U.S. patent application number 10/259430 was filed with the patent office on 2003-05-01 for olfactory receptor expression libraries and methods of making and using them.
Invention is credited to Krautwurst, Dietmar, Reed, Randall R., Yau, King-Wai.
Application Number | 20030082615 10/259430 |
Document ID | / |
Family ID | 22344837 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082615 |
Kind Code |
A1 |
Reed, Randall R. ; et
al. |
May 1, 2003 |
Olfactory receptor expression libraries and methods of making and
using them
Abstract
This invention provides novel libraries of olfactory receptor
odorant/ligand-binding domains and methods of making and using
them. The invention also provides libraries of vectors and cells
comprising these nucleic acid constructs. The compositions and
methods of the invention are used to identify novel ligand-binding
domains for olfactory neuron odorant receptors and their ligands.
Thus, the compositions and methods of the invention can be used to
generate novel odorants, to screen for toxic odorants, or to
manipulate an animal's olfactory response.
Inventors: |
Reed, Randall R.;
(Baltimore, MD) ; Yau, King-Wai; (Towson, MD)
; Krautwurst, Dietmar; (Bergholz Rebhrucke, DE) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
22344837 |
Appl. No.: |
10/259430 |
Filed: |
September 30, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10259430 |
Sep 30, 2002 |
|
|
|
09465901 |
Dec 17, 1999 |
|
|
|
6492143 |
|
|
|
|
60112605 |
Dec 17, 1998 |
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/91.2; 536/24.3 |
Current CPC
Class: |
G01N 2500/20 20130101;
G01N 33/5058 20130101; C12N 15/8509 20130101; A01K 2267/0393
20130101; G01N 33/5008 20130101; A01K 2217/05 20130101; B01J
2219/00707 20130101; G01N 33/6872 20130101; C40B 40/08 20130101;
A01K 2217/075 20130101; A01K 2227/10 20130101; C07K 2319/32
20130101; G01N 33/5014 20130101; C07K 14/705 20130101; C40B 40/06
20130101; C12Q 1/6876 20130101; B01J 2219/00722 20130101; C07K
2319/00 20130101; G01N 33/566 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Claims
What is claimed is:
1. An amplification primer sequence pair for amplifying a nucleic
acid encoding an olfactory receptor ligand-binding region
comprising a first primer comprising a sequence
5'-GGGGTCCGGAG(A/G)(C/G)
(A/G)TA(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3' (SEQ ID NO:1) and a
second primer comprising a sequence
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)
(C/T)T(A/C/G/T)TT(C/T)(C/T)T-3' (SEQ ID NO:2).
2. The amplification primer sequence pair of claim 1, wherein the
receptor ligand-binding region comprises olfactory receptor
transmembrane domains II through VII.
3. A method for generating nucleic acid sequence that encodes a
ligand-binding region of an olfactory receptor, the method
comprising amplification of a nucleic acid using a primer pair as
set forth in claim 1.
4. The method of claim 3, wherein the amplified nucleic acid is
genomic DNA, mRNA or cDNA derived from olfactory neurons or
olfactory epithelium.
5. The method of claim 3, wherein the amplification comprises the
following conditions and steps in the following order: about one
cycle at about 94.degree. C. for about 2 min; and about 30 cycles
of about 45.degree. C. to about 65.degree. C. for about 1 min,
followed by about 72.degree. C. for about one min. followed by
about 94.degree. C. for about 1 min.
6. The method of claim 5, wherein the PCR amplification further
comprises the following conditions and steps in the following
order: about one cycle of about 45.degree. C. to about 65.degree.
C. for about 10 min; and about one cycle of about 72.degree. C. for
about 10 min.
7. A kit for amplification of olfactory receptor sequences
comprising the primer pair of claim 1.
8. A library of olfactory receptor ligand-binding regions
consisting essentially of olfactory receptor transmembrane domain
regions II through VII, II through VI, III through VII, or III
through VI.
9. The library of claim 8, wherein the olfactory receptor
ligand-binding regions are generated by polymerase chain reaction
using degenerate primer pairs.
10. A library of chimeric nucleic acid sequences comprising the
following domains in 5' to 3' order: a nucleic acid encoding an
amino terminal plasma membrane translocation domain; a nucleic acid
encoding a first transmembrane domain; and a nucleic acid encoding
an olfactory receptor ligand-binding region, wherein the chimeric
nucleic acid sequence encodes a 7-transmembrane polypeptide that
can transverse a plasma membrane seven times.
11. The library of claim 10, wherein the amino terminal plasma
membrane translocation domain comprises a sequence as set forth in
SEQ ID NO:3.
12. The library of claim 10, wherein the first transmembrane
receptor is a 7-transmembrane receptor region I domain.
13. The library of claim 12, wherein the 7-transmembrane receptor
transmembrane region I domain comprises a sequence as set forth in
SEQ ID NO:4.
14. The library of claim 10, wherein the olfactory receptor
ligand-binding region comprises olfactory receptor transmembrane
domain regions II through VII, II through VI, III through VII, or
III through VI.
15. The library of claim 10, wherein the olfactory receptor
ligand-binding regions are generated by polymerase chain reaction
using degenerate primer pairs.
16. The library of claim 14, wherein the nucleic acid sequence
encoding the transmembrane domain regions II through VII is
generated by polymerase chain reaction (PCR) amplification of
nucleic acid using a first primer comprising a sequence
5'-GGGGTCCGGAG(A/G)(C/G)(A/G)TA(A/G/T)-
AT(A/G/P)A(A/G/P)(A/G/P)GG-3' (SEQ ID NO:1) and a second primer
comprising a sequence 5' -GGGGCTGCA
GACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/G/T)TT(C/T)(- C/T)T-3' (SEQ ID
NO:2).
17. The library of claim 15, wherein the PCR-amplified nucleic acid
is genomic DNA, mRNA or cDNA derived from olfactory neurons or
olfactory epithelium.
18. The library of claim 10, wherein the ligand-binding region
comprising transmembrane domains II through VII is an amino acid
sequence encoded by a nucleic acid selected from the group
consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45 and SEQ ID NO:47, or an amino acid sequence selected from the
group consisting of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ
ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ
ID NO:46, SEQ ID NO:48.
19. The library of claim 15, wherein the PCR amplification
comprises the following conditions and steps in the following
order: about one cycle at about 94.degree. C. for about 2 min;
about 30 cycles of about 55.degree. C. for about 1 min, followed by
about 72.degree. C. for about one min. followed by about 94.degree.
C. for about 1 min; about one cycle of about 55.degree. C. for
about 10 min; and about one cycle of about 72.degree. C. for about
10 min.
20. The library of claim 10, further comprising a carboxy terminal
7-transmembrane receptor transmembrane region VII domain.
21. The library of claim 20, wherein the 7-transmembrane receptor
transmembrane region VII domain comprises a sequence as set forth
in SEQ ID NO:5.
22. A library of nucleic acid sequences comprising the following
domains in 5' to 3' order a nucleic acid encoding an amino terminal
plasma membrane translocation domain comprising a sequence as set
forth in SEQ ID NO:3, a nucleic acid encoding a transmembrane
region I domain comprising a sequence as set forth in SEQ ID NO:4,
a nucleic acid sequence generated by polymerase chain reaction
(PCR) amplification of mRNA or cDNA derived from olfactory
epithelium using a first primer comprising a sequence
5'-GGGGTCCGGAG(A/G)(C/G)T(A/G)A(A/G/T)AT
(A/G/P)A(A/G/P)(A/G/P)GG-3' (SEQ ID NO:1) and a second primer
comprising a sequence
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)C/T)T(A/C/G/T) TT(C/T)C/T)T-3'
(SEQ ID NO:2), and a nucleic acid encoding a 7-transmembrane
receptor transmembrane region VII domain comprising a sequence as
set forth in SEQ ID NO:5.
23. An expression vector comprising a nucleic acid sequence derived
from a library of nucleic acid sequences as set forth in claim 8 or
claim 10.
24. A transformed or isolated infected cell comprising a nucleic
acid sequence derived from a library of nucleic acid sequences as
set forth in claim 8 or claim 10 or an expression vector as set
forth in claim 23.
25. A transgenic animal comprising a nucleic acid sequence derived
from a library of nucleic acid sequences as set forth in claim 8 or
claim 10 or an expression vector as set forth in claim 23.
26. The transgenic animal of claim 22, wherein the expression
vector is a mammalian expression vector that can be expressed in
olfactory epithelium or olfactory neurons.
27. A library of recombinant polypeptides translated or derived
from the library of nucleic acids as set forth in claim 8 or claim
10.
28. An isolated polypeptide isolated or derived from the library of
polypeptides as set forth in claim 27.
29. A method of determining whether a test compound specifically
binds to a mammalian olfactory receptor comprising the following
steps: (i) expressing a nucleic acid derived from a nucleic acid
library as set forth in claim 8 or claim 10 under conditions
permissive for translation of the nucleic acid to a receptor
polypeptide; (ii) contacting the translated polypeptide with the
test compound; and (iii) determining whether the test compound
specifically binds to the polypeptide.
30. A method of determining whether a test compound specifically
binds to a mammalian olfactory transmembrane receptor comprising
the following steps: (i) contacting a cell stably or transiently
transfected with a nucleic acid derived from a nucleic acid library
as set forth in claim 8 or claim 10; (ii) culturing the cell under
conditions permissive for translation of the nucleic acid to a
receptor polypeptide with the test compound; and (iii) determining
whether the test compound specifically binds to the receptor
polypeptide.
31. The method of claim 30, wherein the receptor polypeptide is
expressed as a transmembrane receptor with a ligand binding site on
the cell's plasma membrane outer surface.
32. The method of claim 30, wherein the specific binding of the
test compound to the polypeptide is determined by measuring a
change in the physiologic activity of the cell, wherein a change in
the cell's activity measured in the presence of the test compound
compared to the cell's activity in the absence of the test compound
provides a determination that the test compound specifically binds
to the polypeptide.
33. The method of claim 32, wherein the measured cell activity is a
change in the calcium ion (Ca.sup.2+) or cAMP concentration in the
cell.
34. The method of claim 33, wherein the calcium ion concentration
is measured by loading the cell with a calcium ion-sensitive
fluorescent dye before contacting the cell with the test
compound.
35. The method of claim 30, wherein the cell is a human cell or a
Xenopus oocyte.
36. A method of determining whether a test compound specifically
binds to a mammalian olfactory transmembrane receptor polypeptide
in vivo comprising the following steps: (i) contacting a non-human
animal stably or transiently infected with a nucleic acid derived
from the library as set forth in claim 8 or claim 10 or an
expression vector as set forth in claim 23 with the test compound;
and (ii) determining whether the animal reacts to the test compound
by specifically binding to the receptor polypeptide, wherein the
specific binding of the test compound to the polypeptide is
determined by measuring a change in a physiologic activity of the
animal, wherein a change in a receptor-encoding vector-infected
animal's activity measured in the presence of the test compound
compared to a bare vector-infected animal's activity in the
presence of the test compound provides a determination that the
test compound specifically binds to the mammalian olfactory
transmembrane receptor polypeptide.
37. The method of claim 36, wherein the measured physiologic
activity is measured by an electroolfactogram.
38. The method of claim 36, wherein the vector is an adenovirus
expression vector.
39. A method of determining whether a test compound is neurotoxic
to an olfactory neuron expressing an olfactory transmembrane
receptor polypeptide comprising the following steps: (i) contacting
an olfactory neuron cell stably or transiently infected with a
nucleic acid derived from a library as set forth in claim 8 or
claim 10 or an expression vector as set forth in claim 23 with the
test compound; and (ii) measuring the physiologic activity of the
cell, wherein a change in the cell's activity measured in the
presence of the test compound compared to the cell's activity in
the absence of the test compound provides a determination that the
test compound is toxic.
40. The method of claim 39, wherein toxicity is indicated by
abnormal calcium ion, cAMP or plasma membrane homeostasis.
41. A peptide domain for the efficient translocation of a newly
translated protein to a plasma membrane comprising an amino acid
sequence as set forth in SEQ ID NO:3 or an amino acid sequence
having conservative amino acid residue substitutions based on SEQ
ID NO:3.
42. The peptide translocation domain of claim 41, wherein the
translocation domain is about 20 amino acids in length.
43. The peptide translocation domain of claim 41, wherein the
polypeptide translocation domain is SEQ ID NO:3.
44. The peptide translocation domain of claim 41, wherein the newly
translated protein is a transmembrane protein.
45. The peptide translocation domain of claim 41, wherein the
transmembrane protein is a 7-transmembrane protein receptor.
46. The peptide translocation domain of claim 45, wherein the
7-transmembrane protein receptor is an olfactory receptor.
47. A hybrid polypeptide comprising an amino terminal amino acid
sequence comprising a peptide translocation domain as set forth in
claim 41 and a second polypeptide sequence, wherein the second
polypeptide sequence is not a rhodopsin polypeptide sequence.
48. The hybrid polypeptide of claim 47, wherein the second
polypeptide sequence is a transmembrane protein.
49. The hybrid polypeptide of claim 48, wherein the transmembrane
protein is a 7-transmembrane protein receptor.
50. The hybrid polypeptide of claim 49, wherein the 7-transmembrane
protein receptor is an olfactory receptor.
51. An isolated or recombinant nucleic acid sequence encoding the
hybrid polypeptide of claim 47.
Description
FIELD OF THE INVENTION
[0001] This invention generally pertains to the fields of cell
biology and medicine. In particular, this invention provides novel
libraries of nucleic acids encoding odorant/ligand-binding domains.
Also provided are libraries of hybrid 7-transmembrane olfactory
receptors comprising these odorant ligand-binding domains. The
compositions and methods of the invention can be used to identify
novel ligand-binding domains for olfactory neuron odorant receptors
and their ligands. Thus, the compositions and methods of the
invention can be used to generate novel odorants and to manipulate
an animal's olfactory response.
BACKGROUND OF THE INVENTION
[0002] A better understanding of the vertebrate olfactory system
would provide improved means to manipulate this process and
possibly prevent disease or injury. For example, means to
manipulate human olfactory neuron odorant receptors from healthy
individuals and from individuals with neuro-psychiatric illnesses
would offer systems for testing possible odorant/ligands for
therapeutic and toxic effects. However, our ability to detect and
discriminate between the thousands of beneficial or toxic odorants
is complicated by the fact that odorant receptors belong to a
multigene family with at least 500 to 1000 members. Furthermore,
each olfactory receptor neuron may express only one, or at most a
few, of these olfactory receptors. Any given olfactory neuron cell
can respond to a small, arbitrary set of odorant-ligands. Odorant
discrimination for a given neuron may depend on the ligand
specificity of the one or few receptors it expresses. Thus, given
this systems' complexity, information about odorant/ligand-receptor
recognition remains meager.
[0003] To analyze odorant/ligand-receptor interactions and their
effects on cell physiology, it is first necessary to identify
specific odorant/ligand(s) and the olfactory receptors to which
they specifically bind. Such analysis requires isolation and
expression of olfactory receptor polypeptides. However, despite the
fact that many putative olfactory receptors have been cloned, only
limited progress has been made in the functional expression of
these receptors because present systems fail to efficiently
translocate these 7-transmembrane proteins to the plasma membrane.
This may be because olfactory receptors are a subclass of
7-transmembrane-domain receptors. For example, expression of one
rat olfactory receptor in insect cells resulted in only a modest
elevation in second messengers when exposed to a mixture of
odorants; responses to single compounds were not seen (Raming
(1993) Nature 361:353-356). The present invention addresses these
and other needs.
SUMMARY OF THE INVENTION
[0004] The present invention provides novel compositions and
methods to generate great numbers, or libraries, of odorant
receptor ligand-binding regions. Also provided are novel chimeric
olfactory receptors that incorporate these libraries of odorant
binding domains. The present invention also provides novel
compositions and methods to efficiently translocate polypeptides to
the plasma membrane surface. Another aspect of the invention is
based on the surprising discovery of a peptide domain that, when
incorporated into a polypeptide, can with great efficiency
"chaperone" or translocate the hybrid protein to the cell plasma
membrane. Combining these two aspects of the invention also
provides expression vectors and cells that efficiently express
these recombinant proteins. Cells and transgenic animals
efficiently expressing libraries of hybrid olfactory receptors can
be used for screening potential beneficial and toxic odorant
molecules.
[0005] The invention provides an amplification primer sequence pair
for amplifying a nucleic acid encoding an olfactory receptor
ligand-binding region comprising a first primer comprising a
sequence 5'-GGGGTCCGGAG(A/G)(C/G)
(A/G)TA(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3' (SEQ ID NO:1) and a
second primer comprising a sequence
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)
(C/T)T(A/C/G/T)TT(C/T)(C/T)T-3' (SEQ ID NO:2). When used to amplify
olfactory receptor nucleic acid sequences, it typically amplifies
the receptor ligand-binding region comprising olfactory receptor
transmembrane (TM) domains II through VII.
[0006] The invention also provides a method for generating nucleic
acid sequence that encodes a ligand-binding region of an olfactory
receptor, the method comprising amplification of a nucleic acid
using the primer pair SEQ ID NO:1 and SEQ ID NO:2. In this method
the amplified nucleic acid can be genomic DNA, mRNA or cDNA derived
from olfactory neurons or olfactory epithelium. The amplification
can be by polymerase chain reaction (PCR), wherein the PCR
amplification comprises the following conditions and steps in the
following order: about one cycle at about 94.degree. C. for about 2
min; and about 30 cycles of about 45.degree. C. to about 65.degree.
C. for about 1 min, followed by about 72.degree. C. for about one
min. followed by about 94.degree. C. for about 1 min. The PCR
amplification protocol can further comprise the following
conditions and steps in the following order: about one cycle of
about 45.degree. C. to about 65.degree. C. for about 10 min; and
about one cycle of about 72.degree. C. for about 10 min.
[0007] Also provides is a kit for amplification of olfactory
receptor sequences comprising primer pairs that can amplify
olfactory receptor transmembrane domain regions II through VII, II
through VI, III through VII, or III through VI, e.g., SEQ ID NO:1
and SEQ ID NO:2 to amplify TM II through VII.
[0008] The invention also provides a library of olfactory receptor
ligand-binding regions consisting essentially of olfactory receptor
transmembrane domain regions II through VII, II through VI, III
through VII, or III through VI, including partial domains, or a
combination of domain sequences. The library of the olfactory
receptor ligand-binding regions can be generated by PCR using
degenerate primer pairs.
[0009] Also provided is a library of chimeric nucleic acid
sequences comprising the following domains in 5' to 3' order: a
nucleic acid encoding an amino terminal plasma membrane
translocation domain; a nucleic acid encoding a first transmembrane
domain; and a nucleic acid encoding an olfactory receptor
ligand-binding region, wherein the chimeric nucleic acid sequence
encodes a 7-transmembrane polypeptide that can transverse a plasma
membrane seven times. The amino terminal plasma membrane
translocation domain comprises an amino acid sequence as set forth
in SEQ ID NO:3 (and encoded by a subsequence of SEQ ID NO:6):
5'
[0010]
5'-GGATCCGGGTTCGCGCCGCCGGCGGGCAGCCGCAAGGGCCGCAGCCATGAACGGGACCGAGGGC
[0011] M N G T E G
[0012] CCAAACTTCTACGTGCCTTTCTCCAACAAGACGGGCGTGGTGGAATTC-3' (SEQ ID
NO:6)
[0013] P N F Y V P F S N K T G V V (SEQ ID NO:3)
[0014] In alternative embodiments, the nucleic acid encoding the
first transmembrane domain can be just a polynucleotide sequence
encoding SEQ ID NO:3, or, SEQ ID NO:6 (including 45 nucleotides
upstream of the initiation codon) or a subsequence thereof.
[0015] The first transmembrane receptor of the sequences of the
library can be a 7-transmembrane receptor region I domain, or
subsequence thereof, e.g., the sequence between the Eco R1 and Pst
1 sites of the M4-chimeric olfactory receptor of the invention (SEQ
ID NO:4), as schematically represented in FIG. 1A; the full length
sequence of the hybrid receptor has an amino acid sequence as set
forth in SEQ ID NO:55, a nucleic acid that can encode this protein
is SEQ ID NO:54, described below.
[0016] The olfactory receptor ligand-binding regions of the library
can comprise olfactory receptor transmembrane domain regions II
through VII, II through VI, III through VII, or III through VI, or
a combination thereof. These olfactory receptor ligand-binding
regions can be generated by amplification, e.g., PCR, using
degenerate primer pairs. The library's nucleic acid sequence
encoding transmembrane domain regions II through VII can generated
by PCR amplification of nucleic acid using a first primer
comprising a sequence 5'-GGGGTCCGGAG(A/G)(C/G)(A/G)TA(A/G/T)AT(A/G-
/P)A(A/G/P)(A/G/P)GG-3' (SEQ ID NO:1) and a second primer
comprising a sequence 5'-GGGGCTGCA
GACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/G/T)TT(C/T)(C/T- )T-3' (SEQ ID
NO:2). The library can be generated from PCR-amplified nucleic acid
isolated as or derived from genomic DNA, mRNA or cDNA derived from
olfactory neurons or olfactory epithelium.
[0017] Exemplary ligand-binding regions comprising transmembrane
domains II through VII ca be an amino acid sequence encoded by a
nucleic acid selected from the group consisting of SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ
ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,
SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:47, or an
amino acid sequence selected from the group consisting of SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,
SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48.
[0018] To generate the nucleic acids of the library, PCR
amplification can comprise the following conditions and steps in
the following order: about one cycle at about 94.degree. C. for
about 2 min; about 30 cycles of about 55.degree. C. for about 1
min, followed by about 72.degree. C. for about one min. followed by
about 94.degree. C. for about 1 min; about one cycle of about
55.degree. C. for about 10 min; and about one cycle of about
72.degree. C. for about 10 min.
[0019] The library can further comprise a carboxy terminal
7-transmembrane receptor transmembrane region VII domain or
subsequence thereof, e.g., the sequence between the Bsp E1 and Xba
1 sites of the M4-chimeric olfactory receptor of the invention (SEQ
ID NO:6), as schematically represented in FIG. 1A; the full length
sequence of the hybrid receptor has an amino acid sequence as set
forth in SEQ ID NO:55, a nucleic acid that can encode this protein
is SEQ ID NO:54.
[0020] The library of nucleic acid sequences can also comprise the
following domains in 5' to 3' order: a nucleic acid encoding an
amino terminal plasma membrane translocation domain comprising a
sequence as set forth in SEQ ID NO:3, a nucleic acid encoding a
transmembrane region I domain comprising a sequence as set forth in
SEQ ID NO:4, a nucleic acid sequence generated by polymerase chain
reaction (PCR) amplification of mRNA or cDNA derived from olfactory
epithelium using a first primer comprising a sequence
5'-GGGGTCCGGAG(A/G)(C/G)T(A/G)A(A/G/T)AT
(A/G/P)A(A/G/P)(A/G/P)GG-3'0 (SEQ ID NO:1) and a second primer
comprising a sequence
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)C/T)T(A/C/G/T) TT(C/T)C/T)T-3'
(SEQ ID NO:2), and a nucleic acid encoding a 7-transmembrane
receptor transmembrane region VII domain comprising a sequence as
set forth in SEQ ID NO:6.
[0021] Also provided are expression vectors (e.g., plasmids,
viruses) comprising a nucleic acid sequence derived from the
libraries of nucleic acid sequences of the invention. Transformed
or isolated infected cells comprising a nucleic acid sequence
derived from a library of nucleic acid sequences of the invention
or an expression vector of the invention are also provided.
Transgenic non-human animals comprising a nucleic acid sequence
derived from a library of nucleic acid of the invention or an
expression vector of the invention are also provided. In the
transgenic animal, the expression vector can be a mammalian
expression vector that can be expressed in olfactory epithelium or
olfactory neurons.
[0022] The invention also provides a library of recombinant
polypeptides translated or derived from the library of nucleic
acids of the invention. Also provided are polypeptides isolated or
derived from the library of polypeptides of the invention.
[0023] Also provided are methods of determining whether a test
compound specifically binds to a mammalian olfactory receptor
comprising the following steps: expressing a nucleic acid derived
from a nucleic acid library of the invention under conditions
permissive for translation of the nucleic acid to a receptor
polypeptide; contacting the translated polypeptide with the test
compound; and determining whether the test compound specifically
binds to the polypeptide.
[0024] Also provided are methods of determining whether a test
compound specifically binds to a mammalian olfactory transmembrane
receptor comprising the following steps: contacting a cell stably
or transiently transfected with a nucleic acid derived from a
nucleic acid library of the invention; culturing the cell under
conditions permissive for translation of the nucleic acid to a
receptor polypeptide with the test compound; and determining
whether the test compound specifically binds to the receptor
polypeptide. In this method, the receptor polypeptide can be
expressed as a transmembrane receptor with a ligand-binding site on
the cell's plasma membrane outer surface. The specific binding of
the test compound to the polypeptide can be determined by measuring
a change in the physiologic activity of the cell, wherein a change
in the cell's activity measured in the presence of the test
compound compared to the cell's activity in the absence of the test
compound provides a determination that the test compound
specifically binds to the polypeptide. The measured cell activity
can be a change in the calcium ion (Ca.sup.2+) or cAMP
concentration in the cell, which can be measured by loading the
cell with a calcium ion-sensitive fluorescent dye before contacting
the cell with the test compound. In this method any cell can be
used, e.g., a human cell or a Xenopus oocyte.
[0025] Also provided are methods of determining whether a test
compound specifically binds to a mammalian olfactory transmembrane
receptor polypeptide in vivo comprising the following steps:
contacting a non-human animal stably or transiently infected with a
nucleic acid derived from the library of the invention or an
expression vector of the invention with the test compound; and
determining whether the animal reacts to the test compound by
specifically binding to the receptor polypeptide, wherein the
specific binding of the test compound to the polypeptide is
determined by measuring a change in a physiologic activity of the
animal, wherein a change in a receptor-encoding vector-infected
animal's activity measured in the presence of the test compound
compared to a bare vector-infected animal's activity in the
presence of the test compound provides a determination that the
test compound specifically binds to the mammalian olfactory
transmembrane receptor polypeptide. In this method, the measured
physiologic activity can be measured by an electroolfactogram. The
vector can be a recombinant virus, e.g., an adenovirus expression
vector.
[0026] The invention also provides a method of determining whether
a test compound is neurotoxic to an olfactory neuron expressing an
olfactory transmembrane receptor polypeptide comprising the
following steps: contacting an olfactory neuron cell stably or
transiently infected with a nucleic acid derived from a library as
set forth in claim 8 or claim 10 or an expression vector as set
forth in claim 23 with the test compound; and measuring the
physiologic activity of the cell, wherein a change in the cell's
activity measured in the presence of the test compound compared to
the cell's activity in the absence of the test compound provides a
determination that the test compound is toxic. In this method
toxicity can be indicated by abnormal calcium ion, cAMP or plasma
membrane homeostasis.
[0027] Also provided are peptide domains for the efficient
translocation of a newly translated protein to a plasma membrane
comprising an amino acid sequence as set forth in SEQ ID NO:3 or an
amino acid sequence having conservative amino acid residue
substitutions based on SEQ ID NO:3. Translocation domains within
the scope of the invention include amino acid sequences
functionally equivalent to the exemplary translocation domain of
the invention SEQ ID NO:3. The peptide translocation domain can be
at least about 20 amino acids in length, at least about 30 amino
acids in length or at least about 40 amino acids in length. The
peptide translocation domain can have a sequence as set forth in
SEQ ID NO:3, or, be encoded by a nucleic acid comprising a sequence
as set forth in SEQ ID NO:6. The newly translated protein can be a
transmembrane protein, e.g., a 7-transmembrane protein receptor,
e.g., an olfactory receptor.
[0028] The invention also provides a hybrid (chimeric) polypeptide
comprising an amino terminal amino acid sequence comprising a
peptide translocation domain of the invention and a second
polypeptide sequence, wherein the second polypeptide sequence is
not a rhodopsin polypeptide sequence. The second polypeptide
sequence can be a transmembrane protein, e.g., a 7-transmembrane
protein receptor, e.g., an olfactory receptor. Also provides are
isolated or recombinant nucleic acid sequences encoding these
hybrid polypeptides. For example, an exemplary chimeric polypeptide
of the invention and a polynucleotide that encodes this hybrid,
described in the Example below and schematically represented in
FIG. 1A as the insert from BamH1 to XbaI, have the amino acid (SEQ
ID NO:55) and nucleic acid (SEQ ID NO:54) sequence, respectively
(restriction enzyme sites are also indicated):
1 BamHI GGATCCGGGTTCGCGCCGCCGGCGGGCAGCCGCAAGGGCCGCAGCCATG-
AACGGGACCGAGGGC (SEQ ID NO:54) M N G T E G (SEQ ID NO:55) EcoRI
CCAAACTTCTACCTGCCTTTCTCCAACAAGACGG- GCGTGGTGGAATTCCCCGGTCAGAACTACA
P N F Y V P F S N K T G V V E P P G Q N Y S
GCACCATATCAGAATTTATCCTCTTTG- GTTTCTCAGCCTTCCCACACCAGATGCTCCCTGCTCT
S T I S E F I L F G F S A F P H Q M L P A L
GTTCCTGCTCTACTTGCTGATGTATTTGTTCACTCTTCTGGGGAACCTGGTCATCATGGCTGCT F
L L Y L L M Y L F T L L G N L V I M A A PstI BspEI
ATCTGGACAGAACATAGACTGCAGAC- ACCCATCCGGAAAGGAGCTGAAGAATGCTATAATTAAA
I W T E H R L Q S G K E L K N A I I K XbaI
AGCTTCCACAGGAATGTCTGTCAACAAAGTATCTAAG- TGTCAGTTCTGTCTAGA S F H R N
V C Q Q S I STOP
[0029] A further understanding of the nature and advantages of the
present invention is realized by reference to the remaining
portions of the specification, the figures and claims.
[0030] All publications, GenBank deposited sequences, ATCC
deposits, patents and patent applications cited herein are hereby
expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIGURE 1A shows a schematic of a mammalian expression
construct of the invention comprising a translocation domain of the
invention and an odorant/ligand-binding domain generated by
degenerate PCR primers, as described in detail in Example 1,
below.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides novel compositions and
methods to efficiently translocate newly translated polypeptides to
the plasma membrane surface. This aspect of the invention is based
on the surprising discovery of peptide domains (e.g., SEQ ID NO:3)
that, when incorporated into the amino terminus of a polypeptide
coding sequence, can with great efficiency "chaperone" or
"translocate" the hybrid ("fusion") protein to the cell plasma
membrane. This "translocation domain" was initially derived from
the amino terminus of the human rhodopsin receptor polypeptide, a
7-transmembrane receptor. Thus, the translocation domain of the
invention is particularly efficient in translocating
7-transmembrane fusion proteins to the plasma membrane. For
example, the mouse olfactory receptor M4 (see, e.g., Qasba (1998)
J. Neurosci. 18:227-236) expressed in a mammalian cell line is
inefficiently translocated to the cell. In contrast, when a
translocating domain of the invention (SEQ ID NO:3) was spliced to
the amino terminus of the M4 olfactory receptor polypeptide, cell
surface expression of the newly translated protein increased from
undetectable levels to 10% or more of the total expressed protein
(as determined by confocal microscopic imaging with antibodies that
recognize the carboxyl terminus of the M4 receptor). Furthermore,
subsequent functional expression studies demonstrated that no
responses could be observed upon addition of extracellular ligand
unless the translocation domain of the invention (SEQ ID NO:3) was
included to effect surface localization.
[0033] The invention also provides novel means to generate
libraries of odorant/ligand-binding regions of olfactory receptor
proteins. Great numbers of these ligand-regions can be generated by
amplification (e.g., by polymerase chain reaction (PCR)) of nucleic
acid from olfactory neurons and epithelium using degenerate primer
pairs. The primer pairs selectively amplify the
odorant/ligand-binding regions of olfactory receptor proteins. The
odorant/ligand-binding regions can comprise transmembrane domain II
through VII, III through VII, III through VI, II through VI, or
combinations or variation thereof, of the 7-transmembrane olfactory
receptor (see below for detailed discussion). Thus, amplification
of, e.g., genomic DNA, or message or cDNA from olfactory neurons,
using the degenerate primers of the invention can generate great
numbers, or "libraries," of odorant/ligand-binding region encoding
nucleic acid.
[0034] The odorant/ligand-binding region-amplifying degenerate
primers of the invention are at least about 17 base pair residues
in length. Amplification conditions can vary; however, lower
temperature conditions (e.g., below about 55.degree. C., usually
not lower than about 45.degree. C.) will generate libraries of
greater complexity and higher temperatures (e.g., over about
55.degree. C.) will generate libraries of less complexity.
[0035] For screening and identification of odorant/ligands that
specifically bind to the domains encoded by the nucleic acid
"libraries" of the invention, the amplified sequences can be
recombinantly spliced into a "framework" polypeptide that is
expressed on the cell surface. If functional studies (including,
e.g., cell signaling responses, e.g., calcium transients) are
desired, 7-membrane polypeptide coding sequences are used as
"donor" regions. In this scheme, the "donor" 7-membrane polypeptide
provides the coding sequence needed to complement the insert, i.e.,
a nucleic acid from an odorant/ligand-binding region library of the
invention. For example, if the amplified odorant/ligand-binding
region is equivalent to transmembrane domain II through VII, the
"donor" provides transmembrane domain I; if the binding region is
transmembrane domain III through VI, the "donor" provides the amino
terminal transmembrane domain I and the carboxy terminal domain
VII; and the like. Any 7-membrane polypeptide coding sequence can
be used as "donor," including olfactory receptor polypeptide;
however, some receptors which depend on long amino-terminal
extensions for ligand recognition and binding (e.g., metabatropic
glutamate, extracellular calcium sensors, GnRH and FSH peptide
hormone receptors) may not produce functional receptors using this
method.
[0036] These constructs can be cloned into expression systems,
e.g., plasmids, vectors, viruses and the like. Any system can be
used, from a minimal transcription unit (e.g., an expression
cassette) to a recombinant virus capable of infecting an animal
(e.g., an engineered adenovirus). These vectors can be used for
functional expression assays in vitro or in vivo to screen large
numbers of putative odorant/ligand molecules or to test for
potential odorant toxicity.
[0037] The efficiency of the odorant-receptor screening systems of
the invention are greatly increased by generating odorant receptor
fusion proteins that can efficiently translocate to the plasma
membrane. These hybrid receptors comprise the polypeptide
translocating domains and the libraries of odorant/ligand-binding
regions of the invention. With this scheme the invention provides
an efficient means to generate and efficiently express thousands of
olfactory receptor binding domains in functional cell and animal
assays for the rapid screening of potential beneficial and toxic
odorant/ligands.
[0038] Both in vitro and in vivo systems can be constructed and
used in the methods of the invention. In vitro screening can
include, e.g., liposome or lipid or planar membrane models. In vivo
screening systems can include, e.g., use of human cells, e.g.,
olfactory neuron cell lines, or infection of animals (e.g., with
virus with sequence encoding chimeric receptor) and transgenic
animals that express the constructs of the invention. Adenovirus
gene transfer vectors are particularly efficient for the transfer
of nucleic acids encoding the hybrid olfactory receptor proteins of
the invention to nasal/respiratory epithelium.
[0039] When human olfactory receptor nucleic acid is amplified, the
in vitro models, cultured cells, and infected and transgenic
animals can be used for screening large numbers of molecules for
their potential as human odorants. The effect of an odorant on
neuronal cell physiology can be also assessed. For example, the
screening systems of the invention can be used to test whether an
odorant/ligand may be potentially toxic (or beneficial) in humans.
Any cell physiologic activity can be measured, e.g., cell death,
cell growth, intracellular calcium ion changes, second messengers
(e.g., G protein activation, cAMP increases), and the like. The
effect of odorant/ligands on apoptotic mechanisms, neuronal growth
characteristics (such as neuron population doubling time and length
of processes), ion exchange and other measurable parameters can
also be used to analyze their potential potency and toxicity.
[0040] Definitions:
[0041] The term "amplifying" and "amplification" as used herein
incorporates its common usage and refers to the use of any suitable
amplification methodology for generating or detecting recombinant
or naturally expressed nucleic acid, as described in detail, below.
For example, the invention provides methods and reagents (e.g.,
specific degenerate oligonucleotide primer pairs) for amplifying
(e.g., by polymerase chain reaction, PCR) naturally expressed
(e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids
of the invention (e.g., odorant/ligand binding sequences of the
invention) in vivo or in vitro.
[0042] The term "7-transmembrane receptor" means a polypeptide
belonging to a superfamily of transmembrane proteins that have
seven domains that span the plasma membrane seven times (thus, the
seven domains are called "transmembrane" or "TM" domains TM I to TM
VII). Olfactory receptors belong to this family. 7-transmembrane
receptor polypeptides have similar and characteristic primary,
secondary and tertiary structures, as discussed in detail
below.
[0043] The term "expression vector" refers to any recombinant
expression system for the purpose of expressing a nucleic acid
sequence of the invention in vitro or in vivo, constitutively or
inducibly, in any cell, including prokaryotic, yeast, fungal,
plant, insect or mammalian cell. The term includes linear or
circular expression systems. The term includes expression systems
that remain episomal or integrate into the host cell genome. The
expression systems can have the ability to self-replicate or not,
i.e., drive only transient expression in a cell. The term includes
recombinant expression "cassettes" which contain only the minimum
elements needed for transcription of the recombinant nucleic
acid.
[0044] As used herein, "isolated," when referring to a molecule or
composition, such as, e.g., an isolated infected cell comprising a
nucleic acid sequence derived from a library of the invention,
means that the molecule or composition (including, e.g., a cell) is
separated from at least one other compound, such as a protein, DNA,
RNA, or other contaminants with which it is associated in vivo or
in its naturally occurring state. Thus, a nucleic acid sequence is
considered isolated when it has been isolated from any other
component with which it is naturally associated. An isolated
composition can, however, also be substantially pure. An isolated
composition can be in a homogeneous state. It can be in a dry or an
aqueous solution. Purity and homogeneity can be determined, e.g.,
using any analytical chemistry technique, as described herein.
[0045] The term "library" means a preparation that is a mixture
different nucleic acid or polypeptide molecules, such as the
library of recombinantly generated olfactory receptor ligand
binding domains generated by amplification of nucleic acid with
degenerate primer pairs, e.g., SEQ ID NO:1 and SEQ ID NO:2, or an
isolated collection of vectors that incorporate the amplified
odorant/ligand binding domains of the invention, or a mixture of
cells each randomly transfected with at least one vector of the
invention.
[0046] The term "nucleic acid" or "nucleic acid sequence" refers to
a deoxy-ribonucleotide or ribonucleotide oligonucleotide in either
single- or double-stranded form. The term encompasses nucleic
acids, i.e., oligonucleotides, containing known analogues of
natural nucleotides. The term also encompasses nucleic-acid-like
structures with synthetic backbones, see e.g., Oligonucleotides and
Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ.
Press (1991); Antisense Strategies, Annals of the N.Y. Academy of
Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992); Milligan
(1993) J. Med. Chem. 36:1923-1937; Antisense Research and
Applications (1993, CRC Press), WO 97/03211; WO 96/39154; Mata
(1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997)
Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid
Drug Dev 6:153-156.
[0047] The term "P" in the sequence is
5'-Dimethoxytrityl-N-benzoyl-2'-deo-
xy-Cytidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,
or equivalent thereof. "P" can be purchased by, e.g., Glen
Research, Sterling, Va., described as "dC-CE Phosphoramidite"
catalog number 10-1010-xx.
[0048] The term "olfactory receptor ligand-binding region" or
"olfactory receptor ligand-binding domain" means a sequence derived
from an olfactory receptor that substantially incorporates
transmembrane domains II to VII (TM II to VII). The domain may be
capable of binding a ligand.
[0049] The term "plasma membrane translocation domain" or simply
"translocation domain" means a polypeptide domain that is
functionally equivalent to the exemplary translocation domain of
the invention (SEQ ID NO:3). Exemplary amino terminal plasma
membrane translocation domain SEQ ID NO:3 was initially derived
from the rhodopsin receptor amino terminus. A protein (e.g., an
olfactory receptor polypeptide) comprising SEQ ID NO:3 as an amino
terminal translocating domain will be transported to the plasma
membrane more efficiently than without the domain (e.g., as
discussed above, M4 receptor expression increased from undetectable
levels to at least 10% of the total expressed protein). "Functional
equivalency" means the domain's ability and efficiency in
translocating newly translated proteins to the plasma membrane as
efficiently as exemplary SEQ ID NO:3 under similar conditions;
relatively efficiencies can be measured (in quantitative terms) and
compared, as described herein. Domains falling within the scope of
the invention can be determined by routine screening for their
efficiency in translocating newly synthesized polypeptides to the
plasma membrane in a cell (mammalian, Xenopus, and the like) with
the same efficiency as the twenty amino acid long translocation
domain SEQ ID NO:3, as described in detail below.
[0050] The "translocation domain," odorant/ligand binding domains,
and chimeric receptors compositions of the invention also include
"analogs," or "conservative variants" and "mimetics"
("peptidomimetics") with structures and activity that substantially
correspond to the exemplary sequences, such as the SEQ ID NO:3
translocation domain. Thus, the terms "conservative variant" or
"analog" or "mimetic" refer to a polypeptide which has a modified
amino acid sequence, such that the change(s) do not substantially
alter the polypeptide's (the conservative variant's) structure
and/or activity, as defined herein. These include conservatively
modified variations of an amino acid sequence, i.e., amino acid
substitutions, additions or deletions of those residues that are
not critical for protein activity, or substitution of amino acids
with residues having similar properties (e.g., acidic, basic,
positively or negatively charged, polar or non-polar, etc.) such
that the substitutions of even critical amino acids does not
substantially alter structure and/or activity. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. For example, one exemplary guideline to
select conservative substitutions includes (original residue
followed by exemplary substitution): ala/gly or ser; arg/lys;
asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro;
his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or
glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr;
thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative
exemplary guideline uses the following six groups, each containing
amino acids that are conservative substitutions for one another: 1)
Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
(see also, e.g., Creighton (1984) Proteins, W.H. Freeman and
Company; Schulz and Schimer (1979) Principles of Protein Structure,
Springer-Verlag). One of skill in the art will appreciate that the
above-identified substitutions are not the only possible
conservative substitutions. For example, for some purposes, one may
regard all charged amino acids as conservative substitutions for
each other whether they are positive or negative. In addition,
individual substitutions, deletions or additions that alter, add or
delete a single amino acid or a small percentage of amino acids in
an encoded sequence can also be considered "conservatively modified
variations."
[0051] The terms "mimetic" and "peptidomimetic" refer to a
synthetic chemical compound that has substantially the same
structural and/or functional characteristics of the polypeptides,
e.g., translocation domains or odorant-ligand binding domains or
chimeric receptors of the invention. The mimetic can be either
entirely composed of synthetic, non-natural analogues of amino
acids, or, is a chimeric molecule of partly natural peptide amino
acids and partly non-natural analogs of amino acids. The mimetic
can also incorporate any amount of natural amino acid conservative
substitutions as long as such substitutions also do not
substantially alter the mimetic's structure and/or activity. As
with polypeptides of the invention which are conservative variants,
routine experimentation will determine whether a mimetic is within
the scope of the invention, i.e., that its structure and/or
function is not substantially altered. Polypeptide mimetic
compositions can contain any combination of non-natural structural
components, which are typically from three structural groups: a)
residue linkage groups other than the natural amide bond ("peptide
bond") linkages; b) non-natural residues in place of naturally
occurring amino acid residues; or c) residues which induce
secondary structural mimicry, i.e., to induce or stabilize a
secondary structure, e.g., a beta turn, gamma turn, beta sheet,
alpha helix conformation, and the like. A polypeptide can be
characterized as a mimetic when all or some of its residues are
joined by chemical means other than natural peptide bonds.
Individual peptidomimetic residues can be joined by peptide bonds,
other chemical bonds or coupling means, such as, e.g.,
glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S),
tetrazole (CN.sub.4--), thiazole, retroamide, thioamide, or ester
(see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone
Modifications," Marcell Dekker, NY). A polypeptide can also be
characterized as a mimetic by containing all or some non-natural
residues in place of naturally occurring amino acid residues;
non-natural residues are well described in the scientific and
patent literature.
[0052] As used herein, "recombinant" refers to a polynucleotide
synthesized or otherwise manipulated in vitro (e.g., "recombinant
polynucleotide"), to methods of using recombinant polynucleotides
to produce gene products in cells or other biological systems, or
to a polypeptide ("recombinant protein") encoded by a recombinant
polynucleotide. "Recombinant means" also encompass the ligation of
nucleic acids having various coding regions or domains or promoter
sequences from different sources into an expression cassette or
vector for expression of, e.g., inducible or constitutive
expression of a fusion protein comprising a translocation domain of
the invention and a nucleic acid sequence amplified using a primer
of the invention.
[0053] The term "transmembrane domain" means a polypeptide domain
that can completely span the plasma membrane. The general secondary
and tertiary structure of transmembrane domains, particular the
seven transmembrane domains of 7-transmembrane receptors such as
olfactory receptors, are well known in the art. Thus, primary
structure sequence can be designed or predicted based on known
transmembrane domain sequences, as described in detail, below. One
such exemplary domain is the 7-transmembrane receptor transmembrane
region I domain comprising a sequence as set forth in SEQ ID
NO:4.
[0054] Generation and Genetic Engineering of Nucleic Acids
[0055] This invention provides novel PCR primers for the
amplification of nucleic acids encoding olfactory receptor ligand
binding regions and libraries of these nucleic acids. The invention
also provides novel libraries of expression vectors that are used
to infect or transfect cells for the functional expression of these
libraries. As the genes and vectors of the invention can be made
and expressed in vitro or in vivo, the invention provides for a
variety of means of making and expressing these genes and vectors.
One of skill will recognize that desired phenotypes for altering
and controlling nucleic acid expression can be obtained by
modulating the expression or activity of the genes and nucleic
acids (e.g., promoters, enhancers and the like) within the vectors
of the invention. Any of the known methods described for increasing
or decreasing expression or activity can be used for this
invention. The invention can be practiced in conjunction with any
method or protocol known in the art, which are well described in
the scientific and patent literature.
[0056] General Techniques
[0057] The nucleic acid sequences of the invention and other
nucleic acids used to practice this invention, whether RNA, cDNA,
genomic DNA, vectors, viruses or hybrids thereof, may be isolated
from a variety of sources, genetically engineered, amplified,
and/or expressed recombinantly. Any recombinant expression system
can be used, including, in addition to mammalian cells, e.g.,
bacterial, yeast, insect or plant systems.
[0058] Alternatively, these nucleic acids can be synthesized in
vitro by well-known chemical synthesis techniques, as described in,
e.g., Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol.
47:411-418; Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997)
Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.
Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang
(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;
Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
Double stranded DNA fragments may then be obtained either by
synthesizing the complementary strand and annealing the strands
together under appropriate conditions, or by adding the
complementary strand using DNA polymerase with an appropriate
primer sequence.
[0059] Techniques for the manipulation of nucleic acids, such as,
e.g., generating mutations in sequences, subcloning, labeling
probes, sequencing, hybridization and the like are well described
in the scientific and patent literature, see, e.g., Sambrook, ed.,
MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold
Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);
LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:
HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic
Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0060] Nucleic acids, vectors, capsids, polypeptides, and the like
can be analyzed and quantified by any of a number of general means
well known to those of skill in the art. These include, e.g.,
analytical biochemical methods such as NMR, spectrophotometry,
radiography, electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), and hyperdiffusion chromatography, various immunological
methods, e.g. fluid or gel precipitin reactions, immunodiffusion,
immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked
immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern
analysis, Northern analysis, dot-blot analysis, gel electrophoresis
(e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or
target or signal amplification methods, radiolabeling,
scintillation counting, and affinity chromatography.
[0061] Amplification of Nucleic Acids
[0062] The invention provides oligonucleotide primers that can
amplify nucleic acid encoding an olfactory receptor ligand-binding
region. The nucleic acids of the invention can also be cloned or
measured quantitatively using amplification techniques. Using the
exemplary degenerate primer pair sequences of the invention (see
below), the skilled artisan can select and design suitable
oligonucleotide amplification primers. Amplification methods are
also well known in the art, and include, e.g., polymerase chain
reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS,
ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995),
ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR)
(see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science
241:1077; Barringer (1990) Gene 89:117); transcription
amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA
86:1173); and, self-sustained sequence replication (see, e.g.,
Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta
replicase amplification (see, e.g., Smith (1997) J. Clin.
Microbiol. 35:1477-1491), automated Q-beta replicase amplification
assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and
other RNA polymerase mediated techniques (e.g., NASBA, Cangene,
Mississauga, Ontario); see also Berger (1987) Methods Enzymol.
152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and
4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
[0063] Once amplified, the libraries can be cloned, if desired,
into any of a variety of vectors using routine molecular biological
methods; methods for cloning in vitro amplified nucleic acids are
described, e.g., U.S. Pat. No. 5,426,039. To facilitate cloning of
amplified sequences, restriction enzyme sites can be "built into"
the PCR primer pair. For example, Pst I and Bsp E1 sites were
designed into the exemplary primer pairs of the invention. These
particular restriction sites were chosen because they have a
sequence that, when ligated, are "in-frame" with respect to the
7-membrane receptor "donor" coding sequence into which they are
spliced (the odorant/ligand binding region-coding sequence is
internal to the 7-membrane polypeptide, thus, if it is desired that
the construct be translated downstream of a restriction enzyme
splice site, out of frame results should be avoided; this may not
be necessary if the inserted odorant/ligand binding domain
comprises substantially most of the transmembrane VII region). The
primers can be designed to retain the original sequence of the
"donor" 7-membrane receptor (the Pst I and Bsp E1 sequence in the
primers of the invention generate an insert that, when ligated into
the Pst I/Bsp E1 cut vector, encode residues found in the "donor"
mouse olfactory receptor M4 sequence). Alternatively, the primers
can encode amino acid residues that are conservative substitutions
(e.g., hydrophobic for hydrophobic residue, see above discussion)
or functionally benign substitutions (e.g., do not prevent plasma
membrane insertion, cause cleavage by peptidase, cause abnormal
folding of receptor, and the like).
[0064] Degenerate Primer Design
[0065] The primer pairs of the invention are designed to
selectively amplify odorant/ligand-binding regions of olfactory
receptor proteins. These domain regions may vary for different
odorants; thus, what may be a minimal binding region for one
odorant may be too limiting for a second potential ligand. Thus,
the invention includes amplification of domain regions of different
sizes comprising different domain structures; for example,
transmembrane (TM) domains II through VII, III through VII, III
through VI or II through VI, or variations thereof (e.g., only a
subsequence of a particular domain, mixing the order of the
domains, and the like), of a 7-transmembrane olfactory receptor. As
domain structures and sequence of many 7-membrane proteins,
particularly olfactory receptors, are known, the skilled artisan
can readily select domain-flanking and internal domain sequences as
model sequences to design degenerate amplification primer pairs.
For example, a nucleic acid sequence encoding domain regions II
through VII can be generated by PCR amplification using a primer
pair SEQ ID NO:1 and SEQ ID NO:2 (see FIG. 1). To amplify a nucleic
acid comprising transmembrane domain I (TM I) sequence, a
degenerate primer can be designed from a nucleic acid that encodes
the amino acid sequence LFLLYL 3' (SEQ ID NO:49). Such a degenerate
primer can be used to generate a binding domain incorporating TM I
through TM III, TM I through TM IV, TM I through TM V, TM I through
TM VI or TM I through TM VII).
[0066] To amplify a nucleic acid comprising a transmembrane domain
III (TM III) sequence, a degenerate primer (of at least about 17
residues) can be designed from a nucleic acid that encodes the
amino acid sequence M(A/G)(Y/F)DRYVAI 3' (SEQ ID NO:50 (encoded by
a nucleic acid sequence such as 5'-ATGG(G/C)CT(A/T)TGACCG
(C/A/T)T(AT)(C/T)GT-3' (SEQ ID NO:51)). Such a degenerate primer
can be used to generate a binding domain incorporating TM III
through TM IV, TM III through TM V, TM III through TM VI or TM III
through TM VII.
[0067] To amplify transmembrane domain VI (TM VI) sequence, a
degenerate primer (of at least about 17 residues) can be designed
from nucleic acid encoding an amino acid sequence
TC(glycine/Alanine)SHL (SEQ ID NO:52), encoded by a sequence such
as 5'-AG(G/A)TGN(G/C)(T/A)N(G/C)C(G/A)CANGT-3'- ) 3' (SEQ ID
NO:53), Such a degenerate primer can be used to generate a binding
domain incorporating TM I through TM VI, TM II through TM VI, TM
III through TM VI or TM IV through TM VI).
[0068] Paradigms to design degenerate primer pairs are well known
in the art. For example, a COnsensus-DEgenerate Hybrid
Oligonucleotide Primer (CODEHOP) strategy computer program is
accessible as http://blocks.fhcrc.org/codehop.html, and is directly
linked from the BlockMaker multiple sequence alignment site for
hybrid primer prediction beginning with a set of related protein
sequences, as known olfactory receptor ligand binding regions (see,
e.g., Rose (1998) Nucleic Acids Res. 26:1628-1635; Singh (1998)
Biotechniques 24:318-319).
[0069] Means to synthesize oligonucleotide primer pairs are well
known in the art. "Natural" base pairs or synthetic base pairs can
be used. For example, use of artificial nucleobases offers a
versatile approach to manipulate primer sequence and generate a
more complex mixture of amplification products. Various families of
artificial nucleobases are capable of assuming multiple hydrogen
bonding orientations through internal bond rotations to provide a
means for degenerate molecular recognition. Incorporation of these
analogs into a single position of a PCR primer allows for
generation of a complex library of amplification products. See,
e.g., Hoops (1997) Nucleic Acids Res. 25:4866-4871. Nonpolar
molecules can also be used to mimic the shape of natural DNA bases.
A non-hydrogen-bonding shape mimic for adenine can replicate
efficiently and selectively against a nonpolar shape mimic for
thymine (see, e.g., Morales (1998) Nat. Struct. Biol. 5:950-954).
For example, two degenerate bases can be the pyrimidine base 6H,
8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one or the purine base
N6-methoxy-2,6-diaminopurine (see, e.g., Hill (1998) Proc. Natl.
Acad. Sci. USA 95:4258-4263). Exemplary degenerate primers of the
invention incorporate the nucleobase analog
5'-Dimethoxytrityl-N-benzoyl-2'-deoxy-C-
ytidine,3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite ((the
term "P" in the sequences, see above). This pyrimidine analog
hydrogen bonds with purines, including A and G residues.
[0070] Exemplary primer pairs for amplification of olfactory
receptor transmembrane domains II through VII include:
2 (a)
5'-GGGGTCCGGAG(A/G)(C/G)(A/G)TA(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P-
)GG-3' and (SEQ ID NO:1) 5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/-
T)T(A/C/G/T)TT(C/T)(C/T)T-3'. (SEQ ID NO:2) (b)
5'-GGGGTCCGGAG(A/G)(C/G)T(A/G)A(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3'
and (SEQ ID NO:7)
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/G/T-
)TT(C/T)(C/T)T-3' (SEQ ID NO:8) (c)
5'-GGGGTCCGGAG(A/G)(C/G)T(A/G)A(A/G/T)AT(A/G/C/T)A(A/G/C/T)(A/G/C/T)GG-3'
and (SEQ ID NO:9) 5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C-
/G/T)TT(C/T)(C/T)T-3'. (SEQ ID NO:10)
[0071] Generating Nucleic Acids from Olfactory Receptor-Expressing
Cells
[0072] The invention provides method for generating nucleic acids
that encode ligand-binding regions of olfactory receptors by
amplification (e.g., PCR) of appropriate nucleic acid sequences
using degenerate primer pairs. The amplified nucleic acid can be
genomic DNA from any cell or tissue or mRNA or cDNA derived from
olfactory receptor-expressing cells, e.g., olfactory neurons or
olfactory epithelium.
[0073] Isolation of from olfactory receptor-expressing cells is
well known in the art (cells expressing naturally or inducibly
expressing olfactory receptors can be used to express the hybrid
olfactory receptors of the invention to screen for potential
odorants and odorant effect on cell physiology, as described
below). For example, cells can be identified by olfactory marker
protein (OMP), an abundant cytoplasmic protein expressed almost
exclusively in mature olfactory sensory neurons (see, e.g.,
Buiakova (1996) Proc. Natl. Acad. Sci. USA 93:9858-9863). Shirley
(1983) Eur. J. Biochem. 32:485-494, describes a rat olfactory
preparation suitable for biochemical studies in vitro on olfactory
mechanisms. Cultures of adult rat olfactory receptor neurons are
described by Vargas (1999) Chem. Senses 24:211-216. Because these
cultured neurons exhibit typical voltage-gated currents and are
responsive to application of odorants, they can also be used to
express the hybrid olfactory receptors of the invention for odorant
screening (endogenous olfactory receptor can be initially blocked,
if desired, by, e.g., antisense, knockout, and the like). U.S. Pat.
No. 5,869,266 describes culturing human olfactory neurons for
neurotoxicity tests and screening. Murrell (1999) J. Neurosci.
19:8260-8270 describes differentiated olfactory receptor-expressing
cells in culture that respond to odorants, as measured by an influx
of calcium.
[0074] Genetic Engineering of Hybrid Receptor-Encoding
Sequences
[0075] The invention provides hybrid protein-coding sequences
comprising polypeptide-encoding nucleic acids fused to the
translocation sequences of the invention. Also provided are hybrid
olfactory receptors comprising the translocation motifs and
odorant/ligand-binding domains of olfactory receptors. These
nucleic acid sequences can be operably linked to transcriptional or
translational control elements, e.g., transcription and translation
initiation sequences, promoters and enhancers, transcription and
translation terminators, polyadenylation sequences, and other
sequences useful for transcribing DNA into RNA. In construction of
recombinant expression cassettes, vectors, transgenics, of the
invention, a promoter fragment can be employed to direct expression
of the desired nucleic acid in all tissues. Olfactory cell-specific
transcriptional elements can also be used to express the fusion
polypeptide receptor of the invention, including, e.g., a 6.7 kb
region upstream of the M4 olfactory receptor coding region. This
region was sufficient to direct expression in olfactory epithelium
with wild type zonal restriction and distributed neuronal
expression for endogenous olfactory receptors (Qasba (1998) J.
Neurosci. 18:227-236). Receptor genes are normally expressed in a
small subset of neurons throughout a zonally restricted region of
the sensory epithelium. The transcriptional or translational
control elements can be isolated from natural sources, obtained
from such sources as ATCC or GenBank libraries, or prepared by
synthetic or recombinant methods.
[0076] The invention provides fusion proteins comprising the
translocation motif of the invention. However, these fusion
proteins can also comprise additional element for, e.g., protein
detection, purification, or other applications. Detection and
purification facilitating domains include, e.g., metal chelating
peptides such as polyhistidine tracts or histidine-tryptophan
modules or other domains that allow purification on immobilized
metals; maltose binding protein; protein A domains that allow
purification on immobilized immunoglobulin; or the domain utilized
in the FLAGS extension/affinity purification system (Immunex Corp,
Seattle Wash.).
[0077] The inclusion of a cleavable linker sequences such as Factor
Xa (see, e.g., Ottavi (1998) Biochimie 80:289-293), subtilisin
protease recognition motif (see, e.g., Polyak (1997) Protein Eng.
10:615-619); enterokinase (Invitrogen, San Diego Calif.), and the
like, between a translocation domain of the invention (for
efficient plasma membrane expression) and the rest of the newly
translated polypeptide may be useful to facilitate purification.
For example, one construct can include a polypeptide-encoding
nucleic acid sequence linked to six histidine residues followed by
a thioredoxin, an enterokinase cleavage site (see, e.g., Williams
(1995) Biochemistry 34:1787-1797), and an amino terminal
translocation domain. The histidine residues facilitate detection
and purification while the enterokinase cleavage site provides a
means for purifying the desired protein(s) from the remainder of
the fusion protein. Technology pertaining to vectors encoding
fusion proteins and application of fusion proteins are well
described in the scientific and patent literature, see e.g., Kroll
(1993) DNA Cell. Biol., 12:441-53.
[0078] Cloning and Construction of Expression Vectors
[0079] The invention provides libraries of expression vectors
comprising the olfactory binding domain-encoding sequences of the
invention. These nucleic acids may be introduced into a genome or
into the cytoplasm or a nucleus of a cell and expressed by a
variety of conventional techniques, well described in the
scientific and patent literature. See, e.g., Roberts (1987) Nature
328:731; Berger (1987) supra; Schneider (1995) Protein Expr. Purif.
6435:10; Sambrook, Tijssen or Ausubel. Product information from
manufacturers of biological reagents and experimental equipment
also provide information regarding known biological methods. The
vectors can be isolated from natural sources, obtained from such
sources as ATCC or GenBank libraries, or prepared by synthetic or
recombinant methods.
[0080] The nucleic acids of the invention can be expressed in
expression cassettes, vectors or viruses which are stably or
transiently expressed in cells (e.g., episomal expression systems).
Selection markers can be incorporated into expression cassettes and
vectors to confer a selectable phenotype on transformed cells and
sequences. For example, selection markers can code for episomal
maintenance and replication such that integration into the host
genome is not required. For example, the marker may encode
antibiotic resistance (e.g., chloramphenicol, kanamycin, G418,
bleomycin, hygromycin) or herbicide resistance (e.g.,
chlorosulfuron or Basta) to permit selection of those cells
transformed with the desired DNA sequences (see, e.g.,
Blondelet-Rouault (1997) Gene 190:315-317; Aubrecht (1997) J.
Pharmacol. Exp. Ther. 281:992-997). Because selectable marker genes
conferring resistance to substrates like neomycin or hygromycin can
only be utilized in tissue culture, chemoresistance genes are also
used as selectable markers in vitro and in vivo.
[0081] Structure of Seven-Transmembrane Receptors
[0082] The invention provides a chimeric nucleic acid sequence
encoding an odorant/ligand binding domain within any
7-transmembrane polypeptide. 7-transmembrane receptors belong to a
superfamily of transmembrane (TM) proteins having seven domains
that transverse a plasma membrane seven times. Each of the seven
domains spans the plasma membrane (TM I to TM VII). Because
7-transmembrane receptor polypeptides have similar primary
sequences and secondary and tertiary structures, structural domains
(e.g., TM domains) can be readily identified by sequence analysis.
For example, homology modeling, Fourier analysis and helical
periodicity detection can identify and characterize the seven
domains within a 7-transmembrane receptor sequence. Fast Fourier
Transform (FFT) algorithms can be used to assess the dominant
periods that characterize profiles of the hydrophobicity and
variability of analyzed sequences. To predict TM domains and their
boundaries and topology, a "neural network algorithm" by "PHD
server" can be used, as done by Pilpel (1999) Protein Science
8:969-977; Rost (1995) Protein Sci. 4:521-533. Periodicity
detection enhancement and alpha helical periodicity index can be
done as by, e.g., Donnelly (1993) Protein Sci. 2:55-70. Other
alignment and modeling algorithms are well known in the art, see,
e.g., Peitsch (1996) Receptors Channels 4:161-164; Cronet (1993)
Protein Eng. 6:59-64 (homology and "discover modeling");
http://bioinfo.weizmann.ac.il/.
[0083] Olfactory Gene and Receptors
[0084] The library sequences of the invention include receptor
sequences that correspond to TM ligand-binding domains, including,
e.g., TM II to VII, TM II to VI, TM III to VII, and TM III to VII,
that have been amplified (e.g., PCR) from mRNA of or cDNA derived
from, e.g., olfactory receptor-expressing neurons or genomic DNA.
Olfactory (or "odorant") receptors belong to the 7-transmembrane
receptor superfamily; however they are also recognized as a
distinct family of receptors. Olfactory receptors are
G-protein-coupled receptors (Raming (1993) Nature 361:353-356).
Genes encoding the olfactory receptors are active primarily in
olfactory neurons (Axel (1995) Sci. Amer. 273:154-159). Individual
olfactory receptor types are expressed in subsets of cells
distributed in distinct zones of the olfactory epithelium (Breer
(1994) Semin. Cell Biol. 5:25-32). The human genome contains
thousands of genes that encode a diverse repertoire of olfactory
receptors (Rouquier (1998) Nat. Genet. 18:243-250; Trask (1998)
Hum. Mol. Genet. 7:2007-2020).
[0085] Identifying Olfactory Receptor TM Domain Structures and
Sequences
[0086] The invention provides libraries of olfactory receptor
odorant/ligand-binding TM domain sequences. These sequence can
include a various TM domains or variations thereof, as describe
above. These sequences can be derived from any 7-transmembrane
receptor. Because these polypeptides have similar primary sequences
and secondary and tertiary structures, the seven domains can be
identified by various analyses well known in the art, including,
e.g., homology modeling, Fourier analysis and helical periodicity
(see, e.g., Pilpel (1999) supra), as described above. Using this
information sequences flanking the seven domains can be identified
and used to designed degenerate primers for amplification of
various combinations of TM regions and subsequences for use in the
compositions and methods of the invention.
[0087] Measuring Changes in Physiologic Activity Due to Olfactory
Receptor-Ligand Binding
[0088] The invention provides methods and compositions for
determining whether a test compound specifically binds to a
mammalian olfactory receptor in vitro or in vivo. The invention
also provides methods and compositions for determining whether a
test compound is neurotoxic to an olfactory neuron expressing an
olfactory transmembrane receptor polypeptide. Any aspect of cell
physiology can be monitored to assess the effect of odorant/ligand
binding to a chimeric olfactory receptor of the invention.
[0089] Olfactory receptors are normally located on the specialized
cilia of olfactory neurons. These receptors bind odorants and
initiate the transduction of chemical stimuli into electrical
signals. This process can involve a G protein-coupled activation of
an adenylyl cyclase, which leads to a rise in cAMP and consequently
the opening of cyclic nucleotide-activated, non-selective cation
channels. These open channels produce a cation influx that results
in the depolarization of the olfactory neuron. Another olfactory
transduction mechanism can also include the generation of IP.sub.3
and the opening of IP.sub.3-activated channels on the ciliary
plasma membrane. Electro-olfactograms can measure the mass response
of sensory neurons in the olfactory epithelium (discussed
below).
[0090] Cell Culture Assays
[0091] The invention provides methods and compositions for
expressing the chimeric olfactory receptors of the invention in
cells to screen for odorants that can specifically bind and the
effect (e.g., biochemical or electrophysiological) of such binding
on cell physiology. Any cell expression system can be used, e.g.,
mammalian cell expression systems. Cells that normally express
olfactory receptors can be used, particularly to study the
physiological effect of an odorant on a cell. Isolation and/or
culturing of such cells and their transformation with chimeric
olfactory receptor-expressing sequences of the invention can be
done with routine methods, as described above. See, e.g.,
description of cultured neurons that exhibit typical voltage-gated
currents and are responsive to application of odorants. Vargas
(1999) supra; olfactory neurons from rats (Coon (1989) Proc. Natl.
Acad. Sci. USA 86:1703-1707). However, the neurotoxicity of various
agents to humans could be more accurately determined using cultured
human neurons than cultured non-human neurons.
[0092] To evaluate electrophysiologic effects of ligand binding to
cell-expressed chimeric receptor, patch-clamping of individual
cells can be done. Patch-clamp recordings of the olfactory receptor
cell membrane can measure membrane conductances. Some conductances
are gated by odorants in the cilia and depolarize the cell through
cAMP- or IP3-sensitive channels, depending on the species. Other
conductances are activated by membrane depolarization and/or an
increased intracellular Ca2+ concentration. See, e.g., Trotier
(1994) Semin. Cell Biol. 5:47-54.
[0093] Changes in calcium ion levels in the cell after exposure of
the cell to known or potential odorant/ligands can be accomplished
by a variety of means. For example, cells can be pre-loaded with
reagents sensitive to calcium ion transients, e.g., Fura-2 (see,
e.g., Rawson (1997) J. Neurophysiol. 77:1606-1613; Restrepo (1996)
J. Neurobiol. 30:37-48). Measurement of calcium transients is
described in detail in Example 1, below. For example, Kashiwayanagi
(1996) Biochem. Biophys. Res. Commun. 225:666-671 measured both of
inositol 1,4,5-trisphosphate induces inward currents and Ca2+
uptake in frog olfactory receptor cells.
[0094] Other physiologic mechanisms can also be measured, e.g.,
plasma membrane homeostasis parameters (including lipid second
messengers), cellular pH changes (see, e.g., Silver (1998) Methods
Cell Biol. 56:237-251), G proteins (see, e.g., Quartara (1997)
Neuropeptides 31:537-563); cAMP, and the like.
[0095] Non-Human Animal Assays
[0096] The invention also provides non-human animals expressing one
or more hybrid olfactory receptor sequences of the invention,
particularly human olfactory receptor sequences. Such expression
can be used to determine whether a test compound specifically binds
to a mammalian olfactory transmembrane receptor polypeptide in vivo
by contacting a non-human animal stably or transiently infected
with a nucleic acid derived from the library of the invention with
a test compound and determining whether the animal reacts to the
test compound by specifically binding to the receptor
polypeptide.
[0097] Use of the translocation domains of the invention in the
fusion polypeptides generates a cell expressing high levels of
olfactory receptor. Animals infected with the vectors of the
invention are particularly useful for assays to identify and
characterize odorants/ligands that can bind to a specific or sets
of receptors. Such vector-infected animals expressing libraries of
human olfactory sequences can be used for in vivo screening of
odorants and their effect on, e.g., cell physiology (e.g., on
olfactory neurons), on the CNS (e.g., olfactory bulb activity), or
behavior.
[0098] Means to infect/express the libraries of nucleic acids and
vectors of the invention are well known in the art, as described
above. A variety of individual cell, organ or whole animal
parameters can be measured by a variety of means. For example,
recording of stimulant-induced waves (bulbar responses) from the
main olfactory bulb or accessory olfactory bulb is a useful tool
for measuring quantitative stable olfactory responses. When
electrodes are located on the olfactory bulb surface it is possible
to record stable responses over a period of several days (see,
e.g., Kashiwayanagi (1997) Brain Res. Brain Res. Protoc.
1:287-291). In this study, electroolfactogram recordings were made
with a four-electrode assembly from the olfactory epithelium
overlying the endoturbinate bones facing the nasal septum. Four
electrodes were fixed along the dorsal-to-ventral axis of one
turbinate bone or were placed in corresponding positions on four
turbinate bones and moved together up toward the top of the bone.
See also, Scott (1997) J. Neurophysiol. 77:1950-1962; Scott (1996)
J. Neurophysiol. 75:2036-2049; Ezeh (1995) J. Neurophysiol.
73:2207-2220. In other systems, fluorescence changes in nasal
epithelium can be measured using the dye di-4-ANEPPS, which is
applied on the rat's nasal septum and medial surface of the
turbinates (see, e.g., Youngentob (1995) J. Neurophysiol.
73:387-398). Extracellular potassium activity (aK) measurements can
also be carried out in in vivo. An increase in aK can be measured
in the mucus and the proximal part of the nasal epithelium (see,
e.g., Khayari (1991) Brain Res. 539:1-5).
[0099] The chimeric olfactory receptor of the invention can be
expressed in animal nasal epithelium by delivery with an infecting
agent, e.g., adenovirus expression vector. Recombinant
adenovirus-mediated expression of a recombinant gene in olfactory
epithelium using green fluorescent protein as a marker is described
by, e.g., Touhara (1999) Proc. Natl. Acad. Sci. USA
96:4040-4045.
[0100] Transgenic Non-Human Animals Incorporating Hybrid Olfactory
Receptors
[0101] The invention also provides non-human animals genetically
engineered to express one or more hybrid olfactory receptor
sequences of the invention, particularly human olfactory receptor
sequences. Because the translocation domains of the invention in
the fusion polypeptides generates an animal expressing high levels
of olfactory receptor, these animals and their progeny are
particularly useful for assays to identify and characterize
odorants/ligands that can bind to a specific or sets of
receptors.
[0102] The endogenous olfactory receptor genes can remain
functional and wild-type (native) activity can still be present. In
other situations, where it is desirable that all olfactory receptor
activity is by the introduced exogenous hybrid receptor, use of a
knockout line is preferred. Methods for the construction of
non-human transgenic animals, particularly transgenic mice, and the
selection and preparation of recombinant constructs for generating
transformed cells are well known in the art.
[0103] Construction of a "knockout" cell and animal is based on the
premise that the level of expression of a particular gene in a
mammalian cell can be decreased or completely abrogated by
introducing into the genome a new DNA sequence that serves to
interrupt some portion of the DNA sequence of the gene to be
suppressed. Also, "gene trap insertion" can be used to disrupt a
host gene, and mouse embryonic stem (ES) cells can be used to
produce knockout transgenic animals (see, e.g., Holzschu (1997)
Transgenic Res 6: 97-106). The insertion of the exogenous sequence
is typically by homologous recombination between complementary
nucleic acid sequences. The exogenous sequence is some portion of
the target gene to be modified, such as exonic, intronic or
transcriptional regulatory sequences, or any genomic sequence which
is able to affect the level of the target gene's expression; or a
combination thereof. Gene targeting via homologous recombination in
pluripotential embryonic stem cells allows one to modify precisely
the genomic sequence of interest. Any technique can be used to
create, screen for, propagate, a knockout animal, e.g., see Bijvoet
(1998) Hum. Mol. Genet. 7:53-62; Moreadith (1997) J. Mol. Med.
75:208-216; Tojo (1995) Cytotechnology 19:161-165; Mudgett (1995)
Methods Mol. Biol. 48:167-184; Longo (1997) Transgenic Res.
6:321-328; U.S. Pat. Nos. 5,616,491; 5,464,764; 5,631,153;
5,487,992; 5,627,059; 5,272,071; and, WO 91/09955, WO 93/09222, WO
96/29411, WO 95/31560, and WO 91/12650.
[0104] The nucleic acid libraries of the invention can also be used
as reagents to produce "knockout" human cells and their
progeny.
[0105] Kits
[0106] The invention provides kits that contain degenerate primer
pairs of the invention. cDNA libraries from olfactory epithelium
can also be included. The kits can contain recombinant adenoviruses
comprising a single construct or libraries of expression vectors of
the invention. The kit can also contain replication-competent
cells, such as 293 cells. The kit can contain instructional
material teaching methodologies, e.g., means to amplify nucleic
acid, infect animals, and the like.
[0107] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
EXAMPLES
[0108] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Identification of Odorant/Ligands for Olfactory Receptors with
Binding Sites Generated by PCR Amplification with Degenerate
Primers by Functional Expression of Libraries of the Receptors in
Human Cell Lines
[0109] The following example sets forth the generation of an
expression plasmid library containing a large and diverse
repertoire of nucleic acids encoding odorant/ligand binding regions
comprising transmembrane (TM) II-VII regions of mouse olfactory
receptor sequences. From this library, 80 chimeric receptors were
tested against 26 odorants after transient transfection into the
human cell line HEK-293. Three receptors were identified that
responded to micromolar concentrations of carvone, (-) citronellal
and limonene, respectively.
[0110] A PCR-based amplification strategy taking advantage of the
homology shared among olfactory receptors at the beginning of TM II
and the end of TM VII was used to generate a library containing a
large number of olfactory-receptor sequences. The structure of the
overall construct, pCMV-Rho/M4.sub.NC, is shown in FIG. 1A. The
degenerate oligonucleotides are flanked by the coding sequences for
the appropriate regions of the mouse M4 olfactory receptor
(described by, e.g., Qasba (1998) J. Neurosci. 18:227-236).
[0111] Making a Chimeric Receptor Cassette and Vector for
Eukaryotic Expression
[0112] Chimeric receptor expression vectors were assembled from a
pBK-CMV plasmid (Stratagene, San Diego, Calif.) modified such that
the lac Z sequences between nucleotides 1098 and 1300 were deleted.
A PCR fragment consisting of 45 nucleotides upstream of the bovine
rhodopsin initiation codon and the first 60 nucleotides of the
coding region (designated "rho-tag" in FIG. 1A) was introduced
between the BamHI and EcoRI sites. Restriction fragments
corresponding to the first 57 amino acids (the N-terminus to TM II,
EcoRI/PstI restriction sites) (SEQ ID NO:1) and to the last 22
amino acids (BspEI/XbaI restriction sites) (SEQ ID NO:2) of the
mouse M4 olfactory receptor were cloned into the rhodopsin-tag
("rho-tag") vector. The resulting vector (designated
pCMV-Rho/M4.sub.NC) possesses unique PstI and BspEI sites at the
beginning of TM II and the end of TM VII, respectively (see FIG.
1A).
[0113] PCR Amplification
[0114] The mouse olfactory-receptor transmembrane II-VII library
was amplified using PCR. The PCR reaction mixture contained: Taq
and Pfu polymerase (Stratagene, San Diego, Calif.) 0.5U each, 0.2
mM dNTP, 1 .mu.M of each primer (degenerate oligonucleotides SEQ ID
NO:1 and SEQ ID NO:2) and either 100 ng mouse genomic DNA
(.beta..sub.2-adrenergic receptor sequence), 10 ng plasmid template
DNA, or 50 to 100 ng 1.sup.st strand cDNA template prepared from
mouse C57BL/6J olfactory epithelium. One amplification protocol was
one cycle of 2 min at 94.degree. C.; 30 cycles at (55.degree. C.,
72.degree. C., 94.degree. C.), 1 min each; 1 cycle at (55.degree.
C., 72.degree. C.), 10 min. A second amplification protocol was 1
cycle for 2 min at 94.degree. C.; 34 cycles at (45.degree. C.,
72.degree. C., 94.degree. C.), 1 min each; and 1 cycle at
(45.degree. C., 72.degree. C.), 10 min. The second procotol, having
a lower hybridization temperature (45.degree. C. versus 55.degree.
C.) generated an equally diverse library of binding domains. A
library of PCR products of about 0.7 kilobase was generated.
[0115] Analysis of Amplified Odorant/Ligand-Binding Sequence
Library
[0116] Sequencing and sequence identity analysis of 26 randomly
chosen PCR amplification products was performed. Deduced amino acid
sequences were aligned by the ClustalW algorithm using default
values established by DNAstar alignment software (DNASTAR, Inc.,
Madison, Wis.) (see, e.g., Burland (2000) Methods Mol. Biol.
132:71-91).
[0117] This analysis revealed that all but three of the sequenced
odorant/ligand binding region inserts were distinct
polypeptide-coding receptor sequences. Exemplary odorant/ligand
binding region nucleic acid sequences generated by amplification of
Mus musculus cDNA, and the respective deduced amino acid sequences,
include
3 (a) 1 agtgtcttat ccattctgga tatgggctat gtcaccacca cagtgcccca
gatgctggta (SEQ ID NO:11) 61 catctggtct gtaagaagaa gaccatatcc
tatgttggat gtgtggctca gatgtacatc 121 ttcctgatgc tgggaatcac
cgagtcttgg ctgtttgcaa tcatggccta tgataggtat 181 gtggccattt
gccatcccct cagatacaaa gtcatcatga gtcctttgct gcgcgggtca 241
ctggtagcct tctgtgggtt ctggggtatc acctgtgccc tgatatatac tgtttctgct
301 atgattcttc cctactgtgg ccccaatgag atcaaccact tcttctgtga
agtgcctgct 361 gtcctgaagc tggcctgcgc agacacctct cccaatgacc
aggtagactt catcctaggc 421 tttatccttc ttttggtccc actctccctc
atcattgttg tctacatcaa tatctttgct 481 gctatcttga gaatccgttc
aactcaaggg aggatcaagg ccttctccac ctgtgtgtcc 541 cacatcactg
tggtcaccat gttctccatc ccgtgtatgg ttatgtatat gaggcctggc 601
tctgagtcct ccccagaaga ggacaagaag ttggctctgt tctacaacgt catctctgcc
661 ttcctcaac with a deduced amino acid sequence
SVLSILDMGYVTTTVPQMLVHLVCKKKTISYVGCVAQMYIFLML (SEQ ID NO:12)
GITESWLFAIMAYDRYVAICHPLRYKVIMSPLLRGSLVAFCGFWGITCALIYTVSAMI
LPYCGPNEINHFFCEVPAVLKLACADTSPNDQVDFILGFILLLVPLSLIIVVYINIFA
AILRTRSTQGRIKAFSTCVSHITVVTMFSIPCMVMYMRPGSESSPEEDKKLALFYNVI SAFLN
(b) 1 tgcaacctgg ccaccatgga cattgtgtgc accccctctg tgattcctaa
ggccctgatt (SEQ ID NO:13) 61 ggcctagtgt ctgaagaaaa caccatctcc
ttcaaaggat gcatggctca gctcttcttt 121 cttctgtggt ccttgtcttc
ggagctgctg ctgctcacgg tcatggccta tgaccgctat 181 gtggccatct
gctttcccct gcactacagc tctagaatga gcccacagct ctgtggggcc 241
ctggccgtgg gtgtatggtc catctgtgct gtgaatgcat ctgtgcacac tggcctgatg
301 acacggctgt cattctgtgg ccccaaggtc atcacccact tcttctgtga
gattccccca 361 ctcctcctgc tttcctgtag tcccacatac attaatagcg
ttatgacact tgtggcagat 421 gccttttatg ggtgcatcaa ctttgtgcta
accttgttat cctatggctg catcattgcc 481 agtgttctgc gcatgcgttc
tgctgagggc aagaggaagg ccttttctac ctgttcatcc 541 cacctcatcg
tggtctcagt gtactactca tctgtgttct gtgcctatgt cagtcctgcc 601
tccagctaca gcccagaaag aagcaaagtt acctccgtgc tgtactcgat cctcagccca
661 accctgaac with a deduced amino acid sequence
CNLATMDIVCTPSVIPKALIGLVSEENTISFKGCMAQLFFLLWS (SEQ ID NO:14)
LSSELLLLTVMAYDRYVAICFPLHYSSRMSPQLCGALAVGVWSICAVNASVHTGLMTR
LSFCGPKVITHFFCEIPPLLLLSCSPTYINSVMTLVADAFYGCINFVLTLLSYGCIIA
SVLRMRSAEGKRKAESTCSSHLIVVSVYYSSVFCAYVSPASSYSPERSKVTSVLYSIL SPTLN
(c) 1 tgcaacctgg ccaccatgga tattatctgc acctcctctg tgctgcccaa
ggcgctggtt (SEQ ID NO:15) 61 ggtctactat ctgaggaaaa caccatctcc
tttaaagggt gcatggccca gctcttcttc 121 cttgtgtggt ccttgtcttc
agagctgctg ctgctcacag tcatggccta tgaccgctat 181 gtggccatct
gctttcccct gcactacagc tctagaatga gcccacagtt gtgtggggct 241
ctggccatgg gtgtatggtc catctgtgct ctgaatgcat ctatcaacac tggtctgatg
301 acacggctgt cattctgtgg acccaaggtc atcacccact tcttctgtga
gattccccca 361 ctccttctgc tctcctgtag ccccacatac gtaaacagca
ttatgactct aatagcagat 421 gtcttctatg gaggcatcaa ttttgtgctt
accttactat cctatggctg catcattgcc 481 agcatcctgc gcatgcgttc
tgctgagggc aagaggaagg ccttttctac ctgctcatcc 541 cacctcatcg
tggtctctgt gtactactca tctgtgttct gtgcctatgt cagccctgca 601
tccagctata gcccagaaag aagcaaagtt acctctgtgt tgtactcatt cctcagccca
661 accctgaac with a deduced amino acid sequence
CNLATMDIICTSSVLPKALVGLLSEENTISFKGCMAQLFFLVWS (SEQ ID NO:16)
LSSELLLLTVMAYDRYVAICFPLHYSSRMSPQLCGALAMGVWSICALNASINTGLMTR
LSFCGPKVITHFFCEIPPLLLLSCSPTYVNSIMTLIADVFYGGINFVLTLLSYGCIIA
SILRMRSAEGKRKAFSTCSSHLIVVSVYYSSVFCAYVSPASSYSPERSKVTSVLYSFL SPTLN
(d) 1 gccacccttt cctgtgttga catcctcttc acctccacca cagtgcccaa
ggccctagtg (SEQ ID NO:17) 61 aacatccaca cccaaagcag gacaatctcc
tatgcaggat gcctggtcca gctctatttt 121 ttcctgactt ttggagacat
ggacatcttt ctcctggcca caatggccta tgaccgcttt 181 gtagctattt
gtcaccctct ccactatagg atgatcatga gcttccagcg ctgctcactc 241
ttagtgacag tctgttggac ccttacaacc gttgtggcca tgacacacac cttcctcata
301 ttccggctct ccttctgctc tcagaaggtc attccagact tcttctgtga
cctgggaccc 361 ctaatgaaga tcgcttgctc tgaaacccgg atcaatgagc
ttgtgcttct cttcctgggg 421 ggtgcagtca tcttaatccc ctttttgctc
atccttatgt cttatatccg cattgtttca 481 gccatcctca gggtcccttc
tgcccaagga aggcgtaagg ccttttctac ctgtgggtcc 541 cacctttctg
tggtggccct attctttggg actgtgataa gggcttatct atgtccttca 601
tcctcttcct ctaactcagt ggtagaggac acagcagcag ctgtcatgta tacagtggtg
661 actcccgtgc tgaac with a deduced amino acid sequence
ATLSCVDILFTSTTVPKALVNIHTQSRTISYAGCLVQLYFFLTF (SEQ ID NO:18)
GDMDIFLLATMAYDRFVAICHPLHYRMIMSFQRCSLLVTVCWTLTTVVAMTHTFLIFR
LSFCSQKVIPDFFCDLGPLMKIACSETRINELVLLFLGGAVILIPFLLILMSYIRIVS
AILRVPSAQGRRKAFSTCGSHLSVVALFFGTVIRAYLCPSSSSSNSVVEDTAAAVMYT VVTPVLN
(e) 1 agtcagctct ccctcatgga cctcatgctg gtctgtaaca ttgtgccaaa
gatggcagtc (SEQ ID NO:19) 61 aacttcctgt ctggcaggaa gtccatctct
tttgccggct gtggcataca aatcggattt 121 tttgtctctc ttgtgggatc
agagggtctc ttgttaggac tcatggctta tgatcgctat 181 gtggccatta
gccacccact tcactatccc attctcatga gccaaaaggt ctgtctccag 241
attgctggaa gttcctgggc ttttgggatc cttgatggaa taattcagat ggtggcagcc
301 atgagcctgc cctactgtgg ctcacggtat atagatcact tcttctgtga
agtgccggct 361 ttactgaagc tggcctgtgc agacacctcc cttttcgaca
ccctgctctt tgcttgctgt 421 gtctttatgc tgcttcttcc tttctcgatc
attgtgactt cctatgctcg catcttgggg 481 gctgtgctcc gtatgcactc
tgcccagtcc cgaaaaaagg ccctggccac ttgttcctcc 541 cacctgacag
ctgtctctct cttctacggg gcagcaatgt tcatctacct gaggccaagg 601
cgatatcgcg ctcctagcca tgacaaagtt gtctcaatct tctacacagt tcttactcct
661 atgctcaac with a deduced amino acid sequence
SQLSLMDLMLVCNIVPKMAVNFLSGRKSISFAGCGIQIGFFVSL (SEQ ID NO:20)
VGSEGLLLGLMAYDRYVAISHPLHYPILMSQKVCLQIAGSSWAFGILDGIIQMVAANS
LPYCGSRYIDHFFCEVPALLKLACADTSLFDTLLFACCVFMLLLPFSIIVTSYARILG
AVLRMHSAQSRKKALATCSSHLTAVSLFYGAAMFIYLRPRRYRAPSHDKVVSIFYTVL TPMLN
(f) 1 tacaaccttt cattgtctga catgggcttt agcagcacca caatccccaa
aatgctgata (SEQ ID NO:21) 61 aacttgcatg cacataagag atccacaaca
tatgctgaat gcctaactca ggtatctttc 121 tttattcttt ttgggtgtat
ggacagcttt ctactggcag tgatggcata tgaccgatgg 181 gtggccattt
gtcaccctct acactaccaa gtcattctga atccttgtcg gtgtagatat 241
ttggttgtaa tgtcattttg tatcagtctc attgattcac aggtgcactg ctttatggtg
301 tcacaactaa cattttgtac taatatagaa atccctcatt tcttctgtga
tgttccagaa 361 cttgtaaaac ttgcttgttc taacactact atcaatgaca
tagccatgtt tctttcaagc 421 atcattgttg gattcctccc tgcctcagga
atattttact cctactataa aattacttct 481 tctattttta gagttccatc
actgttaggg aaatataaag ccttctctac ctgtggatct 541 cacctgtcag
ttgtttgcct attttatgga acaggtatag gagtttacct cagttccaca 601
gtttctggtt cttccaggga aagtatggta gcttcggtaa tgtatacaat ggtggttcct
661 atgatgaac with a deduced amino acid sequence
YNLSLSDMGFSSTTIPKMLINLHAHKRSTTYAECLTQVSFFILF (SEQ ID NO:22)
GCMDSFLLAVMAYDRWVAICHPLHYQVILNPCRCRYLVVMSFCISLIDSQVHCFMVSQ
LTFCTNIEIPHFFCDVPELVKLACSNTTINDIAMFLSSIIVGFLPASGIFYSYYKITS
SIERVPSLLGKYKAFSTCGSHLSVVCLFYGTGIGVYLSSTVSGSSRESMVASVMYTMV VPMMN
(g) 1 agtcagctct ccctcatgga cctcatgctg gtctgtaaca ttgtgccaaa
gatggcagtc (SEQ ID NO:23) 61 aacttcctgt ctggcaggaa gtccatctct
tttgccggct gtggcataca aatcggattt 121 tttgtctctc ttgtgggatc
agagggtctc ttgttaggac tcatggctta tgatcgctat 181 gtggccatta
gccacccact tcactatccc attctcatga gccaaaaggt ctgtctccag 241
attgctggaa gttcctgggc ttttgggatc cttgatggaa taattcagat ggtggcagcc
301 atgagcctgc cctactgtgg ctcacggtat atagatoact tcttctgtga
agtgccggct 361 ttactgaagc tggcctgtgc agacacctcc cttttcgaca
ccctgctctt tgcttgctgt 421 gtctttatgc tgcttcttcc tttctcgatc
attgtgactt cctatgctcg catcttgggg 481 actgtgctcc gtatgcactc
tgcccagtcc cgaaaaaagg ccctggccac ttgttcctcc 541 cacctgacag
ctgtctctct cttctacggg gcagcaatgt tcatctacct gaggccaagg 601
cgatatcgcg ctcctagcca tgacaaagtt gtctcaatct tctacacagt tcttactcct
661 atgctcaac with a deduced amino acid sequence
SQLSLMDLMLVCNIVPKMJWNFLSGRKSISFAGCGIQIGFFVSL (SEQ ID NO:24)
VGSEGLLLGLMAYDRYVAISHPLHYPILMSQKVCLQIAGSSWAFGILDGIIQMVAAMS
LPYCGSRYIDHFFCEVPALLKLACADTSLFDTLLFACCVFMLLLPFSIIVTSYARILG
AVLRMHSAQSRKKALATCSSHLTAVSLFYGAAMFIYLRPRRYRAPSHDKVVSIFYTVL TPMLN
(h) 1 tctaatctgt cctttgtgga catctgcttc acttccacca ctgttccaca
gatgctggta (SEQ ID NO:25) 61 aacattcaca cacaaagcaa ggccatcacc
tatgcaggct gcatcatcca aatgtacttc 121 ttactgcttt tttcagggtt
agacatcttt ctgctgactg tgatggccta tgaccgctat 181 gtggccatct
gtcaccccct gcattacatg atcatcatga gcacaagacg ctgtggattg 241
atgattctgg catgctggat tataggtgtt ataaattccc tgttacacac ctttttggtg
301 ttacggctgt cattctgcac aaacttggaa atcccccatt ttttctgtga
acttaatcaa 361 gttgtacacc aggcctgttc tgacaccttt cttaatgata
tggtaattta cattacagct 421 atgctactgg ctgttggccc cttctctggt
atcctttact cttactctag gatagtatcc 481 tccatttgtg caatctcctc
agtgcagggg aagtacaaag cattttccac ctgtgcatct 541 cacctctcag
ttgtctcctt attttattgc accctcctgg gagtgtacct cagctctgct 601
gtgacccaaa actcacatgc tactgcaaca gcttcattga tgtacactgt ggtcaccccc
661 atgctgaac with a deduced amino acid sequence
SNLSFVDICFTSTTVPQMLVNIHTQSKAITYAGCIIQMYFLLLF (SEQ ID NO:26)
SGLDIFLLTVMAYDRYVAICHPLHYMIIMSTRRCGLMILACWIIGVINSLLHTFLVLR
LSECTNLEIPHFFCELNQVVHQACSDTFLNDMVIYITAMLLAVGPFSGILYSYSRIVS
SICAISSVQGKYKAFSTCASHLSVVSLFYCTLLGVYLSSAVTQNSHATATASLMYTVV TPMLN
(i) 1 agtcagctct ccctcatgga cctcatgctg gtctgtaaca ttgtgccaaa
gatggcagtc (SEQ ID NO:27) 61 aacttcctgt ctggcaggaa gtccatctct
tttgccggct gtggcataca aatcggattt 121 tttgtctctc ttgtgggatc
agagggtctc ttgttaggac tcatggctta tgatcgctat 181 gtggccatta
gccacccact tcactatccc attctcatga gccaaaaggt ctgtctccag 241
attgctggaa gttcctgggc ttttgggatc cttgatggaa taattcagat ggtggcagcc
301 atgagcctgc cctactgtgg ctcacggtat atagatcact tcttctgtga
agtgccggct 361 ttactgaagc tggcctgtgc agacacctcc cttttcgaca
ccctgctctt tgcttgctgt 421 gtctttatgc tgcttcttcc tttctcgatc
attgtgactt cctatgctcg catcttgggg 481 gctgtgctcc gtatgcactc
tgcccagtcc cgaaaaaagg ccctggccac ttgttcctcc 541 cacctgacag
ctgtctctct cttctacggg gcagcaatgt tcatctacct gaggccaagg 601
cgatatcgcg ctcctagcca tgacaaagtt gtctcaatct tctacacagt tcttactcct
661 atgctcaac with a deduced amino acid sequence
SQLSLMDLMLVCNIVPKMAVNFLSGRKSISFAGCGIQIGFFVSL (SEQ ID NO:28)
VGSEGLLLGLMAYDRYVAISHPLHYPILMSQKVCLQIAGSSWAFGILDGIIQMVAAMS
LPYCGSRYIDHFFCEVPALLKLACADTSLFDTLLEACCVFMLLLPFSIIVTSYARILG
AVLRMHSAQSRKKALATCSSHLTAVSLFYGAAMFIYLRPRRYRAPSHDKVVSIFYTVL TPMLN
(j) 1 tgtgccctct ccatctctga gattttctac acctttgcca tcatcccacg
catgttggct (SEQ ID NO:29) 61 gacctgctca ccacacttca ctccatcgcc
tttctggcct gtgccagcca gatgttcttc 121 tccttcacat ttggcttcac
ccattccttt ctactcaccg tcatgggcta tgaccgctac 181 gtggccatct
gtcacccact gagatacaat gtgctcatga gcccccgtgg ctgtgcctgc 241
ctggtagcct ggtcctgggt tggtggatca ttcatgggga cagtggtgac gacagccatt
301 ttcaacctca cattctgtgg acccaatgag atccaccatt ttacttgtca
tgttccacct 361 ctattgaagt tggcatgcgg agagaatgta ctggaggtgg
caaagggtgt agaaatagtg 421 tgcatcacag ccctcctggg ctgctttctc
ctcatcctcc tctcatatgc cttcattgtg 481 gttaccatct tgaagatacc
atcagctgag ggtcggcaca aggctttctc cacatgtgca 541 tcccacctca
cagtggtggt tgtacattat ggctttgctt ctgtcattta cctcaagcct 601
aagggcccca agtctctgga aggagatact ctgatgggca tcacctacac agtcctcacc
661 cccttcctta gt atgctcaac with a deduced amino acid sequence
CALSISEIFYTFAIIPRMLADLLTTLHSIAFLACASQMFFSFTF (SEQ ID NO:30)
GFTHSFLLTVMGYDRYVAICHPLRYNVLMSPRGCACLVAWSWVGGSEMGTVVTTAIFN
LTFCGPNEIHHFTCHVPPLLKLACGENVLEVAKGVETVCITALLGCFLLILLSYAFIV
VTILKIPSAEGRHKAESTCASHLTVVVVHYGEASVIYLKPKGPKSLEGDTLMGITYTV LTPFLS
(k) 1 tgcaacttag cgaccatgga tattatctgc acctcctctg tactgcccaa
ggcgctggtt (SEQ ID NO:31) 61 ggtctactgt ctgaggaaaa caccacctcc
ttcaaagggt gcatgactca gctcttcttt 121 cttgtgtggt ctggatcctc
tgagctgctg ctgctcacag tcatggccta tgaccgctat 181 gtggccatct
gtttgcccct gcattacagc tctaggatga gtccacagct ctgtgggacc 241
tttgccgtgg gtgtatggtc catctgcgca ctaaatgcat ctatcaacac tggtctgatg
301 acacggctgt cattctgtgg ccccaaggtc atcacccact tcttctgtga
gattccccca 361 ctcctcctgc tctcctgtag tcctacatat ataaatagcg
ttatgactct tgtggcagat 421 gccttttatg gaggcatcaa ttttttactt
accttgctat cctatggctg catcattgcc 481 agcatcctgc gcatgcgttc
tgctgagggc aagaggaagg ccttttctac ctgctcatcc 541 cacctcattg
tggtctctgt gtactactca tctgtgttct gtgcctatgt cagccctgct 601
tctagctaca gcccagaaag aagcaaagtt tcctcagtgc tgtactcagt cctcagccca
661 accctcaac with a deduced amino acid sequence
CNLATMDIICTSSVLPKALVGLLSEENTTSFKGCMTQLFFLVWS (SEQ ID NO:32)
GSSELLLLTVMAYDRYVAICLPLHYSSRMSPQLCGTFAVGVWSICALNASINTGLMTR
LSFCGPKVITHFFCEIPPLLLLSCSPTYINSVMTLVADAEYGGINFLLTLLSYGCIIA
SILRMRSAEGKRKAFSTCSSHLIVVSVYYSSVFCAYVSPASSYSPERSKVSSVLYSVL SPTLN
(l) 1 gccaaccttt ccttcgttga tgtctgcttc accaccaatc tcatccccag
gctcctggct (SEQ ID NO:33) 61 ggccatgtgg ctggaacaag gaccatctct
tatgtccact gcctaactca gacgtacttc 121 ctgatttctt ttgccaatgt
ggacaccttt ctgctggctg ccatggccct ggacagattt 181 gtggccatat
gctacccact acagtaccac accatcatca ccccccagct ctgtgtgggg 241
ctggcagccg ttgtgtggat gtgctctgcc ctcatctctc tgatgcacac actcctcatg
301 agcagactga gtttctgctc ctccatcccg gagatctctc acttctactg
tgatgcttac 361 ctgctcatga agttggcctg ttcagacaca cgagtcaatc
aacttgtctt cctgggagct 421 gtggtcctct ttgtggcccc ctgcattctc
attgtggtct cttatgtccg aatcaccatg 481 gtggtcctcc agatcccctc
tgcaaagggc cggcacaaga cattttccac atgtagctca 541 cacttgtctg
tggtcactct gttctatggc acagtactgg gtatctatat acgacctcca 601
gactccttct ccacccagga cacggtagcc accatcatgt atactgtggt tacccccatg
661 ctgaac with a deduced amino acid sequence
ANLSFVDVCFTTNLIPRLLAGHVAGTRTISYVHCLTQTYFLISF (SEQ ID N0:34)
ANVDTFLLAAMALDRFVAICYPLQYHTIITPQLCVGLAAVVWMCSALISLMHTLLMSR
LSECSSIPEISHFYCDAYLLMKLACSDTRVNQLVFLGAVVLFVAPCILIVVSYVRITM
VVLQIPSAKGRHKTFSTCSSHLSVVTLFYGTVLGIYIRPPDSFSTQDTVATIMYTVVT PMLN (m)
1 tgcaacctgg ctaccacgga tattgtgtgc acctcctctg tgattcctaa ggccctgatt
(SEQ ID NO:35) 61 ggcctagtat ctgaggaaaa catcatcacc ttcaagggat
gtatggccca gctcttcttc 121 cttgcatggg caacatccgc agagctgttg
ctgctcacgg tcatggccta tgaccgctat 181 gtggctatct gctttcccct
acactacagc tctaggatga gcccacagct ctgtggagca 241 ctggccgtgg
gtgtatggtc catcagtgct gtgaatgcat ctgtgcacac tggcctgatg 301
acacggctgt cattctgtgg acccaaggtc atcacccact tcttctgtga gataccccca
361 ctcctcctgc tctcctgtag ttccacatac attaatagtg ttatgacact
tgtggcagat 421 gtctttctgg gaggcatcaa cttcatgtta accctgttat
cttatggctt catcattgcc 481 agcatcctgc gcatgcgttc tgctgagggc
aagaggaagg ccttttctac ctgctcatcc 541 cacctcatcg tggtttctgt
gtactactca tctctgttct gtgcctatat cagccctgct 601 tctagctaca
gcccagaaag aagcaaagtt tcctcagtgc tgtactcagt cctcagccca 661
accctcaac with a deduced amino acid sequence
CNLATTDIVCTSSVIPKALIGLVSEENIITFKGCMAQLETLAWA (SEQ ID NO:36)
TSAELLLLTVMAYDRYVAICEPLHYSSRMSPQLCGALAVGVWSISAVNASVHTGLMTR
LSFCGPKVITHEFCEIPPLLLLSCSSTYINSVMTLVADVFLGGINFMLTLLSYGFIIA
SILRMRSAEGKRKAFSTCSSHLIVVSVYYSSLECAYISPASSYSPERSKVSSVLYSVL SPTLN
(n) 1 agcaacctgg cttttgttga tttctgctac tcctctgtca ttacacctaa
gatgcttggg (SEQ ID NO:37) 61 aatttcttgt atagcaaaaa tgccatatcc
ttcaatgcat gtgctgccca gttaggctgc 121 tttctcacat ttatggtatc
agagtgcttg ctcctggctt ccatggcata tgatagatat 181 gcagcaattt
gtaaccctct attgtatatg gtcacaatgt ctcctggaat ctgcattcag 241
cttgtagttg tgccctatag ctatagtttc ctcatggcat tgattcacac tcttctaacc
301 ttccgcctat cctattgcca ttctaatatc atcaatcact tctactgtga
tgacatgcct 361 cttctcaggc taacttgctc agatactcac tacaagcagc
tgtctatttt ggcctgtgct 421 ggaatcacat tcatttcttc tgttctgatt
gtttctgtat cctacatgtt cattatttct 481 gccattctga ggatgcgctc
agctgaagga agacggaaag ccttttccac ctgtagctct 541 cacatgatgg
cagtgagcat attctatgga actcttatct ttatgtactt acagccgagc 601
tctgaccatt ctcttgatac agataagatg gcctctgtct tctacacagt gatcatcccc
661 atgttgaac with a deduced amino acid sequence
SNLAFVDFCYSSVITPKMLGNFLYSKNAISFNACAAQLGCFLTF (SEQ ID NO:38)
MVSECLLLASMAYDRYAAICNPLLYMVTMSPGICIQLVVVPYSYSFLMALIHTLLTFR
LSYCHSNIINHFYCDDMPLLRLTCSDTHYKQLSILACAGITEISSVLIVSVSYMEIIS
AILRMRSAEGRRKAFSTCSSHMMAVSIFYGTLIFMYLQPSSDHSLDTDKMASVFYTVI IPMLN
(o) 1 agtcacttgt ccttcattga catgatgtac atctcaacca ttgtgcccaa
aatgctagtt (SEQ ID NO:39) 61 gattatcttc tagggcaaag gactatttcc
tttgtgggat gcacagctca acactttcta 121 tacctcaccc tggtgggagc
cgagttcttt cttctgggcc tcatggctta tgatcgttat 181 gtggccatct
gcaacccact gaggtaccct gtcctcatga gccgccggat ctgttggatt 241
atcatagcag gctcctggtt tgggggatct ttggatggct tcctcctcac tccaatcacc
301 atgagttttc ctttctgtag atcacgagag attaaccact tcttctgtga
ggcacctgct 361 gtgctgaagt tggcatgtgc agacacagcc ctctatgaga
cggtgatgta tgtgtgctgc 421 gttctgatgc tgttgattcc tttctctgtg
gttatctcat cctatgcgcg gattctggcc 481 actgtctacc atatgagctc
tgtggaagga aggaagaaag cgtttgctac ctgctcgtct 541 cacatgactg
tggtaacctt gttttatggg gctgccatat acacctatat ggtaccacac 601
tcttaccatt ccccatccca agacaaaatt ttttctgtgt tctataccat tctcacaccc
661 atgctgaac with a deduced amino acid sequence
SHLSFIDMMYISTTVPKMLVDYLLGQRTISFVGCTAQHFLYLTL (SEQ ID NO:40)
VGAEFFLLGLMAYDRYVAICNPLRYPVLMSRRICWIIIAGSWFGGSLDGFLLTPITMS (SEQ ID
NO:40) FPFCRSREINHFFCEAPAVLKLACADTALYETVMYVCCVLMLLIPFSVVISSYARILA
TVYHMSSVEGRKKAFATCSSHMTVVTLFYGAAIYTYMVPHSYHSPSQDKIFSVFYTIL TPMLN
(p) 1 tgcaacttag cgaccatgga tattatctgc acctcctctg tactgcccaa
ggcgctggtt (SEQ ID NO:41) 61 ggtctactgt ctgaggaaaa caccatcccc
ttcaaagggt gcatgactca gctcttcttt 121 cttgtgtggt ctggatcctc
tgagctgctg ctgctcacag tcatggccta tgaccgctat 181 gtggccatct
gtttgcccct gcattacagc tctaggatga gtccacagct ctgtgggacc 241
tttgccgtgg gtgtatggtc catctgcgca ctaaatgcat ctatcaacac tggtctgatg
301 acacggctgt cattctgtgg ccccaaggtc atcacccact tcttctgtga
gattccccca 361 ctcctcctgc tctcctgtag tectacatat ataaatagcg
ttatgactct tgtggcagat 421 gccttttatg gaggcatcaa ttttttactt
accttgctat cctatggctg catcattgcc 481 agcatcctgc gcatgcgttc
tgctgagggc aagaggaagg ccttttctac ctgctcatcc 541 cacctcatcg
tggtctctgt gtactactca tctgtgttct gtgcctatat cagtcctggt 601
tccagctaca gcccagaaag aagcaaattt acctcggttt tgtactcagt cctcagccca
661 accctcaac with a deduced amino acid sequence
CNLATMDIICTSSVLPKALVGLLSEENTIPFKGCMTQLFFLVWS (SEQ ID NO:42)
GSSELLLLTVMAYDRYVAICLPLHYSSRMSPQLCGTFAVGVWSICALNASINTGLMTR
LSFCGPKVITHFFCEIPPLLLLSCSPTYINSVMTLVADAFYGGINFLLTLLSYGCIIA
SILRMRSAEGKRKAFSTCSSHLIVVSVYYSSVFCAYISPGSSYSPERSKFTSVLYSVL SPTLN
(q) 1 gccaacctct ccagtgtcga cattagtgct ccatctgtca ttgtccccaa
ggcattggtg (SEQ ID NO:43) 61 aatcatatgt tgggaagcaa gtccatctct
tacacggggt gtatgaccca gatctatttc 121 ttcatcacat tcaacaatat
ggatggcttc ctcctgagtg tgatggccta tgaccgctat 181 gtggccatct
gtcaccctct ccactacacc atgatgatga gacccagact ctgtgtcctc 241
ctggtggcca tatcatgggc catcacaaac ctgcatgctc tcttgcatac tctcctcatg
301 gttcgactca ccttctgttc ccacaatgca gtgcaccact tcttctgtga
cccctaccct 361 atcctgaagc tctcttgttc tgacaccttc atcaatgacc
tgatggtctt caccattggt 421 ggattggtat ttatgactcc atttacatgc
attattgttt cctatgccta catcttctct 481 aaggttctga agttaaaatc
tgcccatgga ataaggaaag ccctgtcgac gtgtgggtct 541 cacctcactg
tggtctccct cttctatggg gcgatcctgg gcatctatat gcacccttca 601
tctacataca cagtgcagga cacagtggcc accgtcatct tcacagtagt gacacccatg
661 gtcaac accctcaac with a deduced amino acid sequence
ANLSSVDISAPSVIVPKALVNHMLGSKSISYTGCMTQIYFFITF (SEQ ID NO:44)
NNMDGFLLSVMAYDRYVAICHPLHYTMMMRPRLCVLLVAISWAITNLHALLHTLLMVR
LTFCSHNAVHHFFCDPYPILKLSCSDTEINDLMVFTIGGLVFMTPETCIIVSYAYIFS
KVLKLKSAHGIRKALSTCGSHLTVVSLFYGAILGIYMHPSSTYTVQDTVATVIFTVVT PMVN (r)
1 agtcacttgg ccttcacgga catctctttc tcatctgtca cagctccaaa gatgctcatg
(SEQ ID NO:45) 61 aatatgctga cacatagcca atccatctca catgctgggt
gtgtttccca aatatatttt 121 ttcttattgt ttgggtgtat tgacaacttc
cttctgactt ccatggccta tgacaggtat 181 gtggccatct gccaccctct
gcattatacc actatcatga gtcaaagcct ctgtgttctg 241 ctagtgatgg
tgtcctgggc attttcctct tctaatggcc ttgtgcatac tcttctcttt 301
gctcgtctct ctctttttag agacaacact gtccaccatt ttttctgtga tctctctgct
361 ttgctgaagc tgtccagctc agacactact atcaatgaac tagtaatcct
cactttagca 421 gtggtggtca tcactgtacc attcatatgc atcctggttt
cttatggcca catgggggcc 481 actatcctaa gaactccatc catcaagggt
atctgcaaag ccttgtccac atgtggttct 541 catctctgtg tagtttcttt
atattatgga gccattattg ggttatattt tttcccctcc 601 tccaataata
ctaatgataa agatgtcata gtagctgtgt tgtacactgt ggttacaccc 661
atgctgaat accctcaac with a deduced amino acid sequence
SHLAFTDISFSSVTAPKMLMNMLTHSQSISHAGCVSQIYFFLLF (SEQ ID NO:46)
GCIDNFLLTSMAYDRYVAICHPLHYTTIMSQSLCVLLVMVSWAFSSSNGLVHTLLFAR
LSLFRDNTVHHFFCDLSALLKLSSSDTTINELVILTLAVVVITVPFICILVSYGHMGA
TILRTPSIKGICKALSTCGSHLCVVSLYYGAIIGLYFFPSSNNTNDKDVIVAVLYTVV TPMLN
(s) 1 atggcgaaca gcactactgt tactgagttt attttgctgg ggctgtcaga
tgcctgtgag (SEQ ID NO:47) 61 ctgcaggtgc tcatattcct gggctttctc
ctgacctact tcctcattct gctgggaaac 121 ttcctcatca tcttcatcac
ccttgtggac aggcgccttt acacccccat gtattacttc 181 ctccgcaact
ttgccatgct ggagatctgg ttcacctctg tcatcttccc caagatgcta 241
accaacatca tcacaggaca taagaccatc tccctactag gttgtttcct ccaagcattc
301 ctctatttct tccttggcac cactgagttc tttctactgg cagtgatgtc
ctttgacagg 361 tatgtggcca tttgtaaccc tttgcgttat gccaccatta
tgagcaaaag agtctgtgtc 421 cagcttgtgt tttgctcatg gatgtctgga
ttgcttctca tcatagttcc tagttcaatt 481 gtatttcagc agccattctg
tggcccaaac atcattaatc atttcttctg tgacaacttt 541 ccacttatgg
aactcatatg tgcagatact agcctggtag agttcctggg ttttgttatt 601
gccaatttca gcctcctggg cactctggct gtgactgcca cctgctatgg ccacattctc
661 tataccattc tacacattcc ttcagccaag gagaggaaga aagccttctc
aacttgctcc 721 tctcatatta ttgtggtgtc tctcttctac ggcagctgta
tcttcatgta tgtccggtct 781 ggcaagaatg gacaggggga ggatcataac
aaggtggtgg cattgctcaa cactgtagtg 841 acacccacac tcaacccctt
catctacact ctgaggaaca agcaggtgaa gcaggtattt 901 agggaacacg
taagcaagtt ccaaaagttc agccagacgt gaaccctcaac with a deduced amino
acid sequence MANSTTVTEFILLGLSDACELQVLIFLGFLLTYFLILLGNFLII (SEQ ID
NO:48) FITLVDRRLYTPMYYFLRNFAMLEIWFTSVIFPKMLTNIITGHKTISLLGCFLQAFLY
FFLGTTEFFLLAVMSFDRYVAICNPLRYATIMSKRVCVQLVFCSWMSGLLLIIVPSSI
VFQQPFCGPNIINHFFCDNFPLMELICADTSLVEFLGFVIANFSLLGTLAVTATCYGH
ILYTILHIPSAKERKKAFSTCSSHIIVVSLFYGSCIFMYVRSGKNGQGEDHNKVVALL
NTVVTPTLNPFIYTLRNKQVKQVFREHVSKFQKFSQT
[0118] Although each insert shared some sequence homology of
previously characterized olfactory receptors, the sequenced
receptors were all new members of the olfactory receptor family and
were distributed broadly (shown in bold-type in FIG. 1B) across a
similarity dendrogram. Also depicted in FIG. 1B are ten previously
cloned olfactory receptors (see, e.g., Buck (1991) Cell
65:175-187), shown in italics in FIG. 1B, designated I3, I8, I14,
I15, I9, F5, F3, F12, F6, and I7. Thus, the arrayed receptor
plasmid inserts represented a diverse library of olfactory receptor
sequences amenable to expression studies, described below.
[0119] Chimeric Vector Construction
[0120] PCR products were digested with PstI and BspEI restriction
enzymes before size fractionation, purification and ligation into
the pCMV-Rho/M4.sub.NC vector (see FIG. 1A). The vector ligation
products were transformed into E. coli and 480 clones were placed
in 96-well plates. PCR screening revealed that >95% of the
clones carried inserts of the expected size. Pools of cells from a
single column of the plates (8 wells) were grown in a 50 ml culture
and plasmid DNA prepared. Insert-containing vectors containing: the
5'-untranslated region of the rhodopsin gene, which included its
coding region for the initiation methionine and the next 19
residues; joined to a full-length cDNA for a mouse olfactory
receptor (M4 or I-C6), under the control of the CMV promoter, were
also prepared. The full-length coding region of olfactory receptors
mI7 and I-C6 were obtained by screening a mouse (129 SV/J) genomic
phage (.lambda.FIX-II) library (2.times.10.sup.6 independent
clones) using .sup.35P-labeled DNA fragments (of TMII through VII
sequence) of the respective receptors under stringent conditions
(hybridized at 0.2.times. SSC at 65.degree. C.). DNA fragments
encoding the full-length receptors were cloned into pBluescript
(Stratagene) and sequenced.
[0121] Culture and Transient Transfection of Human Cells Expressing
Olfactory Receptor
[0122] HEK-293 cells (obtained from the ATCC) were grown in DMEM
supplemented with 10% fetal bovine serum, penicillin (100U/ml),
streptomycin (100 .mu.g/ml) and L-glutamine (2 mM) in 5% CO.sub.2.
Before transfection, the cells were seeded onto
poly-L-lysine-coated 10.5.times.35.times.0.17 mm glass coverslips
(Bellco) placed in the 60 mm culture dishes. Calcium
phosphate-mediated transfections were performed in a 60 mm dish
with 3 to 4 .mu.g of receptor construct DNA, 1 .mu.g of pCIS
G.alpha.15 and G.alpha.16 expression vector (Offermanns (1995)
supra). 2 .mu.g of pBluescript carrier DNA, and 0.3 .mu.g of pRSV-T
antigen expression vector (Gorman (1990) DNA and Protein Eng. Tech.
2:3-9). After 5 to 7 hr incubation, the cells were washed once with
PBS containing 0.5 mM EDTA and 10% DMSO, then with PBS before
continuing growth in regular media for 40-50 hr.
[0123] Expression of Receptors on the Cell Surface for Functional
Ligand-Binding Assays
[0124] Efficient screening of expressed olfactory receptors with a
large number of ligands by functional analysis requires a robust
and sensitive assay system. Although the established role of cAMP
in olfactory signaling offers a biochemical approach involving
measurement of cAMP production in response to odorant stimulation,
an alternative, rapid assay is to co-express the cloned olfactory
receptors with G protein G.alpha..sub.15,16 subunits (see, e.g.,
Offermanns (1995) J. Biol. Chem. 270:15175-15180), which can
promiscuously couple 7-transmembrane domain receptors that normally
signal through other second messengers to the PIP.sub.2 pathway. In
this reporter system, (olfactory) receptor activation leads to the
generation of an IP.sub.3-mediated increase in intracellular
Ca.sup.2+, which can be measured at the single-cell level with high
sensitivity and good temporal resolution using the dye FURA-2 and
radiofluorometric imaging. These attributes were able to compensate
for the low transfection efficiency in transient expression systems
that would hinder more traditional biochemical assays.
[0125] A construct with the TM II-VII region from the
.beta..sub.2-adrenergic receptor inserted in the pCMV-Rho/M4.sub.NC
vector (Rho/M4.sub.NC-.beta..sub.2 TM II-VII) was co-transfected
with G.alpha..sub.15,16 into HEK-293 cells. Immunocyto-chemical
localization of vector-encoded, newly translated polypeptide with a
B6-30 antibody against the rhodopsin tag (directed against the
N-terminal 15 residues of rhodopsin, see Hargrave (1986 Exp. Eye
Res. 42:363-373) was performed. Transfected HEK 293 cells were air
dried and fixed in ice-cold methanol for 10 min. The fixed cells
were blocked with 1.5% goat serum in PBS for 30 minutes and then
incubated for 1 hour in PBS containing 0.03% goat serum and a
1:1000 dilution of the B6-30. After washing with PBS, a
FITC-coupled, polyclonal anti-mouse antibody (Vector) was used to
visualize the rhodopsin-tagged protein. Images of fluorescent cells
were obtained on a Zeiss 510 confocal microscope with excitation at
488 nm. Results of the localization experiments indicated that a
significant portion of the expressed protein appeared to be
localized to the plasma membrane (10% or more of total expressed
protein). These results demonstrate that the rhodopsin
N-terminus-derived "translocation domain" of the invention, when
expressed in the chimeric receptors, was the cause of the efficient
translocation of the chimeric receptor molecules to the plasma
membrane.
[0126] These transfected cells were then tested for their ability
to functionally respond to ligand-receptor binding. The ligand, the
adrenergic agonist isoproterenol, was "bath" applied to the
transfected cells and single cell Ca.sup.2+-imaging was performed.
Cells were pre-loaded with the Ca.sup.2+-sensitive fluorescent dye
FURA-2 AM (Molecular Probes) by bathing in serum-free DMEM
containing 4 .mu.M of the membrane permeant chemical for 1 hr at
37.degree. C., then washed with a standard bath solution (130 mM
NaCl, 2 mM CaCl.sub.2, 5 mM KCl, 10 mM glucose, 10 mM Na.HEPES/pH
7.4 at room temperature). For each experiment, a glass coverslip
with FURA-2 loaded HEK 293 cells was introduced into an
open-topped, longitudinal microperfusion chamber (300 .mu.l bath
volume mounted on a Zeiss Axiovert 135 microscope equipped with an
F Fluar 40.times./1.30 oil-immersion lens. The cells were
superfused with test solutions typically for 30 to 40 seconds (5
ml/application) and washed out with 5 ml of bath solution at the
end of each application. Each test solution was freshly diluted and
manually applied with a micropipette into the chamber. Because of
this manual procedure, there could be several seconds of delay in
actual application from electronic tick marks used to define the
beginning of application in each graph. At the same time, the
solution flow might not be completely laminar. In most cases, the
onset of Ca2+ rise in response to a specific solution occurred
within 15 seconds of the beginning of solution application, though
longer delays were sometimes observed. Acetylcholine was applied at
the end of each experiment at 10 .mu.M for 15-20 seconds.
Ratiometric Ca.sup.2+ measurements were performed as described by
Grynkiewicz (1985) 260:3440-3450, with modifications using the
Zeiss/Attofluor-Ratiovision imaging system. At 5-second intervals,
the cells were sequentially illuminated for less than 100 ms, first
at 340 nm and then at 380 nm. Fluorescence emission at 510 nm was
monitored for each excitation wavelength via an intensified CCD
camera. Averaged pixel intensities within 40 to 100 regions of
interest, corresponding to 40 to 100 individual cells, were
digitized and stored on a computer. Attofluor-Ratiovision software
(Atto Instruments) was used to determine the Ca.sup.2+-dependent
fluorescence signal expressed as the F.sub.340/F.sub.380 ratio.
Signals from all responding cells, or all cells (negative controls)
were averaged and displayed as a function of time.
[0127] Isoproterenol bath application resulted in a transient
increase in intracellular Ca.sup.2+ in the transfected cells. The
Ca.sup.2+ transient induced by isoproterenol was dependent on
cotransfection with the G.alpha..sub.15,16 subunits. Cells
transfected with the G protein subunits alone produced a small
response to isoproterenol, presumably due to some endogenous
.beta.-adrenergic receptors on their surface. However, odorants
such as heptanal (7-al) and octanal (8-al) had no effect.
[0128] A second application of isoproterenol frequently failed to
elicit a response, possibly suggesting a rapid desensitization of
the G.alpha..sub.15,16-mediated signal transduction pathway.
Although its mechanism is unclear, this rapid desensitization was a
frequent observation with this expression system. HEK-293 cells
have intrinsic muscarinic receptors coupled to the PIP.sub.2
pathway via endogenous G proteins. The rise in intracellular
Ca.sup.2+ upon activation of this pathway by bath-applied
acetylcholine (10 .mu.M) served as a control in this system.
[0129] As a second test example, a Rho/M4.sub.NC-ratI7 TM II-VII
chimeric construct was generated and co-expressed with
G.alpha..sub.15,16 in HEK-293 cells. A Ca.sup.2+ transient was
observed in the transfected cells in response to 10 .mu.M octanal.
The transfected cell responded to 30 .mu.M, but not 10 .mu.M, of
heptanal (a shorter aldehyde than octanal). The response to octanal
also required the presence of G.alpha..sub.15,16.
[0130] As with the .beta..sub.2-adrenergic receptor,
desensitization often occurred after a positive response. For
example, little or no effect was observed upon a second application
of octanal, even at 30 .mu.M. A similar response profile was
obtained with a construct in which the translocation domain of the
invention (rhodopsin N-terminus) was fused to the full-length rat
I7 odorant/ligand region encoding sequence. This chimeric receptor
responded to octanal even at 1 .mu.M. The ligand specificity was
not absolute; a small response was also observed to 30 .mu.M
heptanal (similar to an in vivo finding by Zhao (1998) Science
279:237-242). Sometimes, the delay between the start of odorant
application and the beginning of Ca.sup.2+ rise could be more than
30 seconds (e.g., the first response to octanal). The reason for
this relatively long delay is unknown, but it could have arisen
from a non-linear, thresholding mechanism. Additional experiments
in which successive applications of two odorants were separated by
periods as long as 5 minutes, however, removed any possible
confusion with respect to which odorant triggered a given
response.
[0131] The above results validate the HEK-293 cell expression of
cloned olfactory receptor sequences as a screening system for
identifying unknown odorants. They also demonstrate that
odorant/ligands are binding to the 7-transmembrane domain region TM
II-VII of an olfactory receptor to produce a physiologic response
(in these experiments, measured by Ca.sup.2+ transients).
[0132] Identification of Cognate Ligand-Receptor Pairs for the
Cloned Receptor Library
[0133] The 7-transmembrane domain region TM II-VII expressing
vector libraries of the invention were expressed in this cell
expression system. Various odorant were screened for their ability
to generate a physiologic response in the form of a calcium
transient, as above. Eighty plasmid clones arrayed in microtiter
plates were pooled into 10 groups of eight constructs each, and
co-transfected with G.alpha..sub.15,16 into HEK-293 cells. After
pre-loading with FURA-2, the transfected cells were screened
sequentially against each of 26 odorants: Hedione, (-) carvone, (+)
carvone, (+) citronellal, (-) citronellal,
2-methyl-4-propyl-1,3-oxalthia- ne, methylsalicylate, pyrrolidine,
quinoleine, lyral, cyclohexanone, acetophenone,
2-methoxy-3-methyl-pyrazine, pyrazine, 2-methoxypyrazine,
isovalieric acid, isobutyric acid, triethylamine, citralva, (+)
limonene, 6-aldehyde, 7-aldehyde, 8-aldehyde, 9-aldehyde,
10-aldehyde, and 11-aldehyde (Firmenich, S. A., Princeton, N.J.).
The odorants were stored under nitrogen. Stock solutions of the
odorants were made up fresh each day in DMSO and diluted 1000-fold
into the standard bath solution to give the indicated
concentrations approximately 10 seconds before application in a
given experiment.
[0134] All of the (twenty-six) odorants were applied at 10 .mu.M to
induce a Ca.sup.2+ response as described previously. Three sample
"pools" (a mixture of clones) produced transient increases in
Ca.sup.2+ in response to the application of (-) carvone, (-)
citronellal and (+) limonene, respectively. The lack of response of
one pool to (+) carvone could reflect desensitization resulting
from the positive response to (-) carvone occurring immediately
before, or, alternatively, a stereo-specificity in ligand
recognition. This desensitization could also have obscured the
response to subsequent odorant applications; nonetheless, a second
response to (-) carvone could still be elicited. The absence of
response to (+) citronellal for another pool apparently results
from a genuine stereo-specificity in ligand recognition, because
there was no prior positive response that would lead to
desensitization. The lack of responses to the subsequent odorants
was confirmed by additional experiments with the same set of
odorants but (-) where citronellal was applied last.
[0135] Next, 8 individual clones from each of these three tested
pools were isolated and tested for their ability to encode receptor
binding domains with specificity for the odorants identified above.
Three responsive chimeric olfactory receptors were isolated; they
were designated I-D3 (carvone), I-C6 (citronellal) and I-G7
(limonene). Further experiments indicated that the I-D3 receptor
was responsive to both (+) and (-) carvone). The I-C6 receptor
appeared to be selective for the (-) stereoisomer of citronellal.
Finally, the I-G7 receptor responded to both (+) and (-) limonene
at the same concentration of 10 .mu.M, though perhaps not as well
to the (-) isomer. For each of the three isolates, control
experiments indicated that the specific responses required the
presence of G.alpha..sub.15,16 (as discussed above).
[0136] To determine if these physiologic responses were caused by
ligand interaction with a full-length 7-membrane receptor, a
genomic clone of the entire I-C6 receptor coding sequence was
isolated and used to make a chimeric molecule incorporating the
tranlocation domain of the invention (the "rhodopsin tag"
sequence). The full-length I-C6 receptor retained the same
stereo-selectivity as a chimeric receptor construct whose only I-C6
sequence was the transmembrane domains II through VII (i.e., the
odorant/ligand binding domain). Both recombinantly expressed
receptors preferred the (-) isomer of citronellal; it also showed
high sensitivity, responding to this chemical even at 1 .mu.M. The
stereo-specificity was not absolute, however, in that (+)
citronellal was also able to elicit a response when applied at 30
.mu.M and 100 .mu.M. By comparison, carvone and limonene elicited
no responses from this receptor even at 100 .mu.M. Five
structurally related compounds besides (-) and (+) citronellal were
also tested (+/- citral, (-) citronellyl bromide, (-) citral
demethyl acetal, (-) citronellic acid and (-) citronellol), all at
30 .mu.M. Among these, only 30 .mu.M (-) citronellyl bromide
elicited a small response. This compound differs from (-)
citronellal by the substitution of a bromine for the oxygen atom in
the aldehyde functional group. The lack of response to (-)
citronellal may be due to desensitization resulting from the
positive response to 30 .mu.M (+) citronellal immediately before.
Finally, in control experiments lacking G.alpha..sub.15,16, no
response was observed to either (-) citronellal or (-) citronellyl
bromide (FIG. 5C). Although these experiments do not quantitate
ligand affinities, they provide a qualitative rank order of potency
for binding and activating the I-C6 receptor:
(-)citronellal>(+)citronellal, citronellyl bromide>28 other
odorants.
[0137] Analysis of Individual Amino Acid Residues on
Receptor-Odorant Binding Specificities
[0138] To establish the functional expression of mouse olfactory
receptors, a Rho/M4.sub.NC-mouse I7 transmembrane II-VII chimeric
receptor was constructed and examined its responsiveness to several
n-aliphatic aldehydes and alcohols. At 10 .mu.M concentrations of
these odorants, the mouse receptor responded only to heptanal. As
discussed above, the rat I7 chimeric receptor responded better to
octanal than to heptanal in identical experiments. This difference
in odorant selectivity was retained by the full-length clones of
the two receptors fused to the translocation domain of the
invention (the rhodopsin tag). The rat and mouse I7 receptors
differ in altogether 15 amino-acid residues, three of which
(K.sub.90E in the 1.sup.st extracellular loop, V.sub.206I in TM V,
and F.sub.290L in TM VII) reside between transmembrane domains II
and VII.
[0139] In light of the critical role of residues in transmembrane V
for ligand binding in the .beta..sub.2-adrenergic receptor, the
role of residue 206 in differential ligand recognition was
examined. Reciprocal valine/isoleucine substitutions were made in
the full-length rat and mouse I7 receptor sequences. These
substitutions were able to switch the ligand preferences of the two
receptors, namely, making the rat I7 receptor preferentially
recognize heptanal and the mouse receptor preferentially recognize
octanal. Interestingly, the nature of these changes, isoleucine
versus valine and heptanal versus octanal, is consistent with
compensatory alterations in the structures of ligand and receptor
that preserve the complementarity between the two. These
observations provide strong evidence for a direct role of residue
206 in the interaction between the I7 receptor and aliphatic
aldehydes. These results also demonstrate that the compositions and
methods of the invention can be used to analyze
odorant/ligand-olfactory receptor interactions on a molecular
level.
[0140] Summary
[0141] The few studies carried out previously on identifying
cognate odorant-olfactory receptor pairs have generally focused on
a single receptor and examined its responsiveness to a large number
of odorants or odorant mixtures. The present invention provides the
means to take a different approach by generating olfactory receptor
libraries to use in the screening of a large number of cloned
receptors simultaneously against a large panel of individual
odorants. In this way, the problem of poor expression, inefficient
folding or weak coupling to second-messenger systems associated
with certain receptors in a heterologous system is avoided.
Moreover, screening multiple receptors against multiple odorants,
greatly increases the probability of identifying responsive
combinations of receptors and odorants. Finally, the apparent
diversity of the receptor sequences should further enhance the scan
of the odor space. The above-described experiments screened 80
clones (not counting the I7 receptor) against 26 odorants. Because
a given odorant should be recognized by at least one member of,
say, a total of 1000 receptors, the chance of encountering an
odorant that is a cognate ligand to 80 receptors should, on
average, be 8% (=80/1000), or 2 positives in a pool of 26 odorants.
This number is close to the number (3) identified experimentally
herein. The receptor library generated with a single pair of
degenerate primers of the invention (the TM II to TM VII amplifying
pair) encompasses a broad range of the olfactory receptor family.
Several hundred distinct sequences are represented in this
exemplary library of the invention.
[0142] The addition of translocation domains of the invention (the
first twenty amino-acid residues of a rhodopsin N-terminal segment,
with some exemplary domains also consisting of a 5'-untranslated
rhodopsin region) to the chimeric olfactory receptors of the
invention facilitated their plasma membrane localization. This
included the full-length I-C6 receptor, where the inclusion of the
translocation domain was necessary in order to observe a response
to (-) citronellal. The different translocation domains of the
invention may be aiding in the translocation process in different
ways; however, the invention is not limited by what structural
contribution may be played by the translocation domain to the newly
translated protein's translocation process.
* * * * *
References