedioxy or dihydrofuryl
ring with the oxygen adjacent to a phenyl carbon; R.sup.3 is H, Et
or joined with R.sup.2 and the phenyl carbons to which R.sup.2 and
R.sup.3 are attached to form an ethylenedioxy or dihydrofuryl ring
with the oxygen adjacent to a phenyl carbon; and R.sup.4, R.sup.5,
and R.sup.6 are independently Me, F, Cl, CH.sub.2OH, or OMe. Other
marker ligands may be selected from the specified formula by one of
skill in the art.
[0049] These compounds as marker ligands are particularly
desirable, because they are nonhazardous at the concentrations
which would be present in consumer products, such as
pharmaceuticals, cosmetics, foods, packaging, etc. Further, very
small concentrations of such ligands in a product, e.g., in the
range of parts per billion to parts per trillion, can be detected
and quantified by the method of this invention.
[0050] One of skill in the art may use molecules in addition to the
molecules described above as marker ligands, provided that the
marker ligand binds at least one of the selected ligand binding
domain(s) of a natural or synthetic ligand-dependent transcription
factor of the detector composition, as described below.
[0051] C. Detector Composition
[0052] A detector composition of the present invention refers to a
composition which comprises one or more "first" nucleotide
sequences that encode one or more natural or synthetic
ligand-dependent transcription factors. The factor(s) are
preferably under the regulatory control of a first promoter. The
composition also contains a "second" nucleotide sequence encoding a
reporter gene that is under the regulatory control of a receptor
response element or a modified or synthetic response element, and a
second promoter. Optionally, the detector composition contains a
"third" nucleotide sequence encoding a coactivator or corepressor.
These nucleotide sequences may be RNA or DNA. In one embodiment,
the DNA sequences making up the detector composition are preferably
incorporated into a cell or cells. More preferably, the detector
composition containing the nucleotide sequences is a stable cell or
cell line.
[0053] The cell(s) of the detector composition can be any
eukaryotic or prokaryotic cell(s). Of the eukaryotic cells,
invertebrate cells (such as insect cells) are preferred because
they confer the most sensitive response to the marker ligands of
the preferred receptors, e.g., the ecdysone receptor. The selection
of other cell and receptor combinations may be made by one of skill
in the art in view of this disclosure. Thus, the ligands of this
invention will have negligible physiological or other effects on
transformed cells, or the whole organism. Therefore, cells can grow
and express the desired product, substantially unaffected by the
presence of the ligand itself. Preferably, in a eukaryotic cell,
the nucleotide sequences of the detector composition are located in
the nucleus.
[0054] In one preferred embodiment, the detector composition
containing the two nucleotide sequences is an insect cell or cells,
such as Spodoptera frugiperda (Sf9). Insect cells tend to be very
sensitive and robust in tolerating numerous receptors for use in
this composition. Other insect cells include the insect cell line,
BRL-AG2, derived from cotton boll weevil, Anthomus grandis as
described in Stiles et al, In Vitro Cell Dev. Biol., 28A:355-363
(1992).
[0055] Another insect cell line L57 derived from the Drosophila
melanogaster Kc cell line may also be employed. Still other insect
cells for use in this composition may be selected by one of skill
in the art.
[0056] In another preferred embodiment, the detector composition is
a yeast cell or cells, such as Saccharomyces cerevisiae. In another
preferred embodiment, the detector composition is Pichia pastoris.
Still another preferred yeast cell for use in this invention is
Pichia methanolica. Still other strains of yeast cells for use in
this invention include, without limitation, a wide variety of
strains of Saccharomyces Candida, Ambrosiozyma, Apiotrichum,
Arthroascus, Hansenula, Kloeckera, Kluyveromyces, Pichia,
Rhodosporidium, Rhodotorula, Schizosaccharomyes, and
Torulaspora.
[0057] For example, in one embodiment, the detector composition is
a mammalian cell.
[0058] Desirable mammalian cells include, without limitation, cells
such as CHO, BHK, MDCK, and various murine cells, e.g., 10T1/2 and
WEHI cells, African green monkey cells, suitable primate cells,
e.g., VERO, COS1, COS7, BSC1, BSC 40, and BMT 10, and human cells
such as WI38, MRC5, A549, human embryonic retinoblast (HER), human
embryonic kidney (HEK), human embryonic lung (HEL), TH1080 cells.
Other suitable cells may include NIH3T3 cells (subline of 3T3
cells), HepG2 cells (human liver carcinoma cell line), Saos-2 cells
(human osteogenic sarcomas cell line), HuH7 cells or HeLa cells
(human carcinoma cell line). In one embodiment, appropriate cells
include the human embryonic kidney 293T cells.
[0059] Neither the selection of the mammalian species providing the
cells nor the type of mammalian cell is a limitation of this
invention.
[0060] Still other compositions of this invention containing the
two nucleotide sequences can be plant cell(s) or algal cell(s),
such as species of the genera, including without limitation,
Blastocrithidia, Cephaleuros, Chlamydomonas, and Chlorella.
[0061] Where the cell is prokaryotic, desirable bacterial cells
include Escherichia coli, Bacillus subtilis, Salmonella
typhimurium, and various species of Pseudomonas, Streptomyces,
Staphylococcus and Shigella or other enterobacteria. However,
eukaryotic cells are preferred.
[0062] In another embodiment, in which the nucleotide sequences of
the detector compositions are RNA molecules, they may also be
incorporated as RNA molecules, preferably in the form of functional
viral RNAs, such as tobacco mosaic virus. Other useful viruses that
may be employed in the compositions of this invention include,
without limitation, vaccinia, adenoviruses, adeno-associated
viruses, baculoviruses, bunyaviruses, coronaviruses, flaviviruses,
hepadnaviruses, herpesviruses and herpes-like viruses,
orthomyxoviruses, papovaviruses, paramyxoviruses, picornaviruses,
poxviruses, reoviruses, retroviruses, and rhabdoviruses.
[0063] The nucleotide sequences present in the cells or viruses
forming the detector compositions are as described below.
[0064] 1. First Nucleotide Sequence(s)
[0065] The first nucleotide sequences contain at least one
ligand-dependent transcription factor, preferably under the
regulatory control of a selected promoter. In one embodiment, a
ligand dependent transcription factor is a nuclear receptor
superfamily protein or a functional fragment thereof. In another
embodiment, the ligand dependent transcription factor is a modified
or synthetic protein having the transcription activating properties
of a nuclear receptor superfamily protein. Members of this
superfamily include, without limitation, a modified or native
steroid/thyroid nuclear receptor superfamily protein, such as the
ecdysone [see Yao, T.P. et al (1993) Nature, 366: 476-479; Yao,
T.-P. et al, (1992) Cell, 71: 63-72], the estrogen, retinoid X,
progesterone, glucocorticoid, vitamin D, retinoic acid, and
peroxisome proliferation receptor proteins. Still optionally, the
transcription factor has a function similar to that of a
steroid/thyroid nuclear receptor superfamily, but is not
conventionally a member of that superfamily. Among such "optional"
transcription factors are those derived from a tetracycline
inducible lac operon, an IPTG inducible receptor protein, a lactone
receptor protein, and an arabinose-inducible protein.
[0066] The ligand dependent transcription factors of the first
nucleotide sequences of the detector composition contain a
transactivation domain, a DNA binding domain ("DBD"), and a ligand
binding domain ("LBD"). Each domain may be optionally separated by
a hinge region of from 50 to about 1000 nucleotides of any
sequence. Preferably, the hinge region is from 100 to about 500
nucleotides. In some embodiments, the hinge region is about 200
nucleotides in length. Each domain may be a naturally occurring
sequence isolated from its native source, or may be a synthetically
or recombinantly constructed sequence or fragment of a native
sequence which has the required function. Preferably, one or more
of the three domains may be chosen from a source different than the
source of the other domains so that a "chimeric" first nucleotide
sequence is optimized for a selected host cell for transactivating
activity, complementary binding of the ligand, and recognition of a
specific response element.
[0067] 2 a. Ligand Binding Domain
[0068] The LBD of the first nucleotide sequences binds specifically
only to a marker ligand of this invention. In one embodiment, the
LBD also contains a transactivation domain or transactivation
function, generally as its carboxy terminal sequence, so that no
separate transactivation domain is necessary. The binding of the
marker ligand to the LBD triggers the binding of the DNA binding
domain to said response element, which activates or suppresses the
expression of the reporter gene, thereby producing or altering a
signal. In one embodiment of the transcription factor, the LBD is
an isolated, native ligand binding domain obtained from a
steroid/thyroid nuclear receptor superfamily member, such as an LBD
from the insect superfamily, e.g., from the ecdysone receptor
protein. Alternatively, the LBD is a synthetic or recombinantly
modified ligand binding domain or fragment of such a domain from a
member of that superfamily. Still alternatively, the LBD is a
completely synthetic sequence which has the function of binding
only to a marker ligand identified above, and triggering the
above-described sequence of events. The LBD, identified from the
sources above, may be isolated, prepared synthetically or
recombinantly. Such LBDs are generally from about 500 to about 1000
nucleotides in length. In some embodiments, the LBDs are about 750
nucleotides in length. Particularly preferred LBD's for this
methods include the ecdysone receptor LBD and the USP. The ecdysone
receptor CfEcR LBD sequence is described in R. Kothapalli et al,
Dev. Genet., 17(4):319-330 (1995); see also Genbank Accession No.
U29531.
[0069] In one embodiment of this invention, a single first
nucleotide sequence contains a single LBD, which binds
preferentially to a single marker ligand and triggers a cascade of
events resulting in a change (e.g., the production, suppression,
enhancement or elimination) of a detectable signal. In another
embodiment, multiple different LBDs are present in the one or more
first nucleotide sequences. For example, two or more LBDs may
associate (e.g., form a homodimer or a heterodimer) to generate a
single transcription factor or receptor for the marker ligand.
[0070] b. DNA Binding Domain
[0071] Binding of the marker ligand in the product to the one or
more LBDs of the detector composition enables the one or more DBDs
of the ligand dependent transcription factors to bind to the
response element of the second nucleotide sequence in an activated
form, thus resulting in a change (e.g., expression or suppression)
of the exogenous reporter gene. In one embodiment of the
transcription factor, the DBD is an isolated, native DNA binding
domain obtained from a steroid/thyroid nuclear receptor superfamily
member, such as a DBD from the insect superfamily, e.g., from the
ecdysone receptor protein. Alternatively, the DBD is a synthetic or
recombinantly modified DNA binding domain or fragment of such a
domain from a member of that superfamily. Still alternatively, the
DBD may be a DNA binding domain from another transcription factor.
The DBD may be a DNA binding domain of a yeast cell, e.g., the GAL4
DBD or a DNA binding domain from a virus or a DNA binding domain
from a plant cell. Other DNA binding domains which may be used as
the DBD in the nucleotide sequences of this invention include the
DNA binding domain of LexA or a DNA binding domain from a bacterial
LacZ gene. Still other selections for this domain include an
artificial zinc finger region. Still alternatively, the DBD is a
completely synthetic sequence which, in the presence of marker
ligand binding to the LBD, mediates the above-described sequence of
events. Synthetic or recombinant analogs, combinations or
modifications of any of the above sources of native DBDs may also
be included as the DBD of the present invention.
[0072] In one embodiment, a single first nucleotide sequence
contains a single DBD. In another embodiment one or more first
nucleotide sequences contain more than one DBD. The DBD may be
heterologous to the one or more LBDs in the one or more first
nucleotide sequences. Alternatively, the DBD may be homologous to
the LBD(s). Still alternatively, where two or more DBDs are present
in the first nucleotide sequences, the DBDs may be the same or
different DBDs. The DBD domain may be either native, modified, or
chimeras of different DNA binding domains of heterologous receptor
proteins. The DBD, identified from the sources above, may be
isolated, prepared synthetically or recombinantly. Such DBDs are
preferably from about 100 to about 1000 nucleotides in length. In
one embodiment, the DBD domain is about 750 nucleotides in length.
As one example, an ecdysone-derived DBD is characterized by the
presence of two cysteine zinc fingers between which are two amino
acid motifs, the P-box and the D-box, which confer specificity for
ecdysone response elements. One particularly preferred DBD for this
method is the CfEcR DBD, described in R. Kothapalli et al, Dev.
Genet., 17(4):319-330 (1995); see also Genbank Accession No.
U29531. Still other DBD sequences are known in the art.
[0073] C. Transactivation Domain
[0074] The transactivation domain of the ligand dependent
transcription factor amplifies a conformational change in the
ligand dependent transcription factor, when the marker ligand in
the product binds to the LBD in the detector composition. The
transactivation domain may be the carboxy terminal sequence of an
LBD, which supplies an activation function. Alternatively, the
transactivation domain may be a sequence independent of the LBD.
One or more transactivation domains may be present on one or more
of the first nucleotide sequences. The transactivation domain may
be a native, modified, or chimera of different transactivation
domains of heterologous receptor proteins. Useful transactivation
domains may be derived from a steroid/thyroid hormone nuclear
receptor activation domain, a synthetic or chimeric activation
domain, a polyglutamine activation domain, a basic or an acidic
amino acid activation domain, a viral activation domain, a plant
virus activation domain, or the VP16, GAL4, NF-kB, or BP64
activation domains. Similarly, such domains may be prepared by
modifying any of the above domains, or by employing a fragment of
any of the activation domains. In the first nucleotide sequence of
this invention, more than one activation domain may be employed to
increase the strength of activation.
[0075] A useful transactivation domain is generally a nucleotide
sequence from about 16 to about 500 nucleotides in length.
Preferably, more than one activation domain is employed in the
first nucleotide sequence to increase the strength of activation.
Transactivation domains, identified from the sources above, may be
isolated, prepared synthetically or recombinantly. Particularly
preferred transactivation domains for use in the method of this
invention are that of VP16, disclosed in P. E. Pellett et al, Proc.
Natl. Acad. Sci., USA, 82(17):5870-5874 (1985), as well as others
known to those of skill in the art. See, also Swiss Prot. Accession
No. PO4486 for the sequence of the VP16 transactivation domain.
[0076] d. The Promoter
[0077] The first nucleotide sequences also optionally contain a
promoter which regulates the expression of at least one of the
ligand dependent transcription factors in the selected cell or
virus. In the method of this invention, one of skill in the art may
readily select the promoter used to drive the expression of the
ligand dependent transcription factor, according to a desired end
result, i.e., to control the timing and location of expression. The
term "promoter" means a specific nucleotide sequence recognized by
RNA polymerase. The promoter sequence is the site at which
transcription can be specifically initiated under proper
conditions. A wide number of promoters may be selected to regulate
expression of the transcription factor of the first nucleotide
sequence, e.g., constitutive promoters, inducibly regulated
promoters, tissue-specific promoters (expressed only in a
particular type of cells) or promoters specific to certain
developmental states of an organism.
[0078] However, preferably, the promoter for the first nucleotide
sequence(s) is a constitutive promoter. Examples of constitutive
promoters which may be selected include, without limitation, the
retroviral Rous sarcoma virus LTR promoter, the cytomegalovirus
promoter, the SV40 promoter, the dihydrofolate reductase promoter,
the a-actin promoter, the phosphoglycerol kinase promoter, the EF1
promoter, the T7 polymerase promoter, the ecdysone insect promoter,
the skeletal -actin promoter, the myosin light chain 2A promoter,
the dystrophin promoter, the muscle creative kinase promoter,
synthetic muscle promoters, the liver promoter, the hepatitis B
virus core promoter, the alpha-fetoprotein promoter, the actin
promoter, the IE1 promoter, the IR2 and other baculovirus
promoters, the HSP70 promoter, the 35S plant promoter, the CSV
plant promoter, the yeast GAL1 promoter, the yeast ADH1 promoter,
and the yeast MET25 promoter. A presently preferred promoter is the
baculovirus IE1 promoter present in the sequence of AcMNPV
described in M. D. Ayres et al, Virol., 202(2):586-605 (1994); see
also Genbank Accession No. NC001623. Other promoter sequences may
be selected from among those sequences known in the art.
[0079] Preferably in the first nucleotide sequence(s) the promoter
is located 5' to the LBD, which is located 5' to the DBD, which is
located 5' to the transactivation domain. These domains may be
linked directly to each other by fusing the 3' nucleotide of the
promoter to the 5' nucleotide of the LBD, and the 3' nucleotide of
the LBD to the 5' nucleotide of the DBD, etc. Alternatively, where
the transactivation domain is a part of the LBD, the LBD is linked
directly or through a spacer to only a DBD. In still another
alternative, one "first" nucleotide sequence contains an LBD and
DBD and another "first" nucleotide sequence contains the same LBD
and DBD or a different LBD and DBD, with an optional
transactivation domain, whereby the LBDs and DBDs and
transactivation domains of the multiple "first" nucleotide
sequences form a single transcription factor as a homodimer or
heterodimer. The multiple LBD, DBD and transactivation domains may
be present on a single nucleotide sequence or on multiple separate
nucleotide sequences. Alternatively, each of these domains is
preferably separated by an optional spacer of about 16 to about 30
nucleotides. For example, from 6 to about 10 glycine amino acid
residues may be used as the spacer, e.g., from 18 to about 30
nucleotides.
[0080] The first nucleotide sequences are desirably single-stranded
DNA or RNA;
[0081] alternatively, they may be double-stranded DNA or RNA. The
first sequences may be prepared in the form of straight chains, but
are preferably circular plasmids. In still another embodiment, one
first sequence may be on one plasmid, another first sequence may be
on the same plasmid or on a different plasmid; and the second
nucleotide sequence may be on yet another plasmid. Yet a further
embodiment provides that the first nucleotide sequence(s) and the
second nucleotide sequence are present on the same plasmid,
provided that they are separated by one or more transcriptional
blockers or polyA sequences. Still other embodiments of these
sequences may employ sequences that have their origins in viruses,
bacteriophages, and other molecules.
[0082] 2. Second Nucleotide Sequence
[0083] The second nucleotide sequence of the detector composition
encodes a reporter gene that is under the regulatory control of a
receptor response element (e.g., native, modified or synthetic),
and a second promoter.
[0084] a. Response Element
[0085] The term "response element" ("RE") means one or more
cis-acting DNA elements which confer responsiveness on a promoter
mediated through interaction with the DBD of the ligand dependent
transcription factor of the first nucleotide molecule. In the
presence of a marker ligand in the product, the DBD of the first
nucleotide molecule binds to the RE of this second nucleotide
molecule to initiate or suppress transcription of the downstream
reporter gene and second promoter under the regulation of this
response element.
[0086] The RE may be either palindromic (perfect or imperfect) in
its sequence or composed of sequence motifs or half sites separated
by a variable number of nucleotides. The half sites can be similar
or identical and arranged as either direct or inverted repeats. The
RE is preferably a receptor response element obtained from a
steroid/thyroid nuclear receptor superfamily member, as identified
above, or obtained from other sources as identified for the DBDs,
or prepared synthetically. Particularly preferred REs for this
method are a response element from GAL4, a response element from a
steroid/thyroid hormone nuclear receptor, an artificial zinc
finger, a LexA operon, a lac operon response element, and a
synthetic or recombinantly produced response element that
recognizes a synthetic DBD.
[0087] RE, identified from the sources above, may be isolated,
prepared synthetically or recombinantly, and are generally from
about 10 to about 40 nucleotides in length. Preferably the RE are
about 12 to 36 nucleotides in length. As one specific example, DNA
sequences for RE of the natural ecdysone receptor include:
RRGG/TTCANTGAC/ACYY [Cherbas L., et al, (1991), Genes Dev. 5,
120-131]; AGGTCAN.sub.(n)AGGTCA, where N.sub.(n) can be one or more
spacer nucleotides [D'Avino PP., et al, (1995), Mol. Cell.
Endocrinol, 113, 1-9]; and GGGTTGAATGAATTT [Antoniewski C., et al,
(1994). Mol. Cell Biol. 14, 4465-4474]. In addition, the response
element itself can be modified or substituted with response
elements for other DNA binding protein domains such as the GAL4
protein from yeast [Sadowski, et al (1988) Nature, 335: 563-564] or
LexA protein from E. coli [Brent and Ptashne (1985), Cell, 43:
729-736].
[0088] b. The Reporter Gene
[0089] The reporter gene of the second nucleotide sequence is one
that is capable of producing upon expression, directly or
indirectly, a detectable signal. This gene may be readily selected
from among a wealth of such reporters in the art. For example, the
reporter gene may encode a protein or enzyme that is capable of
producing an optically detectable, or colorimetric signal. The
reporter gene may be a gene encoding a fluorescent or luminescent
protein. Alternatively, the reporter gene may encode a protein that
interacts with a substrate to produce a detectable signal. The
reporter gene may cause directly or indirectly a red or blue shift
in the emission or absorption spectrum of the detectable signal
(e.g., UV, visible, NMR, etc). The protein encoded by the reporter
gene may be an enzyme that can catalyze a detectable signal.
[0090] Several specific examples of such reporter genes include
those that encode green fluorescent protein, luciferase,
.beta.-galactosidase, blue fluorescent protein, and secreted
alkaline phosphatase. Preferably, the signal is generated by the
reporter itself or by the interaction between the reporter and its
substrate. As known by one of skill in the art, most reporter
enzymes have more than one substrate. The reaction between the
reporter enzyme and the substrate is a signal detectable by visual
detection, microscopic detection, ultraviolet light detection,
electrical detection, change in capacitance, hybridization,
infrared detection, fluorescence detection and nuclear magnetic
resonance. One of skill in the art may readily select from among a
wide variety of known and commercially available reporter genes and
substrates fitting these descriptions. See, also, the examples
below.
[0091] C. The Second Promoter
[0092] The promoter of the second nucleotide sequence controls,
with the response element, the expression of the reporter gene. The
promoter may be the same as the promoter in the first nucleotide
molecule of the detector compositions. Preferably the second
promoter is a different, inducible promoter. As an inducible
promoter, the second promoter regulates the inducible expression of
the reporter in the selected cell and initiates or suppresses
transcription of the reporter gene only in the presence of a
complex formed by the marker ligand, the ligand-dependent
transcription factor, and the response element. More preferably,
the second promoter is inducible by the response element, e.g., an
ecdysone RE. In one embodiment, the second promoter is an
RE-inducible, minimal promoter, such as the alcohol dehydrogenase
minimal promoter or a synthetic TATA box. Most preferred promoters
are ecdysone inducible promoters. Still other exemplary inducible
promoters, include, without limitation, the zinc-inducible sheep
metallothionine promoter, the dexamethasone-inducible mouse mammary
tumor virus promoter, the tetracycline-repressible promoter, the
tetracycline-inducible promoter, the RU486-inducible promoter, and
the rapamycin-inducible promoter.
[0093] The function of the promoter is to modify the expression of
the reporter gene, so as to induce a change in the detectable
signal. For example, in certain embodiments, the second promoter
turns "on" expression of the reporter gene. In other embodiments,
the second promoter turns "off" expression of the reporter gene. In
still other embodiments, the second promoters induces a change in
the expression of the reporter gene, e.g., its suppresses or
reduces expression or it enhances expression.
[0094] Preferably in the second nucleotide molecule the RE is
located 5' to the second promoter, which is located 5' to the
reporter gene. In one embodiment, the EcR RE are linked to a
synthetic TATA box, which is linked to the reporter gene. These
elements of the second nucleotide sequence may be linked directly
3' nucleotides to 5' nucleotides. Alternatively, each of these
domains is preferably separated by an optional spacer of about 16
to about 30 nucleotides. For example, from 6 to about 10 glycine
amino acid residues may be used as the spacer, e.g., from 18 to
about 30 nucleotides. This second nucleotide sequence may be
single-stranded RNA or DNA or double-stranded RNA or DNA. The
second sequence may be prepared in the form of a straight chain, or
preferably as a circular plasmid. In still another embodiment, the
first sequence may be on one plasmid, and the second nucleotide
sequence may be on a another plasmid. Yet a further embodiment,
provides that the first nucleotide sequence and the second
nucleotide sequence are present on the same plasmid, provided that
they are separated by a transcriptional blocker or polyA sequence.
Still other embodiments of these sequences may employ sequences
that have their origins in viruses, bacteriophages, and other
molecules.
[0095] 3. The Optional Cofactor
[0096] An optional part of the detector composition is a nucleotide
sequence encoding a cofactor. Among the cofactors that may be
useful or necessary depending upon the cell type into which the
detector compositions is transfected include proteins generally
known as coactivators (also termed adapters or mediators) or
corepressors (also known as repressors, silencers, or silencing
mediators).
[0097] Coactivators do not bind sequence-specifically to DNA and
are not involved in basal transcription. A coactivator in the
detector composition of this invention interacts with the
ligand-dependent transcription factor to activate expression of the
reporter gene. They may exert their effect on transcription
activation through various mechanisms, including stimulation of
DNA-binding of activators, by affecting chromatin structure, or by
mediating activator-initiation complex interactions. Examples of
such coactivators include RIP140, TIF 1, RAP46/Bag-1, ARA70,
SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the
promiscuous coactivator C response element B binding protein,
CBP/p300. For review, see CK Glass et at, Curr. Opin. Cell Biol.
9:222-232 (1997).
[0098] Corepressors may be required to effectively inhibit
transcriptional activation in the absence of marker ligand. A
corepressor in the detector composition of this invention interacts
with the ligand-dependent transcription factor to repress
expression of the reporter gene. These corepressors may interact
with the unliganded ecdysone receptor to silence the activity at
the response element. Current evidence suggests that binding of
ligand changes the conformation of the receptor, which results in
release of the corepressor and recruitment of the above described
coactivators, thereby abolishiing their silencing activity.
Examples of corepressors include N-CoR and SMRT. For a review, see
K. B. Horwitz et al. Mol Endocrinol., 10: 1167-1177 (1996).
[0099] In the absence of such cofactors endogenous within the cell
to be transfected with the detector composition, a nucleotide
sequence encoding one or more cofactors may be added exogenously to
the cell as part of the detector composition. In one embodiment, a
sequence encoding a cofactor may be included as part of a first or
second nucleotide sequence of the detector composition. The
cofactor may be placed under the control of a regulated or
unregulated promoter (constitutive or inducible, as described
above) or the nucleotide sequence may be engineered to be expressed
by the first or second promoters described above. In another
embodiment, the cofactor sequence may be placed on a separate
"third" nucleotide sequence, e.g., on a separate straight chain or
circular plasmid. As yet a third alternative, a selected cofactor
protein may be added to the product or portion or extract of the
product as a separate step in the method described herein.
[0100] 4. Preparation of the Detector Compositions
[0101] Once the individual component domains and regions of the
first and second nucleotide molecules (and optional third
nucleotide molecule or sequence) are selected as discussed above,
the nucleotide sequences useful in the methods of the invention may
be prepared conventionally by resort to known chemical synthesis
techniques, e.g., solid-phase chemical synthesis, such as described
by Merrifield, J. Amer. Chem. Soc., 85:2149-2154 (1963), and J.
[0102] Stuart and J. Young, Solid Phase Peptide Synthelia, Pierce
Chemical Company, Rockford, Ill. (1984), or detailed in the
examples below. Alternatively, the nucleotide sequences useful in
the method of this invention may be prepared and assembled by known
recombinant DNA techniques and genetic engineering techniques, such
as polymerase chain reaction, by cloning and expressing within a
host microorganism or cell a DNA fragment carrying the
above-identified nucleic acid sequences, etc. [See, e.g., Sambrook
et al., Molecular Cloning. A Laboratory Manual., 2d Edit., Cold
Spring Harbor Laboratory, New York (1989); Ausubel et al. (1997),
Current Protocols in Molecular Biology, John Wiley & Sons, New
York]. These first and second molecules may be separate straight
chain or circular nucleotide constructs. Alternatively, they may be
circular plasmids. Still alternatively, they may be located on a
single molecule. As another alternative, two or more proteins made
from first nucleotide sequences may associate to form homodimers or
heterodimers to generate a single transcription factor.
[0103] Once prepared, these molecules may be introduced into a
selected cell by any conventional means, such as, for example,
transfection, electroporation, liposome delivery, membrane fusion
techniques, high velocity DNA-coated pellets, viral infection and
protoplast fusion. Alternatively, if the molecules are prepared as
RNA molecules, they may be designed as part of a selected virus by
conventional recombinant techniques. For insect and plant cells
only a first nucleotide sequence containing an LBD, DBD and
transactivation domain and a second nucleotide sequence may be
desired to perform this method. For mammalian cells and yeast
cells, at least two "first" nucleotide sequences and a second
nucleotide sequence may be desired, optionally with a cofactor, to
perform this method.
[0104] It is preferred for ease of use of the present invention,
that the cells or viruses of the detector compositions be prepared
in live, unfrozen or live, lyophilized form. Any of the
above-identified cells, once transfected with the first and second
nucleotide molecules, may be lyophilized by conventional means,
such as those taught in conventional texts.
[0105] Even more preferably, the lyophilized cell or viral detector
composition is immobilized on a solid support. Among suitable solid
supports include microcells, microcapsules, microtiter plates,
beads, and biochips. Useful supports include those described in
International Patent Publication WO99/27351, published Jun. 3,
1999; or International Patent Publication WO99/27140, published
Jun. 3, 1999; U. S. Pat. No. 6,096,273; International Patent
Publication WO00/14197, published Mar. 16, 2000, among others. Such
solid supports for immobilizing the cells or lyophilized cells of
the detector compositions may be selected from among the many known
types available commercially; and methods for adhering the cells or
viruses to the supports are provided by the manufacturers of the
supports. In yet another embodiment, the support may be an
adhesive, e.g., for application to solid products.
[0106] D. Performance of the Method
[0107] The method of the present invention thus relies on the
presence of a marker ligand in or on a selected product of the
present invention. To detect the presence or quantify the marker
ligand in the product, the product is contacted with one or more
detector compositions as defined above. If the marker ligand is
applied to the solid product, rather than a liquid product, it may
be extracted from the solid product. For ease of use, it is
preferred that the detector composition comprise an immobilized
transfected cell or RNA virus as described above. For example, when
a sample of product is placed on a detector composition immobilized
on a biochip, the marker ligand binds preferentially to the LBD in
the immobilized cell. This binding causes a conformational change
in the ligand dependent transcription factor which causes the DBD
to complex with the RE on the second nucleotide molecule. The
complex formed between the DBD and the RE activates the second
promoter, which modulates the expression of the exogenous reporter
gene.
[0108] The order in which the various components bind to each
other, that is, marker ligand to LBD-DBD-transactivation domain
sequence and DBD to response element, is not critical. However, the
presence of the marker ligand is absolutely required to either turn
on expression of the reporter gene or turn off expression of a
reporter gene or change the expression of the reporter gene,
thereby creating a detectable signal or detectable change in the
signal indicative of the presence of the marker ligand. If no
marker ligand is present in the product, no change of signal is
detectable.
[0109] E. A Detection Kit
[0110] The components of this method are readily adaptable into a
kit that contains one or more detector compositions suitable for
detecting the marker ligand in a liquid or solid product, suitable
vessels for containing sample and/or a plurality of detector
compositions of the invention in an environment suitable for
preserving the detectable properties of the signals generated by
the reporter gene or reporter gene system, and/or suitable
substrates for interaction with the reporter gene product to
produce a detectable signal. The kit of the present invention can
contain either the same or different detector compositions, whereby
a plurality of samples can be examined with the same detector
compositions or with multiple detector compositions. These kits can
additionally contain reagents necessary to culture the cells, if
necessary; and/or reagents necessary to reactivate the lyophilized
cells or lysates; instructions for performing a detection assay,
substrates to which the detector composition has been pre-adsorbed
in a lyophilized state, diluents and buffers, indicator charts for
signal comparisons, disposable gloves, decontamination
instructions, applicator sticks or containers, and sample
preparator cups. For convenience, it is preferable, to provide
frozen, lyophilized or otherwise preserved cells which, after
reactivation, express the detector compositions of the invention.
The kits of the invention can contain intact cells constitutively
expressing the reporter proteins of the invention or cells only
expressing the reporter protein after induction or expressing as
long as no inhibitor is added. Respective inducers or inhibitors
are preferably also included in these kits. Further, the kits
preferably include the solutions required for reactivating
preserved expression systems. The kits preferably also contain
necessary buffer substances or culture media, as far as this is
required due to the expression system used.
[0111] Kits of the invention are useful for the rapid screening of
a plurality of samples for the presence or absence of marker
ligand. Use according to the invention, therefore, is in accordance
with the methods of the invention already discussed in detail
above, whereby the use of the kits of the invention enable a user
to carry out determination of product source or adulteration in a
simple and rapid manner, with the kit of the invention preferably
providing him with all components required.
F. THE EXAMPLES
[0112] The following examples illustrate several embodiments of the
methods and compositions of this invention. These examples are
illustrative only, and do not limit the scope of the present
invention.
Example 1
The EcR Gene Switch
[0113] An embodiment of the ecdysone gene switch is prepared as a
receptor plasmid (the first nucleotide molecule described above)
and a reporter plasmid (the second nucleotide molecule described
above).
[0114] The receptor plasmid pIE1VP16CfEcRCDEF contained CfEcR CDEF
domains fused to the VP16 activation domain and expressed under the
baculovirus El promoter. This plasmid was constructed in two steps.
To construct vector pIE1VP16, the IE1 promoter region of AcMNPV
(described in Ayres et al, cited above) was amplified using primers
tagged with Nde1 and Bg1II restriction enzyme sties. The amplified
product was cloned into plasmid vector containing the VP16
activation domain (described in Pellett et al, cited above), and
multiple cloning sites, followed by an SV40 polyA signal. CfEcR
CDEF domains (described in Kothapalli et al, cited above) were
amplified using primers tagged with BamHI and XbaI. The amplified
CfEcRCDEF was then cloned into the pIE1VP16 vector.
[0115] The reporter plasmid pMK43.2 contained the 6X ecdysone
response elements from the heat shock protein 27 gene, as described
in Riddihough and Pelham, EMBO J., 6:3729-3734 (1987), cloned
upstream to an alcohol dehydrogenase minimal promoter and an E.
coli .beta.-galactosidase reporter gene. The construction of that
plasmid is described in M. R. Koelle et al, Cell, 67:59-77
(1991).
[0116] The gene switch operates as follows when the two plasmids
are transfected into a host cell. The EcR protein, which in the
receptor plasmid described above is produced constitutively by the
baculovirus IE1 promoter, is expressed in the cytoplasm of the
selected host cell in an inactive form. In an alternative
embodiment, the EcR protein can be placed under the control of a
tissue-specific inducible or developmentally regulated promoter to
control the tiring and location of EcR expression in the host. The
EcR protein migrates to the cell nucleus and binds to a specific
DNA sequence, referred to as the Response Element, which is present
in the reporter plasmid described above. The Response Element is a
unique DNA sequence that is not naturally found in the host's DNA
and is uniquely recognized by the EcR protein. The Response Element
is functionally linked to the regulated gene of interest, which in
this embodiment is the reporter gene .beta.-galactosidase.
[0117] When the cell is contacted by marker ligands, e.g., in a
sample which is exposed to the cell in one of the assays of Example
3 or 4, the marker ligands bind to and activate the EcR receptor to
"switch on" expression of the .beta.-galactosidase gene and thus
produce the protein encoded by that gene. The assays described
below are designed to allow the identification and measurement of
that reporter gene.
Example 2
Preparation of Marked Solutions
[0118] To a 100 ml volumetric flask was added 1.0166g of the marker
ligand, methoxyfenoxide
(N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-3-methoxy-
-2-methylbenzohydrazide). The solution was diluted to 100 mL volume
with absolute ethanol, resulting in Solution A. One milliliter of
Solution A contains approximately 10 mg of marker ligand.
[0119] Solution B was prepared by adding to a 100 ml volumetric
flask 10 mL (.+-.0.04 mL) of Solution A above. The resulting
solutions were each diluted to 100 mL volume with absolute ethanol.
The resulting Solution B contained approximately 1 mg marker
ligand.
[0120] Solution C was prepared by adding to a 100 ml volumetric
flask 10 mL (.+-.0.04 mL) of Solution B. The solution was diluted
to 100 mL volume with absolute ethanol. One ml of the resulting
Solution C contains approximately 0.1 mg marker ligand.
[0121] To prepare sample solutions for testing in the yeast and
insect assays below, 1 mL (.+-.0.12 mL) of stock solution A, B, or
C was added to approximately 100 g of gasoline or vodka, as
summarized in Table 1 below:
1 TABLE 1 Stock Dilution with Concentration Sample # Solution
gasoline or vodka mg/ml 1 A 100.07 g gasoline 6.95 2 B 100.08 g
gasoline 0.69 3 C 100.01 g gasoline 0.07 4 A 100.04 g vodka 11.67 5
B 100.06 g vodka 1.17 6 C 100.03 g vodka 0.12
[0122] After the samples in Table 1 were diluted 1 to 100 in
acetonitrile/water (1/1), the concentration of the marker ligand in
each gasoline and vodka sample was quantified by liquid
chromatography/mass spectrometry (LC/MS) on a HP1000-VG platform.
LC involves gradient separation on a 3.times.50 mm C-18 column
(Polaris Metachem) using an injection volume of 25 .mu.L. The MS
was in signal ion monitoring mode providing specific detection of
marker ligand molecular ions. All HPLC standards were prepared in
acetonitrile/water (1/1). Quantification was performed comparing
analyte concentration (area) and five standard concentrations
(area) which were analyzed before and after each marker ligand set
(A,B, and C for gasoline or vodka). The results are summarized in
Table 2.
2TABLE 2 Std Retention Sam- Conc. Time, ple Type .mu.g/ml minutes
Area Response .mu.g/ml Std Standard 0.012 4.646 1211 1210.613
0.0119 Std Standard 0.12 4.646 11145 11144.99 0.1099 std Standard
1.2 4.646 120026 120026.1 1.2085 std Standard 12 4.646 940122
940122.4 11.9851 wash Blank 4.863 14 13.547 0.0001 1 Analyte 4.646
76515 76514.81 0.7638 2 Analyte 4.61 8257 8256.889 0.0813 3 Analyte
4.61 916 915.831 0.009 4 Analyte 4.646 129204 129203.8 1.3033 5
Analyte 4.646 14591 14590.56 0.1439 6 Analyte 4.646 1545 1544.863
0.0152 wash Blank 4.61 10 9.863 0.0001 std Standard 0.012 4.646
1222 1221.55 0.012 std Standard 0.12 4.646 11546 11545.91 0.1138
std Standard 1.2 4.646 120204 120203.5 1.2103 std Standard 12 4.61
941603 941603.3 12.0119
[0123] These results demonstrate that conventional analytical
chemical detection (e.g., chromatographic methods) can be used to
detect the presence of marker ligand in samples of gasoline and
vodka.
Example 3
Insect Cell-Based Marker Ligand Assay
[0124] An insect cell line, BRL-AG2, was derived from cotton boll
weevil, Anthomus grandis as described in Stiles et al, In Vitro
Cell Dev. Biol., 28A:355-363 (1992). Another insect cell line L57
was prepared as a Drosophila melanogaster Kc cell line modified to
silence the expression of the ecdysone receptor. Each of these
insect cell lines was transfected with the receptor plasmid and
reporter plasmid described in Example 1 above.
[0125] In the insect cell-based marker ligand assay, 200,000 of the
transfected BRL-AG2 cells or the L57 cells described above were
distributed per well of 48-well plates. 0.5 .mu.L of either a
negative control, e.g., DMSO, or a marked product (vodka or
gasoline) of Example 2 was added to the cells. The cells were
maintained in the medium containing the marker ligand solutions of
Example 2 or a positive control (i.e., methoxyfenoxide in alcohol)
or a negative control (alcohol only) for 48 hours at 25.degree. C.
The cells were harvested and resuspended in reporter lysis buffer
(Promega Corporation, Madison, Wis.) for 15 minutes. A 10 .mu.L
aliquot of the buffer containing the resuspended cells was assayed
for .mu.-galactosidase activity using Galacto-Star.TM.
chemiluminescent reporter gene assay system (Tropix Corporation,
Bedford, Mass.). Fold induction was calculated by dividing relative
light units (RLUs) in the presence of the marker ligand with the
RLUs in the absence of the marker ligand.
[0126] The results are tabulated in Tables 3 and 4 below.
3TABLE 3 Detection of Markers in Vodka: Concentration of BRL-AG2
Cells L57 Cells Marker (mg/ml) (Fold Induction) (Fold Induction) 0
1 1 0.00007 151 27 0.00069 182 46 0.00695 202 63
[0127]
4TABLE 4 Detection of Markers in Gasoline: Concentration of BRL-AG2
L57 Cells Marker (mg/ml) (Fold Induction) (Fold Induction) 0 1 1
0.00012 16 1 0.0012 21 23 0.012 31 30
[0128] The cells appeared healthy even at 50 .mu.Ls vodka or
gasoline. There is an approximately 20-50X induction of reporter
activity by marked products. The marked product containing of 0.5
.mu.L per mL of marker ligand is sufficient to observe significant
change in reporter activity. These results demonstrate an increased
sensitivity of at least 10.times. for marker ligand vs. the
chemical methods of Example 3. The marker solutions could be
further diluted in the biological assay because the biological
assay reached saturation at the lowest concentration tested. The
biological assay results mimic the chemical analysis in that the
gasoline samples show a lower concentration of marker ligand. This
indication may be due to error in calculating the marker ligand
concentration in the gasoline samples.
[0129] All references cited above are incorporated herein by
reference. Numerous modifications and variations of the present
invention are included in the above-identified specification and
are expected to be obvious to one of skill in the art. Such
modifications and alterations to the compositions and processes of
the present invention are believed to be encompassed in the scope
of the claims appended hereto.
* * * * *