U.S. patent application number 11/759741 was filed with the patent office on 2008-12-04 for olfactory receptors for isolvaleric acid and related malodorants and use thereof in assays for identification of blockers.
This patent application is currently assigned to Senomyx, Inc.. Invention is credited to Fernando Echeverri, Yi Han, Kun Wang, Sergey Zozulya.
Application Number | 20080299586 11/759741 |
Document ID | / |
Family ID | 29273615 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299586 |
Kind Code |
A1 |
Han; Yi ; et al. |
December 4, 2008 |
OLFACTORY RECEPTORS FOR ISOLVALERIC ACID AND RELATED MALODORANTS
AND USE THEREOF IN ASSAYS FOR IDENTIFICATION OF BLOCKERS
Abstract
A subgenus of olfactory receptors (ORs) that are activated by
isovaleric acid (IVA) are identified as well as assays that utilize
one or more of these ORs. These assays are useful for identifying
potential anti-odorants which may be used in deodorants, air and
carpet fresheners, fabric deodorizers, and other compositions for
camouflaging odor attributable to IVA and related carboxylic
acids.
Inventors: |
Han; Yi; (San Diego, CA)
; Zozulya; Sergey; (San Diego, CA) ; Echeverri;
Fernando; (Chula Vista, CA) ; Wang; Kun; (San
Diego, CA) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W., SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
Senomyx, Inc.
San Diego
CA
|
Family ID: |
29273615 |
Appl. No.: |
11/759741 |
Filed: |
June 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10300846 |
Nov 21, 2002 |
7344845 |
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11759741 |
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60341872 |
Dec 21, 2001 |
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60348371 |
Jan 16, 2002 |
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Current U.S.
Class: |
435/7.21 ;
435/358; 435/365; 435/367; 435/369; 436/501 |
Current CPC
Class: |
G01N 33/502 20130101;
C12N 2510/00 20130101; G01N 2333/4719 20130101; G01N 33/566
20130101; G01N 33/5008 20130101; G01N 33/6893 20130101; C07K 14/705
20130101 |
Class at
Publication: |
435/7.21 ;
436/501; 435/365; 435/367; 435/369; 435/358 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/566 20060101 G01N033/566; C12N 5/06 20060101
C12N005/06 |
Claims
1-58. (canceled)
59. A method of screening for a compound that putatively blocks
isovaleric acid associated malodor comprising: (i) contacting an
isovaleric acid olfactory receptor polypeptide that possesses at
least 95% sequence identity to the polypeptide contained in SEQ ID
NO:24 with at least one compound; (ii) assaying whether said at
least one compound inhibits the binding of said olfactory receptor
polypeptide to isovaleric acid; and (iii) identifying a compound
that putatively blocks isovaleric associated malodor if it inhibits
the said binding of said olfactory receptor polypeptide to
isovaleric acid.
60. The screening method of claim 59 wherein said identified
compound is assessed in a smell test to determine whether said
identified compound blocks or inhibits isovaleric acid associated
malodor.
61. The screening method of claim 59 wherein the olfactory receptor
polypeptide has the sequence contained in SEQ ID NO:24.
62. The method of claim 59 wherein said olfactory receptor
polypeptide is expressed by a cell that also expresses a G
protein.
63. The method of claim 62 wherein the G protein is Galpha15 or
Galpha16.
64. The screening method of claim 62 wherein the cell is selected
from a HEK-293, COS, and a CHO cell.
65. The screening method of claim 62 wherein the cell is bound to a
solid phase.
66. A method of identifying a compound that putatively modulates
isovaleric acid associated malodor comprising: (i) contacting a
cell line that expresses an isovaleric acid receptor polypeptide
that has a sequence that is at least 95% identical to SEQ ID NO:24
with at least one compound; (ii) screening for compounds that block
or inhibit the activity of said olfactory receptor polypeptide; and
(iii) identifying a compound that putatively modulates isovaleric
acid associated malodor if it blocks or inhibits the activity of
said isovaleric acid receptor polypeptide.
67. The method of claim 66 wherein the receptor is expressed by a
cell that additionally expresses a G protein.
68. The method of claim 66 wherein said cell is selected from a
Xenopus oocyte, CHO cell, HEK-293 cell and a HeLa cell.
69. The method of claim 67 wherein the G protein is Galpha15 or
Galpha16.
70. The method of claim 66 wherein said receptor polypeptide has
the sequence contained in SEQ ID NO:24.
71. The method of claim 66 wherein the screening assay selects for
compounds that affect receptor internalization.
70. The method of claim 66 wherein the screening assay selects for
compounds that affect receptor phosphorylation.
71. The method of claim 66 wherein the screening assay selects for
compounds that affect arrestin translocation.
72. The method of claim 66 wherein the assay screens for compounds
that affect G protein activation of said receptor.
73. The method of claim 66 wherein the screening assay selects for
compounds that affect the conformation of said olfactory receptor
polypeptide.
74. The method of claim 73 wherein the screening assay selects for
compounds that alter the susceptibility of said receptor
polypeptide to proteolysis.
75. The method of claim 73 wherein the screening assay detects any
change in conformation using NMR spectroscopy.
76. The method of claim 73 wherein the screening assay detects any
conformational change by fluorescence spectroscopy.
77. The method of claim 66 which screens for compounds that
displace binding of a radioactively or fluorescently labeled ligand
to said olfactory receptor polypeptide.
78. The method of claim 77 which detects the displacement of said
labeled compound by fluorescence polarization or FRET assay.
79. The method of claim 66 which is a high throughput screening
assay.
80. The method of claim 66 wherein the receptor is attached to a
peptide that facilitates surface expression.
81. The method of claim 66 wherein a gene encoding the receptor is
operably linked to a reporter gene.
82. The method of claim 81 wherein the reporter is luciferase, beta
galactosidase, alkaline phosphatase or beta lactamase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application relates to PCT WO 01/68805 A2 and U.S. Ser.
No. 09/809,291, both by Zozulya et al., having a filing date of
Mar. 13, 2001, and both entitled "Human Olfactory Receptors and
Genes Encoding Same." These applications disclose the amino acid
and nucleic acid sequences for a genus of human olfactory receptors
which includes some of the isovaleric acid olfactory receptors
which are the subject of this application. This application claims
priority to U.S. Provisional Application Ser. No. 60/341,872 filed
Dec. 21, 2001 and U.S. Provisional Application Ser. No. 60/348, 371
filed Jan. 16, 2002 both of which are incorporated by reference in
their entirety herein.
BACKGROUND OF INVENTION
[0002] 1. Field of Invention
[0003] The present invention relates to the elucidation of the
isovaleric acid odorant specificity of a number of previously
reported odorant receptors, the specificity of which were
previously not known, and the use of this information in the design
of assays for identifying compounds that mimic, block, modulate
and/or enhance the activity of isovaleric acid odorant receptors.
More specifically the invention relates to the discovery that a
group of murine olfactory receptors (identified as mOR IVA A
through mOR IVA L infra) are activated by isovaleric acid and that
these murine olfactory receptors have counterparts in the human
olfactory receptor repertoire (identified as OR19-04.05 (AOLFR262),
OR19.04.11 (AOLF284 OR09.05.02 (AOLFR297), OR17.03.01 (AOLFR252),
OR11.22.02 (AOLFR108), OR06.02.04 (AOLFR269), OR11.49.11
(AOLFR058), OR11.49.10 (AOLFR022), OR11.39.06 (AOLFR074),
OR11.38.05 (AOLFR346), and OR03.01.01 (AOLFR340 infra. These
receptors and cell lines expressing these receptors or chimeras,
variants, and fragments thereof have application in assays,
especially high throughput assays, for identifying compounds that
activate, block, mimic and/or modulate the activity of isovaleric
acid receptors.
[0004] 2. Description of the Related Art
[0005] The olfactory system provides sensory information about the
chemical composition of the external world. Olfactory sensation is
thought to involve distinct signaling pathways. These pathways are
believed to be mediated by olfactory receptors (ORs). Cells which
express olfactory receptors, when exposed to certain chemical
stimuli, elicit olfactory sensation by depolarizing to generate an
action potential, which is believed to trigger the sensation.
[0006] As such, olfactory receptors specifically recognize
molecules that elicit specific olfactory sensation. These molecules
are also referred to herein as "odorants." Olfactory receptors
belong to the 7-transmembrane receptor superfamily (Buck et al.,
Cell 65:175-87 (1991)), which are also known as G protein coupled
receptors (GPCRs). G protein-coupled receptors mediate
transmembrane signaling which controls many physiological
functions, such as endocrine function, exocrine function, heart
rate, lipolysis, carbohydrate metabolism, neurotransmission vision
and taste perception. The biochemical analysis and molecular
cloning of a number of such receptors has revealed many basic
principles regarding the function of these receptors.
[0007] Genes encoding the olfactory receptors are active primarily
in olfactory neurons (Axel, Sci. Amer., 273:154-59 (1995)).
Individual olfactory receptor types are expressed in subsets of
cells distributed in distinct zones of the olfactory epithelium
(Breer, Semin. Cell Biol., 5:25-32 (1994)). The human genome
contains approximately one thousand genes and pseudogenes that
encode a diverse repertoire of olfactory receptors (Mombaerts,
Annu. Rev. Genomics Hum. Genet., 2:493-510 (2001), Glusman et al.,
Genome Res., 11(5):685-702 (2001); Zozulya et al., Genome Biol.,
2(6):0018 (2001)). It has been demonstrated that members of the OR
gene family are distributed on all but a few human chromosomes.
Through fluorescence in situ hybridization analysis, Rouquier
showed that OR sequences reside at more than 25 locations in the
human genome. Rouquier also determined that the human genome has
accumulated a striking number of dysfunctional OR copies: 72% of
the analyzed sequences were found to be pseudogenes. An
understanding of an animal's ability to detect and discriminate
among the thousand of distinct odorants or tastants, and more
particularly to distinguish, for example beneficial tastants or
odorants from toxic tastants or odorants, is complicated by the
fact that chemosensory receptors belong to a multigene family with
over a thousand members. For instance, there are up to 1,000
odorant receptors in mammals.
[0008] Moreover, each chemosensory receptor neuron may express only
one or a few of these receptors. With respect to odorant receptors,
any given olfactory neuron can respond to a small set of odorant
ligands. In addition, odorant discrimination for a given neuron may
depend on the ligand specificity of the one or few receptors it
expresses. To analyze odorant-receptor interactions and their
effects on olfactory cells, specific ligands and the olfactory
receptors to which they bind are identified. This analysis requires
isolation and expression of olfactory polypeptides, followed by
binding assays.
[0009] Some studies suggest that OR genes can be expressed in
tissues other that the olfactory epithelium, indicating potential
alternative biological roles for this class of chemosensory
receptors. Expression of various ORs has been reported in human and
murine erythroid cells (Feingold 1999), developing rat heart
(Drutel, Receptor Channels, 3(1):33-40 (1995)), avian notochord
(Nef, PNAS, 94(9):4766-7 (1997)) and lingual epithelium (Abe, FES
Letl., 316(3):253-56 (1993)). One well experimentally documented
case also established the existence of a large subset of mammalian
ORs transcribed in testes and expressed on the surface of mature
spermatozoa, thereby suggesting a possible role of ORs in sperm
chemotaxis (Parmenthier, Nature, 355:453-55 (1992); Walensky, Mol.
Med., 1(2):130-41 (1998 Branscomb, Genetics, 156(2):785-97 (2000)).
It was also hypothesized that olfactory receptors might provide
molecular codes for highly specific cell-cell recognition functions
in development and embryogenesis (Dreyer, PNAS, 95(11):9072-77
(1998)).
[0010] The elucidation of a family of receptors encoded in the
human genome which comprise over two hundred and fifty G-protein
coupled receptors that are involved in the perception of odorants
and human smell is disclosed in Senomyx PCT WO 01/68805 A2, having
an international filing date of Mar. 13, 2001, entitle "Human
Olfactory Receptors and Genes Encoding Same" by Zozulya, S., and
U.S. Ser. No. 09/809,291 also by Zozulya, Sergey also filed on Mar.
13, 2001. Additionally, Zozulya et al. recently disclosed in Genome
Biology a repertoire of human olfactory receptors (Zozulya et al.,
"The Human Olfactory Receptor Repertoire", Genome Biology
2(6):research 0018.1-0018.12 (2001)). Both of these patent
applications and this publication by Zozulya are incorporated by
reference in their entirety herein.
[0011] The discovery encompassed by these patent applications and
publication is enormous as it provides a large genus of olfactory
receptors that may be utilized in screens to identify odorant
molecules that activate one or more of these receptors. These
molecules have application, e.g. in cosmetics and compositions for
consumptions. However, these patent applications fail to provide
the odorant specificity of many of these receptors. Particularly,
these patent applications fail to identify whether the genus of
odorant receptors disclosed therein includes receptors having
specificity for isovaleric acid or related carboxylic acid
odorants.
[0012] With respect thereto, it is generally known that the malodor
of human and animal bodily secretions, particularly sweat, is
partially attributable to the production of several unpleasant
smelling organic acids, including particularly isovaleric acid and
3-methyl-2-hexenoic acid (constituents of human sweat), propionic
acid (constituent of foul malodor) and hexenoic acid (a constituent
of pet malodor).
[0013] In order to alleviate such odors, numerous types of
anti-odorant compositions have been developed, and are available in
various forms including deodorants, air fresheners, fabric
fresheners, carpet fresheners, among others. These compositions
contain a variety of active ingredients intended to alleviate
malodor. Some of these constituents function by camouflaging the
malodor by substituting a more pleasant smell, e.g. a perfume. Some
others function by blocking the malodor by a phenomenon known as
cross adaptation with the malodorance constituent.
[0014] The existing technology for developing anti-odorants is
based on psychophysical testing of chemical compounds in order to
identify either specific or general odor blockers or maskants. The
screening strategies based on psychophysical testing of human
subjects, however, have intrinsically very low throughput and can
be subjective and error prone. Additionally, the identification of
some anti-odorants has been accomplished to a limited effect by
applying the conventional principles and strategies of medicinal
chemistry by relying on the structure-activity (SAR) studies of
malodorants and potential malodor-blocking compounds in order to
identify best candidates for psychophysical testing. However, this
approach is generally tedious and is generally only applicable for
a malodorant of interest, especially for refining existing leads
from primary screens of chemical libraries.
[0015] Therefore, it would be beneficial if more broadly applicable
assays could be developed that enable the rapid identification of
anti-odorants that effectively inhibit or block the odor of
isovaleric acid and structurally related malodorants. Ideally, it
would be beneficial if compounds could be identified that totally
and selectively block the perception of the odor of isovaleric acid
and/or related malodorants by mammalian subjects, preferably human
subjects. This goal would ideally be achieved by developing a
greater understanding of the biology of isovaleric acid odor
perception, particularly by elucidating the particular receptors
that are involved in the odor perception of isovaleric acid and
related compounds.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 depicts the multiple full-length sequence alignment
for all murine IVA receptors and their predicted human
counterparts. Conserved amino acid positions are highlighted by
black (>50% identity) or grey (>50% homology) background.
[0017] FIG. 2 depicts the multiple CDR protein sequence alignment
for all murine IVA receptors and their predicted human
counterparts. Conserved amino acid positions are highlighted by
black (>50% identity) or grey (>50% homology) background.
[0018] FIG. 3 depicts the response of a single olfactory neuron to
IVA indicated b changes in fura-2 fluorescence intensity ratios
(340/380 nm). Either high K.sup.+ solution or IVA at either 100
.mu.M or 10 .mu.M were applied. The neuron was washed continuously
between applications of high K.sup.+ and IVA.
BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION
[0019] Thus, one object of the invention is to develop a greater
understanding of the biological processes involved in the
perception of malodor by isovaleric acid and related compounds.
[0020] It is a more specific object of the invention to identify a
subgenus of mammalian olfactory receptors that specifically respond
to isovaleric acid and structurally related odorants.
[0021] More specifically, it is an object of the invention to
identify a subgenus of human olfactory receptors that specifically
respond to isovaleric acid and structurally related compounds.
[0022] It is another specific object of the invention to develop
assays, particularly high throughput screening assays, to identify
compounds that block, inhibit, enhance and/or modulate specific
receptor(s) that are activated by isovaleric acid and structurally
related compounds.
[0023] It is a more specific object of the invention to develop
assays, particularly high throughput screening assays, to identify
compounds that block inhibit, enhance and/or modulate one or more
of a subgenus of mammalian olfactory receptors, preferably human or
murine olfactory receptors, that are activated by isovaleric acid
These assays are especially to be used for identifying compound(s)
that block the malodor of isovaleric acid, a malodorous compound
found in human axillary secretions.
[0024] It is another specific object of the invention to provide
cell lines that stably or transiently express at least one
isovaleric acid olfactory receptor according to the invention or a
fragment, variant or chimera thereof and to use same in assays for
identifying compounds that block, inhibit, modulate and/or enhance
the activation of isovaleric acid receptors by isovaleric acid
and/or structurally related malodorant compounds.
[0025] It is another object of the invention to provide a
microarray of olfactory receptors that respond to isovaleric acid
and/or structurally related malodorant compounds.
[0026] It is another object of the invention to use compounds that
block, inhibit, modulate and/or enhance the activation of
isovaleric acid receptors in compositions intended to camouflage or
block the odor of isovaleric acid and structurally related
compounds, e.g. deodorants, carpet fresheners, air fresheners,
fabric fresheners, e al.
DETAILED DESCRIPTION OF THE INVENTION
[0027] As discussed supra, the present invention hinges on the
elucidation of the odorant specifically of a specific subgenus of
olfactory receptors that respond to isovaleric acid and potentially
to structurally related compounds (such as other aliphatic
carboxylic acids that elicit malodor such as 3-methyl-2-hexanoic
acid, propionic acid, and hexenoic acid.) This goal was achieved by
performing receptor activity assay experiments using a family of
murine olfactory receptors in vitro to identify a subgenus of
murine olfactory receptors that specifically respond to isovaleric
acid. Using these sequences, the human homologs of these murine
olfactory receptors were identified based on sequence similarity of
the amino acid sequences of the murine olfactory IVA receptors to
that of previously reported human olfactory sequences.
Particularly, the present inventors compared the amino acid
sequences of the identified murine isovaleric acid ORs to that of
the amino acid sequences of the repertoire of human ORs disclosed
in Zozulya et al., Genome Biology 2(6):research 0018.1-0018.12
(2001) and Zozulya, S. PCT WO 01/68805 A2, and U.S. Ser. No.
09/809,291 both having an international filing date of Mar. 13,
2001, and all of which are incorporated by reference herein in
their entirety.
[0028] In particular, the murine olfactory receptors for isovaleric
acid were identified using experimental techniques and procedures
similar to those reported by other groups for elucidating the
specificity ("de-orphan") of rodent olfactory receptors (Touhara et
al., Proc. Natl. Acad. Sci., USA 96(7):4040-5 (1999); Malnic et
al., Cell, 96(5):713-23 (1999); and Dulac, C. and Axel, R., Cell
83(2):195-206 (1995).
[0029] Essentially, individual murine olfactory neurons isolated
from an enzymatically and mechanically dissociated preparations of
murine olfactory epithelium. A very mild trypsin dissociation
allows to obtain a single cell suspension in which neurons still
bear their axon and dendrites and can therefore be picked based on
their morphology. The neurons were loaded with a Ca.sup.2+-sensing
fluorescent dye (fura-2) and contacted with isovaleric acid (at
10-100 .mu.M). Single neurons that responded to the isovaleric acid
stimulus, as indicated by transient increases in the concentration
of intracellular Ca2+ were identified in a microscope field and
collected using a suction microcapillary. The identified single
IVA-responding neurons were then subjected to reverse transcription
with (dT).sub.24 containing oligo primers to synthesize cDNA from
poly(A) fraction of cellular mRNA.
[0030] The cDNA was then subjected to polymerase chain reaction
(PCR) using degenerate oligonucleotide primers corresponding to the
highly conserved regions of mammalian olfactory receptors
(disclosed in Zozulya patent application and publication
incorporated by reference) to amplify a fragment of a unique
olfactory receptor expressed in each particular neuron. The
amplified OR fragments were then cloned in a plasmid vector and
their nucleotide sequences elucidated. The experimental procedures
mentioned above are described in more detail in the EXAMPLES
section of this application. The partial OR sequences were extended
to the corresponding predicted full-length or near full-length open
reading frame sequence of the corresponding OR gene by murine
genomic sequence database mining. The extended sequences were
conceptually translated to obtain protein sequences of murine
isovaleric acid receptors. These protein sequences were then used
to query the protein sequence database of human ORs using the BLAST
algorithm to identify the predicted human counterparts of the
murine isovaleric acid receptors based on sequence similarity.
[0031] Additionally, instead of the full-length sequences, the
ligand-binding regions "complementarity determining regions (CDRs)"
(as determined according to Pilpel, Y. and Lanar, D., Protein Sci.
8(5):969-77 (1999)) of the identified murine IVA acid ORs were to
identify the human counterpart ORs based on sequence similarity.
Based on this sequence comparison and analysis, the present
inventors have identified a subgenus of OR genes that should bind
the same or similar compounds, as the identified subgenus of murine
IVA olfactory receptors, namely, these human ORs should be
activated by isovaleric acid and/or structurally related compounds,
e.g. other carboxylic acid odorants.
[0032] In particular, based on the results of the above-described
experiments an sequence analysis, the present inventors have
identified eleven (11) murine olfactory receptors that are
activated by isovaleric acid and eleven predicted human homologs
thereof that should respond similarly to isovaleric acid and/or
structurally related compounds.
[0033] Based on the particular human and murine olfactory receptor
libraries that were screened, the current understanding of this
family of receptors, and using available screening methods it is
believed that the present inventors have identified the full
repertoire of murine and olfactory receptors that are activated by
isovaleric acid. It is anticipated that these receptors, as well as
fragments, chimeras and variants thereof will be well suited for
identifying compounds that block, inhibit modulate and/or enhance
the activation of these receptors. In particular, it is anticipated
that these receptors alone or in combination may be utilized to
screen libraries for compounds that bind these receptors. It is
especially anticipated that these assays may yield commercially
significant malodorants.
[0034] A particular advantage of the subject invention is that it
provides a repertoire of isovaleric acid receptors that likely
recognize isovaleric acid with different affinities and/or
recognize different structural determinants of the isovaleric acid
molecule. Therefore, the subject receptors in combination should be
useful in assays for identifying compounds that are structurally
related to isovaleric acid, some of which may yield effective
malodorants, especially for blocking the malodor of isovaleric acid
and related chemical compounds.
[0035] The predicted DNA and amino acid sequences of the eleven
isovaleric acid murine olfactory receptors expressed in isovaleric
acid-responding olfactory neurons are set forth below:
TABLE-US-00001 mOR IVA A (DNA Sequence) SEQ ID NO:1
ATGGGAAAAGAAAATCACACAGAACTATCACAATTCCTGCTACTGGGTCT
CTCAGATGATCCTAAATTGGAGCCTATTCTTTTCGGGATATTCTTATTTA
TGTACCTGGTCACAGTGCTTGGTAACCTGCTCATCATCCTGGCTGTCAGT
TCTGATTCCCATCTCCAGAACCCCATGTACTTCTTCCTCTCCAACCTCTC
ATTTGTAGACATGTGTTCACTTCTACCACTGTCCCAAAGATGCTGGTGAA
CATCCAGACAAAGAACAAAATATCTCCTACATGCAGTGCCTCACTCAAGT
CTATTTTTTTATGGTGTTTGCTGGAATGGATAATTTCTTACTGACTGTAA
TGGCCTTTGACCGCTTTGTGGCTATTTGTACCCCTTAAACTACACAGTCA
TCATGAACCCTCACTTCTGTTGCTTCCTTGTGCTAATGTGCTGGATTATC
ATTTTATCAGTCTCCCTGTTTCATAGTCTATTAATGAAGCAATTAACTTT
TTCCATGGGTACTGAAATCCCACATTTCTTCTGTGAGTTGGCCAAATTCT
CAGAGTAGCAAGGTCTGATATTCTCATCAATAATATCGCATTATATCTGG
CTACTGCCCTGTTATGTGTGTTTCCTGTCACTGGAATTCTCTTCTCTTAC
TGCAGATTGTCTCCTCCTTATTGAATATGTCTTCAGTAGTCAGCAAGTAT
AGAGCCTTTTCCACCTGTGGATCTCACCTCTGTGTGGTCTGTTTGTTTTA
TGGTACAGCACTGGGGGTTTACCTCAGTTCAGCTGGGACTGATGTTCTCA
AGGAAGCACTATAGCCTCAGTGATGTATACTGTGGTCACTCCTATGCTCA
ACCCATTCATCTACAGCCTGAGGAATAAAGATGTGAAGGGGGCTCTGGTA
AGAATCCTTAAAGTATATTCTTGTCCCTGA mOR IVA A (Amino Acid Sequence) SEQ
ID NO:2 MGKENHTELSQFLLLGLSDDPKLQPILFGIFLFMYLVTVLGNLLIILAVS
SDSHLHNPMYFFLSNLSFVDMCFTSTTVPKMLVNIQTKNKNISYMQCLTQ
VYFFMVFAGMDNFLLTVMAFDRFVAICHPLNYTVIMNPHFCCFLVLMCWI
IILSVSLFHSLLMKQLTFSMGTEIPHFFCELAQILRVASSDILINNIALY
VATALLCVFPVTGILFSYSQIVSSLLNMSSVVSKYRAFSTCGSHLCVVCL
FYGRALGVYLSSAGTDVSQSGSTIASVMYTVVTPMLNPIYSLRNKDVKGA LVRILKVYSCP mOR
IVA B (DNA Sequence) SEQ ID NO:3
ATGGAAGAACACAATCTTACATTAATGACTGAATTCATCCTAATGGGTAT
CAGTGACCACTCTGAATTGCAGGCCCCATTATTTGGGCTGATCCTTGCCA
TATACATGACCTCAATGGTAGGTAATATGGGAATCATTGTTTTAATCACT
TTGGACTCACGCCTGCTAACACCCATGTACTTCTTTATAAAACACCTGGC
TATTACAGATCTTGGAATTCTACAGCTGTGGGACCCAAAATGTTGGAAAA
TTTTGTTGTAGATCAAAATACAATTTCATTTAATCTTTGTGCCACACAAC
TAGCTTTCTTTCTTGTATTCATTGGAGTGAGCTATTCATTCTCTCTGCGA
TGTCCTATGACCGCTATGTGGCCATCTGAAGCCTCTGCTCTACACTGTCC
TCATGTCCCAAAAACTATGTTGGGTTCTTATGTCAATGCCTTATCTCTAC
TGCACATTTGTGTCTCTTGTCATCACAGTGAAGATTTTTACTTCATCCTT
CTGTGGCTACAATGTCATTAACCATTTCTACTGTGAGTGTACCCCTTGCT
GTCTCTACTCTGTTCACATGCAGAGGAAATCGCATTTATTGTTTATGATC
TTTGCAGCTTTTGATTTGATTGTGTCTCTTCTTATTGTTCTGGTATCCTA
CATGTTTATCCTCATAGCAGTTCTCAGGATGAACTCTGCAGAGGGCAGGT
ACAAGGCTTTCTCCACATGTGGGTCCCACCTGACAGTGGTGACAGTGTTC
TATGGTACTTTAATATTTATGTATGTACAACGTCAGTCCAGTCATTCTGA
TGACAATGATAAGGTGTCTTCAATTTTTTACACCCTCGTTATACCCATGC
TGAATCCTTTGATCTATAGTTTGAGGAACAAGGATGTAAAATTTGCCCTA
CATAGGACTTGGAGAAATATTTGTAAGATCTTCCCTTAG mOR IVA B (Amino Acid
Sequence) SEQ ID NO:4
MEEHNLTLMTEFILMGISDHSELQAPLFGLILAIYMTSMVGNMGIIVLIT
LDSRLLTPMYFFIKHLAITDLGYSTAVGPKMLENFVVDQNTISFNLCATQ
LAFFLVFIGSELFILSAMSYDRYVAICKPLLYTVLMSQKLCWVLMSMPYL
YCTFVSLLITVKIFTSSFCGYNVINHFYCDCIPLLSLLGSHAEEIAFIVM
IFAAFDLIVSLLIVLVSYMFILIAVLRMNSAEGRYKAFSTCGSHLTVVTV
FYGTLIFMYVQPQSSHSDDNDKVSSIFYTLVIPMLNPLIYSLFNKDVKFA LHRTWRNICKIFP
mOR IVA C (DNA Sequence) SEQ ID NO:5
ATGACTGAGGACAACTACTCGTTGACAACAGAGTTCATCCTCATAGGATT
CTCAGAGCACCCAGACTTAAAGATACTTCTATTCCTGGTGTTATCTACCA
TCTATCTGGTCACCATGGTGGGGAATCTTGGGCTGGTGGGCTTGATCTAC
ATGGAGCCTCTCTCCACACACCCATGTACATCTTTCTGGGCAACCTGGCT
CTCATGGATTCCTGTTGCTCCTGTGGCATCACTCCTAAGATGCTAGAGAA
GTTTTTTTGTGTGAAGAGAAGGATTTCTCTCTATGAATGCATGGCACAGT
TCTATTTTCTCTGTCTTGCTGAAACTGCAGACTGCTTCCTTCTGGCAGCC
ATGGCCTATGACCGCTATGTGGCCATATGCAACCCTCTGCAGTACCACAC
CATGATGTCCAAGAAGCTCTGCCTTCAAAGACCACAGGAGCCTACATAGC
AGGAAACCTGCATTCCATGATTCACATAGGGTTCTTGTTCAGGTTAATTT
TCTGCAGGTCTCATGTGATCAAGCACTTCTTTTGTGATGTCCTCGCCCTA
TACAGACTCTCATGTGTTGACCCTTATATCAATGAACTGATGATACTCAT
CTTTTCTGGTTCAGTTCAAACCTTTTCCATTATTATAGTCTTGATTCTTA
TTTCTGCATCCTTTTTACTATATTCACAATGAAGTCCAGAGAGGGAAGAA
GCAAAGCCTTATCTACTTGTGCATCCCACTTTCTGTCTGTGTGAATATTC
TATGGTCTCTTCTCTACACATATATTCGACCAAGTTCACTTAATGAAGGG
TATAAAGAATACCTGTTGCTATATTTTATACTCTAGTAATTCCTTTATTA
AACCCGTTTATTTATAGTCTGAGAAATAAAGAAGTAATTAATGTGATGAA
AAGAGCAATGAAGAAAACATTATAA mOR IVA C (Amino Acid Sequence) SEQ ID
NO:6 MTEDNYSLTTEFILIGFSDHPDLKILLFLVLSTIYLVTMVGNLGLVALIY
MEPRLHTPMYIFLGNLALMDSCCSCAITPKMLENFFSVNRRISLYECMAQ
FYFLCLAETADCFLLAAMAYDRYVAICNPLQYHTMMSKKLCLQMTTGAYI
AGNLHSMIHIGFLFRLIFCRSHIKHFFCDVLPLYRLSCVDPYINELMILI
FSGSVQTFSIIIVLISYFCILFTIFTMKSREGRSKALSTCASHFLSVSIF
YGSLLYTYIRPSSLNEGYKDIPVAIFYTLVIPLLNPFIYSLRNKEVINVM KRAMKKRL mOR IVA
D (DNA Sequence) SEQ ID NO:7
ATGGGCAAATTAAACCACACTTATCTGACGGAGTTCATCCCGCTGGGCCT
CTCTTCAGATCATCAGAGTCAGATCCTGCTGTTTGTGGTATTTCTCATCA
TCTACCTGTCACTGTGTTTGGGAACCTGCTCATCATACTCCTCATTCATG
TTGACTCCCGACTTCATACAGCAATGTACTTCTTTCTAAAAATCCTGTCA
TTCAATGATCTCTGTTTTCTACAACAATTGTTCCAAAGATGCTAGTCCAC
TTTCTAGGTGTCAGAAAGACCATTTCATTTGCTGGGTGCTCAGTGCAAAT
GTTTTCTTTCCTCATAATGGGGTGTACAGAAAGCTCTCTTCTGGCAGTCA
TGTCATATGACCGCTACATAGCTGTCTGCAAACCCCTGCACTACTCCACC
ATCATGACACATAAGGTTTGTGTTCTGGTAGTTGTAGGATCCTGGACTAG
TGGAATATTTGTGTCTGTAGTAGATACCTCATTTACTTTATGCTTGACGT
ACCGGGGACCAAATATAATCAATCATTACTTTTGTGAGCCTCCTGCACTC
TTAAAGCTGGCTTCAGAAGAAACCTACACAGCTGAAATGGTCATATTTGC
AATGGGTATAATAATTCTCTTAGGTCCTGTCTCTCTTATCCTTTTCTCCT
ATTGGAATATTATCTCCACTGTGGTTCAAATACAATCAGGTGAGGGGAGG
CTCAAGGTTTTCTCTACCTGCAGTTCCCATTTTATTGTTGTTATCTTCTT
CTATGGCTCAACAATATTTACCTACATGCAGCCAAACTCAAAGAAAATGA
ATGAAAAGGATAAGGTAATCTCGGTATTCTACTCAATAGTAACATCCATG
ATGAACCCATTCATTTATAGCCTAAGGAACAAAGATGTGAAAGGGGCATT
AAAGAAAGTACTTAAAAGAGAGATAAGATAA mOR IVA D (Amino Acid Sequence) SEQ
ID NO:8 MGKLNHTYLTEFILLGLSSDHQTQILLFVVFLIIYLITVFGNLLIILLIH
VDSRLHTPMYFFLKILSFNDLCFSTTIVPKMLVHFLGVRKTISFAGCSVQ
MFSFLIMGCTESSLLAVMSYDRYIAVCKPLHYSTIMTHKVCVLLVVGSWT
SGIFVSVVDTSFTLCLTYRGPNIINHFCEPPALLKLASEETYTAEMVIFA
MGIIILLGPVSLILFSYWNIISTVVQIQSGEGRLKFSTCSSHFIVVIFFY
GSTIFTYMQPNSKKMNEKDKVISVFYSIVTSMMNPFIYSLRNKDVKGALK KVLKREIR mOR IVA
E (DNA Sequence) SEQ ID NO:9
ATGGAAACAGGAAATGAGACTGAGCTTTCAGAATTCTTTCTTCTGGGATT
TTCAGAGAATCAACCTCAAATTCAGCCTGTCATATTTGGACTGTTCCTCT
TCATGTATATATTGACTTTCACTGGAAACCTAGTCATCATCATGGCCATC
ATTGTTGACTGCCACCTGGAGACACCCATGTACCTCTTCCTGTCTAATCT
GTCCTTTGTGGACATCTGCTTCACTTCCACCACTGTTCCACAGATGCTGG
TAAACATTCACAGACAAAGCAAGGCCATCACCTATGCAGGCTGCATCATC
CAGATGTACTTCTTACTGCTTTTTTCAGGGTTAGACATGTTTCTGCTGAC
TGTGATGGCCTATGACCGCTATGTGGCCATCTGTCACCCCCTGCATTACA
TGATCATCATGAGCACAAGACGCTGTGGATTGATGATTCTGGCATGCTGG
ATTATAGGTGTTATAAATTCCCTGTTACACACCTTTTTGGTGTTACGGCT
GTCATTCTGCACAAACTTGGAAATCCCCCATTTTTTCTGTGAACTTAATC
AAGTTGTACACCAGGCCTGTTCTGACACCTTTCTTAATGATATGGTAATT
TACATTACAGCTATGCTACTGGCTGTTGGCCCCTTCTCTGGTATCCTTTA
CTCTTACTCTAGGATAGTATCCTCCATTTGTGCAATCTGCTCAGTGCAGG
GGAAGTACAAAGCATTTTCCACCTGTGCATCTCACCTCTCAGTTGTCTCC
TTATTTTATTGCACCCTCCTGGGAGTGTACCTCAGCTCTGCTGTGACCCA
AAACTCACATGCTACTGGAACAGCTTCATTGATGTACACTGTGGTCACCC
CCATGCTGAATCCCTTCATCTACAGTCTGAGGAACAAAGACATAAAGACA
GCTCTGAAAATCCTGTTAGGGAGTGTAACTAGAAGGAGATCAATGGATTC ACCTTCATAA mOR
IVA E (Amino Acid Sequence) SEQ ID NO:10
METGNDTQLSEFFLLGFSENQPQIQPVIFGLFLFMYILTFTGNLLIIMAI
IVDSHLHTPMYLFLSNLSFVDICFTSTTVPQMLVNIHTQSKAITYAGCII
QMYFLLLFSGLDIFLLTVMAYDRYVAICHPLHYMIIMSTRRCGLMILACW
IIGVINSLLHTFLVLRLSFCTNLEIPHFFCELNQVVHQACSDTFLNDMVI
YITAMLLAVGPFSGILYSYSRIVSSICAISSVQGKYKAFSTCASHLSVVS
LFYCTLLGVYLSSAVTQNSHATATASLMYTVVTPMLNPFIYSLRNKDIKT
ALKILLGSVTRSRSMDSP mOR IVA F (DNA Sequence) SEQ ID NO:11
ATGAGTGTGGGCAATGAGAGCATGTCAGGGGAGTTCATTCTCTTAGGGTT
TTCGATCGGCCATGGCTGGAGCTGCCGCTCTTTGTGGTGTTTCTAGTGTC
CTATATCTGACCATCTTTGGAAATATGATGATCATTCTTGTGTCCCGCCT
GGATTCCAAACTCCACACGCCCATGTACTTTTTGCTCAGTAACCTGTCCT
TGCTGGACCTGTGTAGACCACAAGCACGGTCCCACAGATGCTCATCAACA
TCTGCAGCACCCGGAAGGTGATCAGCTATGGTGGCTGTGTGGCCCAGCTT
TTCATTTTCCTGGCCTTGGGTTCCACAGAATGCTTTCTGGTGGGCGTCAT
GTCCTTTGACAGGTTTGTAGCCATCTGTCGGCCTCTCCACTACTCAGTCA
TCATGCACCAGAGGCGCTGGCTCCAGTTGGCGGCTGCATGTTGGATCAGT
GGCTTCAGCAACTCAGTATTACAGTCTACGTGGACCCTTCAGATGCCACT
GTGTGGACACAAGGAAGTGGACCATTTCTTTTGCGAAGTCCCTGCCCTGC
TCAAGTTGTCCTGTGTGGATAGGACAGCTAATGAAGCAGAGGTGTTCTTG
ATCAGTGTGCTGTTTCTTTTAGCCGTGACCCTCATCCTCATATCATATTC
TTTTATTGTCCAGGCAGTGTTGAGAATAAGATCAGCTGAAGGTCGGCGAA
AGGCATTTGGGACATGTGGGTGCCACCTCATCGTGGTGGTCCTTTTGTAT
GGCACTGCCATCTACATGTATCTGCAGCCACCATCCCCTACTTCCAAGGA
CGGGGGGAAAATGGTGTCTCTCTTTTATGGGATCATCACACCCATGCTGA
ACCCCCTCATCTACACACTCAGGAACAAAGAGGTAAAGGGAGCGTTCAAG
AGGTTGGTGACAAGGATCATCCTGAGTAGAAAATAA mOR IVA F (Amino Acid
Sequence) SEQ ID NO:12
MSVANESISREFILLGFSDRPWLELPLFVVFLVSYILTIFGNMMIILVSR
LDSKLHTPMYFFLTNLSLLDLCYTTSTVPQMLINICSTRKVISYGGCVAQ
LFIFLALGSTECFLLGVMSFDRFVAICRPLHYSVIMGQRRCLQLAAACWI
SGFSNSVLQSTWTLQMPLCGHKEVDHFFCEVPALLKLSCVDTTANEAELF
FISVLFLLIPVTLILISYAFIVQAVLRIRSAEGRRKAFGTCGSHLIVVVL
FYGTAIYMYLQPPSPTSKDRGKMVSLFYGIITPMLNPLIYTLRNKEVKGA FKRLVTRIILSRK
mOR IVA G (DNA Sequence) SEQ ID NO:13
ATGTTCCAAGGAAATCTTTCCGGAGTAACTGAGTTCAATCTTGCTGGTTT
AACAGACAAACCAGGGCTGCAGCTGGCCCTCTTCCTGCTGTTCCTAGGAA
TGTATGTGGTCACAGTGGTGGGGAATCTCAGCATGATCACCCTGATACTA
TTCAGTTCTCAACTACACACACCCATGTATTATTTTCTCAGCAGTCTGTC
CTTCATTGACCTCTCCAGTGCATTGTCATTATTCCCAAAATGTTGGTGAA
CTTTGTGACAGTGCAGAACATCATCTCCTACGCTGAATGTATGACACAGT
TTTGCTTTTTGCTACTTTTTACTATTGCAGAGTGTCACATGTTAGCTGTA
ATGGCATATGACCGGTATGTTGCCATTGTAAGCCCTTGCTTTACAATGCT
GTAATGTCCTATCAAGTTTGTTGCTGGATTATTTGGAGTATATATTATGG
CTTTTGTTGGTGCCACAACTCAAACAGTCTTCAGTTAAAAGTGCATTTTT
GTAAGGGCAATGTAATAAATCATTACTTCTGTGATCTTTCCCCACTCCTG
GAACTCTCTTGTTCTGATACTTTTATTAATGAAGTATTAGCTTTGTGCTT
CAGTGTTTTCAATATCTTTATTCCAACTCTGACAATTCTAAGCTCTTACA
TCTTCATGATAGCCAGCATCCTCCGGATTAAATCCACTGAAGGCAGGTCC
AAAGGCTTCAGCACTTGCAGCTCACACATATCAGCAGTTGCTATATTCTT
TGGATCCCTTGCATTCATGTACCTGCAGCCATCATCAATCAACTCCATGG
ACCAAAGGAAAGTGTCCTCTGTATTTTATACCATTGTCGTGCGCATGCTG
AATCCTTTGATCTACAGCCTGAGGAATAAGGATGTCAAAGTTGCTCTAAA
TAAGTTCCTTGAAAGAATTTTTTGTTGTGAACAAAACTAA mOR IVA G (Amino Acid
Sequence) SEQ ID NO:14
MFQGNLSGVTEFNLAGLTDKPGLQLPLFLLFLGIYVVTVVGNLSMITLIL
FSSQLHTFMYYFLSSLSFIDLCQSIVIIPKMLVNFVTVQNIISYPEGMTQ
FCFLLLFTIAECHMLAVMAYDRYVAICKPLLYNAVMSYQVCSWMIFGVYI
MAFVGATTQTVFMLKVHFCKANVINHYFCDLSPLLELSCSDTFINEVLAL
CFSVFNIFIPTLTILSSYIFlIASILRIKSTEGRSKAFSTGSSHISAVAI
FFGSLAFMYLQPSSINSMDQRKVSSVFYTIVVPMLNPLIYSLRNKDVKVA LNKFLERIFSCEQN
mOR IVA I (DNA Sequence) SEQ ID NO:15
TCTGAGTTTATCCTGTTAGAGGTCCCCATTCAGCCAGAGGATCAAGCTGT
GTAGTTTGCCCTGTTCCTGGCCATGTACCTGACAACTGTGCTGGGGAACC
TGCTCATCATTCTTCTCATTAGGCTGGACTCTCACCTCCACACCCCCATG
TACTTCTTCCTCAGTCACTTGGCCTTCACGGACATCTCTTTGTCATCTGT
CACAGCTCCAAAGATGCTGATGAATATGCTGACACATAGCCAATCCATCT
CACATGCTGGGTGTGTTTCGCAAATATATTTTTTCTTATTGTTTGGGTGT
ATTGACAACTTCCTTCTGACTTCCTGGCCTATGACAGGTATGTGGCCATC
TGCCACCCTCTGCATTATACCACTATCTGAGTCAAAGCCTCTGTGTTGTG
CTAGTGATGGTGTCCTGGGCATTTTCCTCTCTAATGGGGTTGTGCATACT
CTTCTCTTTGCTCGTCTCTCTCTTTTTAGAGACAACACTGTCGACCATTT
TTTCTGTGATCTCTCTGCTTTGCTGAAGCTGTCCAGCTCAGACACTACTA
TCAATGAACTAGTAATCCTCACTTTAGCAGTGGTGGTCATCACTGTACCA
TTCATATGCATCCTGGTTTCTTATGGCCACATGGGGGCCACTATCCAAGA
ACTCCATCGATCAAGGGTATCTGCAAAGCCTTGTCCACATGTGGTTCTCT
CTCTGTGTAGTTTCTTTATATTATGGAGCCATTATTGGGTTATATTTTTT
CCCCCCTCCAATAATACTAATGATAAAGATGTCATAGTAGCTGTGTTGTA
CAGTGTGGTTACACCCATGCTGAATCCGTTTATCTATAGTCTGAGGAATC
GGGATATAAATGGAGCATTGAGAAAGACACTCAGCAGGAGACTGTGTTCA CACTGA mOR IVA I
(Amino Acid Sequence) SEQ ID NO:16
SEFILLELPIQPEDQAVYFALFLAMYLTTVLGNLLIILLIRLDSHLHTPM
YFFLSHLAFTDISFSSVTAPKMLMNMLTHSQSISHAGCVSQIYFFLLFGC
IDNFLLTSMAYDRYVAICHPLHYTTIMSQSLCVLLVMVSWAFSSSNGLVH
TLLFARLSLFRDNTVHHFFCDLSALLKLSSSDTTINELVILTLAVVVITV
PFICILVSYGHMGATILRTPSIKGICKALSTCGSHLCVVSLYYGAIIGLY
FFPSSNNTNDKDVIVAVLYTVVTPMLNPFIYSLRNRDINGALRKTLSRRL CSH mOR IVA J
(DNA Sequence) SEQ ID NO:17
ATGCCTAGAAACAACAACCAGACTACCATCTCTCAGTTGCTCCTCCTGGG
TCTCCCGATCCCCCAAGAGTTTCAGCATCTGTTCTATGCCCTGTTCCTGG
CCATGTACTCACCACTGTCTTGGGGAACCTCATCATCATCATACTCATTC
GACTGGACTCCATCTCCACAGACCCATGTACTTGTTTCTGAGCAACTTGT
GCTTGACTGACCTCTAATTTTCCTCTGTCACAATGCCCAAGTTGCTGCAG
AACATGCAGAGCCAAGTCCTTCAATCCCCTATGCAGGCTGCCTGACACAA
ATGTACTTCCTTTTGTTTTTTGAGATCTTGAGAGCTTCCTCCTTGTGGCC
ATGGCCTATGACCGCTATGTAGCCATCTGCTTCCCTCTTGATTACAGCAG
CATCATGAGCCCGAGGCTCTGTGTGAGGTTGTGCTGCTGTCCTGGTTGCT
GACCATGTCCCATTCCATGCTGCACACTTTCTCTTAACTAGGTTGTCTTT
CTGTGAAAACAATGTGATCCCCCATTTTTTCTGTATCTGTGTGCTCTGCT
GAAGCTGGCCTGCTCTGATATTCACATTAATGAATTGTGATATTGATCAT
AGGAGGGCTTGTTGTTATACTTCCATTTCTACTCATCACAGGTCTTATGC
ACGCATCATCTCCTCCATTGTCAAGGTCCCTTCAACTCAAGGCATCCACA
AGGTCTTCTCCACTTGTGGTTCTCACCTGTCTGTGGTGTCACTGTTCTAT
GGGACAATTATTGGCCTCTACTTATGTCCATCTGCTAATAACTCTACTCT
AAAGACACTGTCATGTCTATGATGTACACCGTGGTAACTGCCATGCTGAA
CCCCTTATCTACAGCCTGAGGAACAGAGACATGAAGGAAGCCCTAAAAAG
AGTGCTTCAAAAGAAAACTATCTTTTGA mOR IVA J (Amino Acid Sequence) SEQ ID
NO:18 MPRNNNQTTISQFLLLGLPIPQEFQHLFYALFLAMYLTTVLGNLIIIILI
RLDSHLHTPMYLFLSNLSFTDLXFSSVTMPKLLQNMQSQVPSIPYAGCLT
QMYFLLFFGDLESFLLVAMAYDRYVAICFPLHYTSIMPRLCVSLVLLSWL
LTMSHSMLHTLLLTRLSFCENNNIPHFFCDLSALLKLACSDIHINELVIL
IIGGLVVILPFLLITVSYARIISSILKVPSTQGIHKVFSTCGSHLSVVSL
FYGTIIGLYLCPSANNSTLKDTVMSMMYTVVTPMLNPFIYSLRNRDMKEA LKRVLQKKTIF mOR
IVA K (DNA Sequence) SEQ ID NO:19
ATGCAAAACCAGAGCTTTGTCACTGAGTTCATACTCTTGGGGCTTTCCCA
GAACCCAAAAGTTGAGAAAATACTGTTTGTTGTATTTTTATTGGTCTATA
TTGCAACTATGGGGGAAACATGATAATTGTGGTGACCATCATCTATAGCG
CTGCACTGTTGAGTTCCCCCATGTACTTCTTCTTAATATTTCTGTCTTTC
CTGGATGCTTGCACTTCCTCTACTGTCACCCCCAAGATGATTGTAGACTT
CTTCTATGAGAGGAAGACCATCTCCTTTGAATGTTGCATCACACAACTGT
TACTAGCCACTTCTTTGCAGGAGTGAGGTGATTATCTTGACATCTATGGC
CTATGACCGCTATGTGGCCATCTGCAACCTCTTCACTAGTCTTCCATCAT
GACCAGGAGGCTCTGTGGCACTCTCGTAATGTGGCCTGGACAGGAGGATT
CTTACATTCTATCACACAAGTTATCTTCACGTTGCAGCTACCCTTCTGTG
GGCCCAAATTTTGATCATTTCATATGTGACTTGTTCCATTACTGCAGCTT
GGGTGGACTGACACACACATTTTTGTCATTTTGGTGTTTGCTAATAGTGG
GTCTCTGCATCATTATCTTCTCCTTGTTGATTGTTTCCTATGGTGTCATC
CTCTTGTGTCTAAGAGGTCACAGCTCAGAAGGACGAAGGAAAGCTCTCTC
AACCTGTGGATCCCATATTACTGTTATGATATTATTCTTTGTCCCATGTA
TGGTAATATATGCACGGCCTTCATCTGCCTTCCTTTGAGAAAAACACACT
TATTTTGCCTCTGTCCTGACACCATTGTTCAATCCTATGGTTTACACTTT
GAGAAATAAAGAAATGAAGAATGCGATCAGGAAAATGTGTAGGAAAATGT
TAGTAGATTCTATAACTTTTAA mOR IVA K (Amino Acid Sequence) SEQ ID NO:20
MQNQSFVTEFILLGLSQNPKVEKILFVVFLLVYIATIGGNMIIVVTIIYS
PALLSSPMYFFLIFLSFLDACTSSTVTPKMIVDFFYERKTISFECCITQL
FTSHFFAGVEVIILTSMAYRYVAICKPLHYSSIMTRRLCGTLVMVAWTGG
FLHSITQVIFTLQLPFCGPNFIDHFIDLFPLLQLACTDTHIFVILVFANS
GSFCIIIFSLLIVSYGVILFSLRGHSSEGRRKALSTCGSHITVMILFFVP
CMLIYARPSSAFSFEKNTLIFASVLTPLFNPMVYTFRNKEMKNAIRKMCR KMLVDSDNF mOR
IVA L (DNA Sequence) SEQ ID NO:21
ATGGGACAGAACCACAATGTCACAGAATTCATTTTTGTGGGTCTTAGTCA
AGATCGTGCTGGGCAAAAAGTATTATTTGTCTTGTTTCACTGACTTACAT
TGTGACAATGTTCGGAAACCTGCTGATTGCACTTACAGTGATTGCCAGCC
CCTCCTTAAACTCCCCAATGTAGTTCTTCCTTGCGTGTCTGTGAGTCCTG
GATGCTCTTTATTGCAATACAATCTCACCAAATTTGATTATAGACTTGTT
ATATAATAAAAAGAATATCTCCTTGAGAGCTTGCATGCTCCAGCTGTTTG
TAGAGCACTTATTTGGAGGTGTTGAGTCTTCCTTCTGGTATTCATGGCCT
ATGATCGCTATGTGGCCATCTGTAAGCCAGTGCACTATTTGACCATCATG
AACCAGAGGGTGTGCATTCTTCTATTGCTGATAGCTGGAGTTGGAGGCAT
CTTACACTCACTGATTCAAGTTCTGACTGTGTATAAACTTCCTTTTTGTG
GTCCCAATGTCATTGATCACTTCATGTGTGACATGAATCAATTACTCGGG
CTTGCATGCACTGACACCTACTTCCTTGGCATCACTGTCATGGCCAATGG
TGGAGTAATCTGTGTGGGAATTTTGACCTTTCTCTTAGTCTCCTATGGAA
TCATTCTAAACTCTCTTAAGACCCACAGTCGGGAAGGAAGACATAAAGCT
CTTTTACCTGCAGTTGTCACATCATGGTTGTTGTCTGCTTTTTTGCTCCC
TGTAGTTTATATATGCTAGAGCTGTCTCCAACTTTCCAGTGGATAAATAT
ATTGCTGTGTTTATACAGTTGTTAGTCCCATGCTGAATCCATTGATATAT
ACCTTGAGAAATTCAGAGATGAAAAACTCTATTAAAAAGCTCTGGTGTCT
CTAAAACTCTAACAACATAA mOR IVA L (Amino Acid Sequence) SEQ ID NQ:22
MGQNHNVTEFIFVGLSQDPAGQKVLFVLFSLTYIVTMFGNLLIALTVIAS
PSLNSPMYFFLAGLSVLDALYCNTISPNLIIDLLYNKKNISFRACMLQLF
VEHLFGGVEVFLLVFAYDRYVAICKPLHYLTIMNQRVCILLLLIAGVGGI
LHSLIQVLTVYKLPFCGPNVIDHFMCDMNQLLGLACTDTYFLGITVMANG
GVICVGIFTFLLVSYGIILNSLKTHSREGRHKALFTCSS
[0036] As noted above, the amino acid and DNA sequences for the
predicted human homolog of the above-identified murine olfactory
receptors for isovaleric acid are disclosed in the Zozulya
publications incorporated by reference in their entirety herein.
(Zozulya, S., PCT WO 01/68805 A2, filed Mar. 13, 2001; Zozulya et
al., Genome Biol. 2(6):research 0018.1-0018.12 (2001)) and are
identified by the following identifiers therein:
OR19.04.05 (AOLFR262);
OR19.04.11 (AOLFR284);
OR09.05.02 (AOLFR299);
OR17.03.01 (AOLFR258);
OR11.22.02 (AOLFR108);
OR06.02.04 (AOLFR269);
OR11.49.11 (AOLFR058);
OR11.49.10 (AOLFR022);
OR11.39.06 (AOLFR074);
OR11.38.05 (AOLFR346); and
OR03.01.01 (AOLFR340).
[0037] The predicted DNA and amino acid sequences for these human
genes is set forth below:
TABLE-US-00002 OR11.22.02 (AOLFR108) (DNA Sequence) SEQ ID NO:23
ATGTGTTCTTTTTTCTTGTGCCAAACAGGTAAACAGGCAAAAATATCAAT
GGGAGAAGAAAACCAAACCTTTGTGTCCAAGTTTATCTTCCTGGGTCTTT
GACAGGACTTGCAGACCCAGATCCTGCTATTTATCCTTTTCCTGATCATT
TATGTGCTGACCCTGCTTGGAAACCAGCTCATCATCATTCTCATCTTCCT
GGATTCTCGCCTTCACACTCCCATGTATTTTTTTCTTAGAAATCTCTCCT
TTGCAGATCTCTGTTTCTCTACAGCATTGTCCCTCAAGTGTTGGTTCACT
TCTTGGTAAAGAGGAAAACCATTTCTTTTTATGGGTGTATGACACAGATA
ATTGTCTTTCTTCTGGTTGGGTGTACAGAGTGTGCGCTGCTGGCAGTGAT
GTCCTATGACCGGTATGTGGCTGTCTGCAAGCCCTGTACTACTCTACCAT
CATGACACAACGGGTGTGTCTCTGGCTGTCCTTCAGTCCTGGGCCAGTGG
GGCACTAGTGTCTTTAGTAGATACCAGCTTTACTTTCCTCTTCCCTACTG
GGGACAGAATATAATCAATCACTACTTTTGTGAACCTCCTGCCCTCGTGA
AGCTGGCTTCCATAGACACTTACAGCACAGAAATGGGCATCTTTTAATGG
GCGTGGTAATCCTCCTGGCCCCTGTCTCCCTGATTCTTGGTTCTTATTGA
ATATTATCTGCACTGTTATGCAGATGGAGTCTGGGGAAGGGAGACTCAAG
GCTTTTTCCACCTGTGGCTCCGATCTTATTGTTGTTGTCCTCTTGTATGG
GTCAGGAATATTCACCTACATGCGACCAAACTCCAAGACTACAAAAGAAC
TGGATAAAATGATATCTGTGTTGTATACAGCGGTGACTCCAATGTTGAAC
CCCATAATTTATAGCTTGAGGAACAAAGATGTCAAAGGGGCTCTCAGGAA
ACTAGTTGGGAGAAAGTGCTTCTCTCATAGGCAGTGA OR11.22.02 (AOLFR108) (Amino
Acid Sequence) SEQ ID NO:24
MCSFFLCQTGKQAKISMGEENQTFVSKFIFLGLSQDLQTQILLFILFLII
YLLTVLGNQLIIILIFLDSRLHTPMYFFLRNLSFADLCFSTSIVRQVLVH
FLVKRKTISFYGCMTQIIVFLLVGCTEGALLAVMSYDRYVAVCKPLYYST
IMTQRVCLWLSFRSWASGALVSLVDTSFTFHLPYWGQNIINHYFCEPPAL
LKLASIDTYSTEMAIFSMGVVILLAPVSLILGSYWNIISTVIQMQSGEGR
LKAFSTCGSHLIVVVLFYGSGIFTYMRPNSKTTKELDKMISVFYTAVTPM
LNPIIYSLRNKDVKGALRKLVGRKCFSHRQ OR11.49.11 (AOLFR058) (DNA Sequence)
SEQ ID NO:25 ATGGAGCTCTGGAACTTCACCTTGGGAAGTGGCTTCATTTTGGTGGGGAT
TCTGAATGACAGTGGGTCTCCTGAACTGCTCTGTGCTACACAATTACAAT
CCTATACTTGTTGGGCCTGATCAGCAATGGCCTACTGCTCGTGGCTATCA
CCATGGAAGCCCGGCTCCACATGCCCATGTACCTCCTGCTTGGGGAGCTC
TCTCTCATGGACCTCCTGTTCACATGTGTTGTCAGTCCCAAGGCCCTTGC
GGACTTTCTGCGCAGAGAAAACACCATCTCCTTTGGAGGCTGTGCCCTTC
AGATGTTCCTGGCACTGACAAGGGTGGTGCTGAGGACGTCCTACTGGCCT
TCATGGCCTATGACAGGTATGTGCCCATTTGTCATGCTCTGACATACATG
ACCCTCATGAGCTCAAGAGCCTGCTGGTCATGGTGGCCACGTCCTGGATC
CTGGCATCCCTAAGTGCCGTAATATATACCGTGTATACCATGCAGTATCC
CTTCTGCAGGGCCCAGGAGATCAGGCATCTTCTCTGTGAGATCCCACAGT
TGCTGAAGGTGGCCTGTGCTGATACCTCCAGATATGAGCTCATGGTATAT
GTGATGGGTGTGACCTTCCTGATTCCCTCTCTTGCTGCTATACTGGGCTC
CTATACACAAATTCTACTCAGTGTGCTCGATATGCCATCAAATGAGGGGA
GGAAGAAAGCCCTTGTCACCTGCTCTTCCCACCTGACTGTGGTTGGGATG
TTCTATGGAGCTGCGACATTCATGTATGTCTTGCCCAGTTCCTTCCACAG
CACCAGACAAGACAACATCATCTCTGTTTTCTACACAATTGTCACTCCAG
CCCTGAATCCACTCATCTACAGCCTGAGGAATAAGGAGGTCATGCGGGCG
TTGAGGAGGGTCCTGGGAAAATACATGCTGCCAGGACACTCCACGCTCTA G OR11.49.11
(AOLFR058) (Amino Acid Sequence) SEQ ID NO:26
MELWNFTLGSGFILVGILNDSGSPELLCATITILYLLALISNGLLLLAIT
MEARLHMPMYLLLGQLSLMDLLFTSVVTPKALADFLRRENTISFGGCALQ
MFLALTMGGAEDLLLAFMAYDRYVAICHPLTYMTLMSSRACWLMVATSWI
LASLSALIYTVYTMHYPFCRACEIRHLLCEIPHLLKVACADTSRYELMVY
VMGVTFLIPSLAAILASYTQILLTVLHMPSNEGRKKALVTCSSHLTVVGM
FYGAATFMYVLPSSFHSTRQDNIISVFYTIVTPALNPLYSLRNKEVMRAL RRVLGKYMLPAHSTL
OR11.49.10 (AOLFR022) (DNA Sequence) SEQ ID NO:27
ATGAGACANNNNAAGAATATNACAGAATTTGTCCTCGTGGGCTTTTCTGA
GGATCGTGGTGTGNNNAAAGCATTATTTGTCATGTTTTTACTCACATACN
NNNNNACAGTGGTGGGGAAGCTGCTCATTGTNGTGGATATTATTGCCAGC
CCTTNNTTGGGTTCCCCAATGTATTTCTTCCTTGCCTGCCTGTCATTTAT
AGATGCTGCATATTCCACTACCATTTCTCCCAAGTTAATTGTAGGCTTAT
TCTGTGATAAAAAGACTATTTCGTTCCAAGGTTGCATGGGCCAGCTATTT
ATAGACCATTTCTTTGGTGGGGCTCAGGTCTTCGTTCTGGTGGTGATGGC
CTGTGATCGGTATGTGGCCATCTGTAAGGCAGTGCACTATTTGACCATCA
TGAATCGAGAGGTTTGCTTCCTCTGTTGGTNNTNNCCATGATTGGAGGTT
TTGTAGATTCTGCGTTTCAAATTGTTGTGTACAGTCTCCCTTTCTGTGGT
CCCNATGTCATTGTTCATTTCAGTTGTGACATGCACCCATTACTGGAACT
GGCATGCACTGACACCTACTTTATAGGCCTCACTGTTGTTGTCAATAGTG
GAGCAATCTGTATGGTCATTTTCAACCTTCTGTTAATCTCCTATGGATCA
TCCTAAGCTGCCTTAAAACTTACAGTCAGGAAAAGAGGGGTAAAGCCTTG
TCTACCTGCAGCTCCGGCAGTACCGTTGTTGTCCTCTTTTTTGTACCCTG
TATTTTCATATATGTTAGACCTGTTTCAAACTTTCCTACTGATAAGTTCA
TGACTGTGTTTTATACCATTATCACACACATGCTGAGTCCTTTAATATAT
ACGTTGAGAAATTCAGAGATGAGAAATGCTATAGAAAAACTCTTGGGTAA
AAAGTTAACTATATTTATTATAGGAGGAGTGTCCGTCCTCATGTAG OR11.49.10
(AOLFR022) (Amino Acid Sequence) SEQ ID NO:28
MRXXNNXTEFVLLGFSQDPGVXKALFVMFLLTYXXTVVGNLLIVVDIIAS
PXLGSPMYFFLACLSFIDAAYSTTISPKLIVGLFCDKKTISFQGCMGQLF
IDHFFGGAEVFLLVVMACDRYVAICKPLHYLTIMNRQVGFLLLVXXMIGG
FVHSAFQIVVYSLPFCGPXVIVHFSCDMHPLLELACTDTYFIGLTVVVNS
GAIGMVIFNLLLISYGVILSSLKTYSQEKRGKALSTCSSGSTVVVLFFVP
GIFIYVRPVSNFPTDKFMTVFYTIITHMLSPLIYTLRNSEMRNAIEKLLG KKLTIFIIGGVSVLM
OR11.39.06 (AOLFR074) (DNA Sequence) SEQ ID NO:29
ATGGAACAACACAATCTAACAACGGTGAATGAATTCATTCTTACGGGAAT
CACAGATATCGCTGAGCTGCAGGCACCATTATTTGCATTGTTCGTCATGA
TCTATGTCATCTCAGTGATGGGCAATTTGGGCATGATTGTCCTCACCAAG
TTGGACTCCAGGTTGCAAACCCCTATGTACTTTTTTCTCAGACATGTGGC
TTTCATGGATCTTGGTATTCAACAACTGTGGGACCCAAAATGTTAGTAAA
TTTTGTTGTGGATAAGAATTAATTTCTTATTATTTTTGTGCAACACAGCT
AGCTTTCTTTCTTGTGTTCATTGGAGTGAACTTTTTATTGTCTGAGCCAT
GTCCTACGACCTCTATGTGGCCATCTGTAACCCTCTGCTATAGACAGTAA
TCATGTCACGAAGGGTATGTCAGGTGCTGGTAGCAATCCCTTACCTCTAT
TGCACATTCATTTCTCTTCTAGTCACCATAAAGATTTTTACTTTATCCTT
CTGTGGCTACAACGTCATTAGTCATTTCTACTGTGACAGTCTCCCTTTGT
TACCTTTGCTTTGTTCAAATACACATGAAATTGAATTGATATTCTATCTT
TGCAGCTATTGATTTGATTTCATCTCTTCTGATAGTTCTTTTATCTTACC
TGCTGATCCTTGTAGCCATTCTGAGGATGAATTCTGCTGGCAGACAAAAG
GCTTTTCTACCTGTGGAGCCCACGTGACAGTGGTCATAGTGTTCTATGGG
ACTTTGCTTTCATGTACGTGCAGCCCAAGTCCAGTCATTCCTTTGACACT
GATAAAGTGGTTCCATATTTTACACCCTGGTTATCCCCATGTTGAATCCC
TTGATCTATAGTTTACGAAACAAAGATGTAAAATATGCCCTACGAAGGAC
ATGGAATAACTTATGTAATATTTTTGTTTAA OR11.39.06 (AOLFR074) (Amino Acid
Sequence) SEQ ID NO:30
MEQHNLTTVNEFILTGITDIAELQAPLFALFLMIYVISVMGNLGMIVLTK
LDSRLQTPIYFFLRHLAFMDLGYSTTVGPKMLVNFVVDKNIISYYFCATQ
LAFFLVFIGSELFILSAMSYDLYVAICNPLLYTVIMSRRVCQVLVAIPYL
YCTFISLLVTIKIFTLSFCGYNVISHIYCDSLPLLPLLGSNTHEIELIIL
IFAAIDLISSLLIVLLSYLLILVAILRMNSAGRQKAFSTCGAHLTVVIVF
YGTLLFMYVQPKSSHSFDTDKVASIFYTLVIPMLNPLIYSLRNKDVHYAL RRTWNNLCNIFV
[0038] Thus, the invention provides a set of mammalian ORs that
respond to IVA, particularly murine and human IVA ORs and the use
thereof for screening of modulators of IVA ORS, e.g. activators,
inhibitors, stimulators, enhancers, agonists, inverse agonists and
antagonists of the IVA ORs of the invention, or fragments or
variants thereof. Such modulators are envisioned to be especially
useful as anti-odorants. These screening methods can be used to
identify high affinity agonists and antagonists of IVA olfactory
receptors.
[0039] Thus, the invention provides assays for IVA olfactory
modulation, where the ORs, or fragments or variants thereof, of the
invention act as direct or indirect reporter molecules for the
effect of modulators on olfactory transduction by IVA. The ORs, or
fragments or variants thereof, can be used in assays, e.g., to
measure changes in ion concentration, membrane potential, current
flow, ion flux, transcription, signal transduction, receptor-ligand
interaction, second messenger concentrations, in vitro, in vivo and
ex vivo. In one embodiment, the IVA ORs, or fragments or variants
thereof, can be used as an indirect reporters via attachment to
second reporter molecules, such as green fluorescent protein (see,
e.g., Mistili et al., Nature Biotech., 15:961-64 (1997)). In
another embodiment, the ORs, or fragments or variants thereof, can
be expressed in host cells, and modulation of olfactory
transduction via OR activity can be assayed by measuring changes in
intracellular Ca.sup.2+ levels.
[0040] Methods of assaying for modulators of olfactory transduction
include in vitro ligand binding assays using the IVA ORs of the
invention, IVA or fragments or variants thereof. More particularly,
such assays can use the ORs; portions thereof such as the
extracellular or transmembrane domains; chimeric proteins
comprising one or more of such domains; oocyte receptor expression;
tissue culture cell receptor expression; transcriptional activation
of the receptor; G protein binding to the receptor; ligand binding
assays; voltage, membrane potential and conductance changes; ion
flux assays; changes in intracellular second messengers such as
cAMP and inositol triphosphate; changes in intracellular Ca.sup.2+
levels; and neurotransmitter release.
[0041] The invention also provides for methods of detecting IVA
olfactory nucleic acid and protein expression, allowing for the
investigation of IVA olfactory receptor transduction regulation and
specific identification of IVA olfactory receptor expressing cells.
The IVA ORs, fragments, and variants of the invention can also be
used to generate monoclonal and polyclonal antibodies useful for
identifying IVA olfactory receptor expressing cells. IVA olfactory
receptor expressing cells can be identified using techniques such
as reverse transcription and PCR amplification of total RNA or poly
(A).sup.+ RNA, northern blotting, dot blotting, in situ
hybridization, RNase protection, S1 digestion, probing DNA
microchip arrays, western blots, and the like.
Identification and Characterization of IVA Olfactory Receptors
[0042] The amino acid sequences of the IVA ORs and polypeptides of
the invention can be identified by putative translation of the IVA
coding nucleic acid sequences. These various amino acid sequences
and the coding nucleic acid sequences may be compared to one
another or to other sequences according to a number of methods.
[0043] For example, in sequence comparison, typically one sequence
acts as a reference sequence, to which test sequences are compared.
When using a sequence comparison algorithm, test and reference
sequences are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, as
described below for the BLASTN and BLASTP programs, or alternative
parameters can be designated. The sequence comparison algorithm
then calculate the percent sequence identities for the test
sequences relative to the reference sequence, based on the program
parameters.
[0044] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of: from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the loc homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970',
by the search for similarity method of Pearson & Lipman, PNAS,
85:2444 (1988), b computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by manual alignment and visual inspection (see, e.g., Current
Protocols in Molecular Biology (Ausubel et al, eds. 1995
supplement))
[0045] A preferred example of an algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al. Altschul et
al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J.
Mol. Biol. 215:403-410 (1990)). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue alignments
or the end of either sequence is reached. The BLAST algorithm
parameters W, T, and X determine the sensitivity and speed of the
alignment. The BLASTN program (for nucleotide sequences) uses as
defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=4
and a comparison of both-strands. For amino acid sequences the
BLASTP program uses as defaults a wordlength of 3, and expectation
(E) of 10 and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff, PNAS, 89:10915 (1989)) alignments (B) of 50, expectation
(E) of 10, M=5, N=-4, and a comparison c both strands.
[0046] Another example of a useful algorithm is PILEUP. PILEUP
creates a multiple sequence alignment from a group of related
sequences using progressive, pairwise alignments to show
relationship and percent sequence identity. It also plots a
so-called "tree" or "dendogram" showing the clustering
relationships used to create the alignment (see, e.g., FIG. 2).
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-60 (1987). The method
used is similar to the method described by Higgins & Sharp,
CABIOS 5:151-153 (1989). The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences The final alignment is achieved by a series of
progressive, pairwise alignments. The program is run by designating
specific sequences and their amino acid or nucleotide coordinates
for regions of sequence comparison and by designating the program
parameters. Using PILEUP, a reference sequence is compared to other
test sequences to determine the percent sequence identity
relationship using the following parameters: default gap weight
(3.00), default gap length weight (0.10), and weighted end gaps.
PILEUP can be obtained from the GCG sequence analysis software
package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.
12:387-395 (1984) encoded by the genes were derived by conceptual
translation of the corresponding open reading frames. Comparison of
these protein sequences to all known proteins in the public
sequence databases using BLASTP algorithm revealed their strong
homology to the members of the mammalian olfactory receptor family,
each of the odorant receptor sequences having at least 50%, and
preferably at least 55%, at least 60%, at least 65%, and most
preferably at least 70%, amino acid identity to at least one known
member of the family.
[0047] The nucleic acid molecules of the present invention are
typically intronless and encode putative IVA OR proteins generally
having lengths of approximately 29 to approximately 400 amino acid
residues that contain seven transmembrane domains, as predicted by
hydrophobicity plotting analysis, indicating that they belong to
the G protein-coupled receptor 7-transmembrane (7.TM.) superfamily,
which includes the subset of taste and olfactory receptors. In
addition to the overall structural similarity, each of the IVA ORs
identified herein has a characteristic sequence signature of an
olfactory receptor. In particular, all the identified sequences
contain very close matches to the following consensus amino acid
motif (Mombaerts, 1999, Pilpel 1999): EFILL (SEQ ID NO:45) before
transmembrane domain 1, LHTPMY (SEQ ID NO:46) in intracellular loop
1, MAYDRYVAIC (SEQ ID NO:47) at the end of transmembrane domain 3
and the beginning of intracellular loop 2, SY at the end of
transmembrane domain 5, FSTCSSH (SEQ ID NO:48) in the beginning of
transmembrane domain 6, and PMLNPF (SEQ ID NO:49) in transmembrane
domain 7. Combination of all the above-mentioned structural
features of the identified genes and encoded proteins strongly
suggests that they represent novel members of the human olfactory
receptor family.
DEFINITIONS
[0048] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise.
[0049] "OR" refers to one or more members of a family of G
protein-coupled receptors that are expressed in olfactory cells.
Olfactory receptor cells can also be identified on the basis of
morphology (see, e.g., Roper, supra), or by the expression of
proteins specifically expressed in olfactory cells. OR family
members may have the ability to act as receptors for olfactory
transduction.
[0050] "IVA OR" refers to a member of the family of G
protein-coupled receptors that is expressed in an olfactory cell,
which receptors bind and/or are activated by isovaleric acid (IVA)
in a binding or activity assay for identifying ligands that bind
and/or activate GPCRs. Such assays are described infra. IVA
receptors herein will include fragments, variants, including
synthetic and naturally occurring, and chimeras that respond to or
bind IVA.
[0051] "OR" nucleic acids encode a family of GPCRs with seven
transmembrane regions that have "G protein-coupled receptor
activity," e.g., they may bind to G proteins in response to
extracellular stimuli and promote production of second messengers
such as IP3, cAMP, cGMP, and Ca.sup.2+ via stimulation of enzymes
such as phospholipase C and adenylate cyclase.
[0052] Topologically, certain chemosensory GPCRs have an
"N-terminal domain;" "extracellular domains;" "transmembrane
domains" comprising seven transmembrane regions, and corresponding
cytoplasmic, and extracellular loops; "cytoplasmic domains," and a
"C-terminal domain" (see, e.g., Hoon et al., Cell, 96:541-51
(1999); Buck & Axel, Cell, 65:175-87 (1991)). These domains can
be structurally identified using methods known to those of skill in
the art, such as sequence analysis programs that identify
hydrophobic and hydrophilic domains (see, e.g., Stryer,
Biochemistry, (3rd ed. 1988); see also any of a number of Internet
based sequence analysis programs, such as those found at
dot.imgen.bcm.tmc.edu). Such domains are useful for making chimeric
proteins and for in vitro assays of the invention, e.g., ligand
binding assays.
[0053] "Extracellular domains" therefore refers to the domains of
OR polypeptides that protrude from the cellular membrane and are
exposed to the extracellular face of the cell. Such domains
generally include the "N terminal domain" that is exposed to the
extracellular face of the cell, and optionally can include portions
of the extracellular loops of the transmembrane domain that are
exposed to the extracellular face of the cell, i.e., the loops
between transmembrane regions 2 and 3, between transmembrane
regions 4 and 5, and between transmembrane regions and 7.
[0054] The "N terminal domain" region starts at the N-terminus and
extends to a region close to the start of the first transmembrane
region. "Transmembrane domain," which comprises the seven
"transmembrane regions," refers to the domain of OR polypeptides
that lies within the plasma membrane, and may also include the
corresponding cytoplasmic (intracellular) and extracellular loops.
The seven transmembrane regions and extracellular and cytoplasmic
loops can be identified using standard methods, as described in
Kyte & Doolittle, J. Mol. Biol., 157:105-32 (1982), or in
Stryer, supra. The general secondary and tertiary structure of
transmembrane domains, in particular the seven transmembrane
domains of G protein-coupled receptors such as olfactory receptors,
are known in the art. Thus, primary structure sequence can be
designed or predicted based on known transmembrane domain
sequences, as described in detail below. These transmembrane
domains are useful for in vitro ligand-binding assays, both soluble
and solid phase.
[0055] "Cytoplasmic domains" refers to the domains of OR
polypeptides that face the inside of the cell, e.g., the "C
terminal domain" and the intracellular loops of the transmembrane
domain, e.g., the intracellular loop between transmembrane regions
1 and 2, the intracellular loop between transmembrane regions 3 and
4, and the intracellular loop between transmembrane regions 5 and
6. "C terminal domain" refers to the region that spans the end of
the last transmembrane domain and the C terminus of the protein,
and which is normally located within the cytoplasm.
[0056] The term "ligand-binding region" or "ligand-binding domain"
refers to sequences derived from a chemosensory receptor,
particularly an olfactory receptor that substantially incorporates
at least transmembrane domains 11 to VII. The ligand-binding region
may be capable of binding a ligand, and more particularly, an
odorant.
[0057] The phrase "functional effects" in the context of assays for
testing compounds that modulate OR family member mediated olfactory
transduction includes the determination of any parameter that is
indirectly or directly under the influence of the receptor, e.g.,
functional, physical and chemical effects. It includes ligand
binding, changes in ion flux, membrane potential, current flow,
transcription, G protein binding, GPCR phosphorylation or
dephosphorylation, signal transduction receptor-ligand
interactions, second messenger concentrations (e.g., cAMP, cGMP
IP3, or intracellular Ca.sup.2+), in vitro, in vivo, and ex vivo
and also includes other physiologic effects such increases or
decreases of neurotransmitter or hormone release.
[0058] By "determining the functional effect" in the context of
assays is meant assays for a compound that increases or decreases a
parameter that is indirectly or directly under the influence of an
OR family member, e.g., functional, physical and chemical effects.
Such functional effects can be measured by any means known to those
skilled in the art, e.g., changes in spectroscopic characteristics
(e.g., fluorescence, absorbance, refractive index), hydrodynamic
(e.g., shape), chromatographic, or solubility properties, patch
clamping, voltage-sensitive dyes, whole cell currents, radioisotope
efflux, inducible markers, oocyte OR gene expression; tissue
culture cell OR expression; transcriptional activation of OR genes;
ligand-binding assays; voltage, membrane potential and conductance
changes; ion flux assays; changes in intracellular second
messengers such as cAMP, cGMP, and inositol triphosphate (IP3);
changes in intracellular calcium levels; neurotransmitter release,
and the like.
[0059] "Inhibitors," "activators," and "modulators" of OR genes or
proteins are used interchangeably to refer to inhibitory,
activating, or modulating molecules identified using in vitro and
in vivo assays for olfactory transduction, e.g., ligands, agonists,
antagonists, and their homologs and mimetics. Inhibitors are
compounds that, e.g., bind to, partially or totally block
stimulation, decrease, prevent, delay activation, inactivate,
desensitize, or down regulate olfactory transduction, e.g.,
antagonists. Activators are compounds that, e.g., bind to,
stimulate, increase, open activate, facilitate, enhance activation,
sensitize, or up regulate olfactory transduction, e.g., agonists.
Modulators include compounds that, e.g., alter the interaction of a
receptor with: extracellular proteins that bind activators or
inhibitor (e.g., odorant-binding proteins, ebnerin and other
members of the hydrophobic carrier family); G proteins; kinases
(e.g., homologs of rhodopsin kinase and beta adrenergic receptor
kinases that are involved in deactivation and desensitization of a
receptor); and arrestins, which also deactivate and desensitize
receptors. Modulators can include genetically modified versions of
OR family members, e.g., with altered activity, as well as
naturally occurring and synthetic ligands, antagonists, agonists,
small chemical molecules and the like. Such assays for inhibitors
and activators include, e.g., expressing OR family members in cells
or cell membranes, applying putative modulator compounds, in the
presence or absence of tastants, e.g., sweet tastants, and then
determining the functional effects on olfactory transduction, as
described above. Samples or assays comprising OR family members
that are treated with a potential activator, inhibitor, or
modulator are compared to control samples without the inhibitor,
activator, or modulator to examine the extent of modulation.
Control samples (untreated with modulators) are assigned a relative
OR activity value of 100%. Inhibition of a OR is achieved when the
OR activity value relative to the control is about 80%, optionally
50% or 25-0%. Activation of an OR is achieved when the OR activity
value relative to the control is 110%, optionally 150%, optionally
200-500%, or 1000-3000% higher.
[0060] The terms "purified," "substantially purified," and
"isolated" as used herein refer to the state of being free of
other, dissimilar compounds with which the compound of the
invention is normally associated in its natural state, so that the
"purified," "substantially purified," and "isolated" subject
comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably
at least 50% or 75% of the mass, by weight, of a given sample. In
one preferred embodiment, these terms refer to the compound of the
invention comprising at least 95% of the mass, by weight, of a
given sample. As used herein, the terms "purified," "substantially
purified," and "isolated" "isolated," when referring to a nucleic
acid or protein, of nucleic acids or proteins, also refers to a
state of purification or concentration different than that which
occurs naturally in the mammalian, especially human, body. Any
degree of purification or concentration greater than that which
occurs naturally in the mammalian, especially human, body,
including (1) the purification from other associated structures or
compounds or (2) the association with structures or compounds to
which it is not normally associated in the mammalian, especially
human, body, are within the meaning of "isolated." The nucleic acid
or protein or classes of nucleic acids or proteins, described
herein, may be isolated, or otherwise associated with structures or
compounds to which they are not normally associated in nature,
according to a variety of methods and processes known to those of
skill in the art.
[0061] As used herein, the term "isolated," when referring to a
nucleic acid or polypeptide refers to a state of purification or
concentration different than that which occurs naturally in the
mammalian, especially human, body. Any degree of purification or
concentration greater than that which occurs naturally in the body,
including (1) the purification from other naturally-occurring
associated structures or compounds, or (2) the association with
structures or compounds to which it is not normally associated in
the body are within the meaning of "isolated" as used herein The
nucleic acids or polypeptides described herein may be isolated or
otherwise associated with structures or compounds to which they are
not normally associated in nature, according to a variety of
methods and processed known to those of skill in the art.
[0062] As used herein, the terms "amplifying" and "amplification"
refer to the use of any suitable amplification methodology for
generating or detecting recombinant of 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 in vivo or
in vitro.
[0063] 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). The families of olfactory and certain taste receptors each
belong to this super-family. 7-transmembrane receptor polypeptides
have similar and characteristic primary, secondary and tertiary
structures, as discussed in further detail below.
[0064] The term "library" means a preparation that is a mixture of
different nucleic acid or polypeptide molecules, such as the
library of recombinantly generated chemosensory, particularly
olfactory receptor ligand-binding domains generated by
amplification of nucleic acid with degenerate primer pairs, or an
isolated collection of vectors that incorporate the amplified
ligand-binding domains, or a mixture of cells each randomly
transfected with at least one vector encoding an olfactory
receptor, i.e. an IVA OR according to the invention.
[0065] 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 analogs 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. Acad. of Sci., Vol. 600,
Eds. Baserga et al. (NYAS 1992); Milligan J. Med. Chem.
36:1923-1937 (1993); Antisense Research and Applications (1993, CRC
Press), WO 97/03211; WO 96/39154; Mata, Toxicol. Appl. Pharmacol.
144:189-197 (1997); Strauss-Soukup, Biochemistry 36:8692-8698
(1997); Samstag, Antisense Nucleic Acid Drug Dev, 6:153-156
(1996)).
[0066] Unless otherwise indicated, a particular nucleic acid
sequence also implicitly encompasses conservatively modified
variants thereof (e.g., degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating, e.g., sequences in which the third position
of one or more selected codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.,
19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-08 (1985);
Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term
nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide, and polynucleotide.
[0067] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0068] The term "plasma membrane translocation domain" or simply
"translocation domain" means a polypeptide domain 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. For
instance, a "translocation domain" may be derived from the amino
terminus of the bovine rhodopsin receptor polypeptide. In one
embodiment, the translocation domain may be functionally equivalent
to an exemplary translocation domain (5'-MNGTEGPNFYVPFSNKTGVV) (SEQ
ID NO:50). However, rhodopsin from any mammal may be used, as can
other translocation facilitating sequences. Thus, the translocation
domain is particularly efficient in translocating 7-transmembrane
fusion proteins to the plasma membrane, and a protein (e.g., an
olfactory receptor polypeptide) comprising an amino terminal
translocating domain will be transported to the plasma membrane
more efficiently than without the domain. However, if the
N-terminal domain of the polypeptide is active in binding, the use
of other translocation domains may be preferred.
[0069] "Functional equivalency" means the domain's ability and
efficiency in translocating newly translated proteins to the plasma
membrane as efficiently as the exemplary translocation domain above
under similar conditions; relatively efficiencies an 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 supra, as described in
detail below.
[0070] The "translocation domain," "ligand-binding domain", and
chimeric receptors compositions described herein also include
"analogs," or "conservative variants" and "mimetics"
("peptidomimetics") with structures and activity that substantially
correspond to the exemplary sequences. 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.
[0071] More particularly, "conservatively modified variants"
applies to both amino acid and nucleic acid sequences. With respect
to particular nucleic acid sequences conservatively modified
variants refers to those nucleic acids which encode identical or
essentially identical amino acid sequences, or where the nucleic
acid does not encode an amino acid sequence, to essentially
identical sequences. Because of the degeneracy of the genetic code,
a large number of functionally identical nucleic acids encode any
given protein.
[0072] For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
polypeptide.
[0073] Such nucleic acid variations are "silent variations," which
are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a polypeptide also
describes every possible silent variation of the nucleic acid. One
of skill will recognize that each codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine, and TGG,
which is ordinarily the only codon for tryptophan) can be modified
to yield a functionally identical molecule Accordingly, each silent
variation of a nucleic acid which encodes a polypeptide is implicit
in each described sequence.
[0074] 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/gin or his; asp/glu; cys/ser; gln/asn; gly/asp;
gly/ala or pro; his/asn or gin; ile/leu or val; leu/ile or val;
lys/arg or gin 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 (I); 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),
Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman
and Company (1984); Schultz and Schimer, Principles of Protein
Structure, Springer-Verlag (1979)). 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."
[0075] 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, ligand-binding domains, or chimeric
receptors of the invention. The mimetic can be either entirely
composed of synthetic, non-natural analogs of amino acids, or may
be 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.
[0076] 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, c ester (see, e.g.,
Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, 7:267-357, "Peptide Backbone Modifications," Marcell
Dekker, NY (1983) A polypeptide can also be characterized as a
mimetic by containing all or some nor natural residues in place of
naturally occurring amino acid residues; non-natural residues are
well described in the scientific and patent literature.
[0077] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and proteins which can be made detectable, e.g., by
incorporating a radiolabel into the peptide or used to detect
antibodies specifically reactive with the peptide.
[0078] A "labeled nucleic acid probe or oligonucleotide" is one
that is bound, either covalently, through a linker or a chemical
bond, or noncovalently, through ionic, van der Waals,
electrostatic, or hydrogen bonds to a label such that the presence
of the probe may be detected by detecting the presence of the label
bound to the probe.
[0079] As used herein a "nucleic acid probe or oligonucleotide" is
defined as a nucleic acid capable of binding to a target nucleic
acid of complementary sequence through one or more types of
chemical bonds, usually through complementary bas pairing, usually
through hydrogen bond formation. As used herein, a probe may
include natural (i.e., A, G, C, or T) or modified bases
(7-deazaguanosine, inosine, etc.). In addition, the bases in a
probe may be joined by a linkage other than a phosphodiester bond,
so long as it does not interfere with hybridization. Thus, for
example, probes may be peptide nucleic acids in which the
constituent bases are joined by peptide bonds rather than
phosphodiester linkages. It will be understood by one of skill in
the art that probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. The probes are
optionally directly labeled as with isotopes chromophores,
lumiphores, chromogens, or indirectly labeled such as with biotin
to which a streptavidin complex may later bind. By assaying for the
presence or absence of the probe, one can detect the presence or
absence of the select sequence or subsequence.
[0080] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0081] A "promoter" is defined as an array of nucleic acid
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element. A promoter also
optionally includes distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0082] 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.
[0083] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0084] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes," Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined
ionic strength pH. The Tm is the temperature (under defined ionic
strength, pH, and nucleic concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating a 42.degree. C., or, 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Such hybridizations and wash steps can be
carried out for, e.g., 1, 2, 5, 10, 15, 30, 60; or more
minutes.
[0085] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially related if the
polypeptides that they encode are substantially related. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. Such hybridizations and wash steps can
be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes.
A positive hybridization is at least twice background. Those of
ordinary skill will readily recognize that alternative
hybridization and wash conditions can be utilized to provide
conditions of similar stringency.
[0086] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0087] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms variable light chain (VL) and variable heavy chain (VH) refer
to these light and heavy chains respectively.
[0088] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0089] An "anti-OR" antibody is an antibody or antibody fragment
that specifically binds a polypeptide encoded by a OR gene, cDNA,
or a subsequence thereof, i.e. an IVA OR according to the
invention.
[0090] The term "immunoassay" is an assay that uses an antibody to
specifically bind an antigen. The immunoassay is characterized by
the use of specific binding properties of a particular antibody to
isolate, target, and/or quantify the antigen.
[0091] The phrase "specifically (or selectively) binds" to an
antibody or, "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein at least two times the background and
do not substantially bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, polyclonal
antibodies raised to an IVA OR subgenus member from specific
species such as rat, mouse, or human car be selected to obtain only
those polyclonal antibodies that are specifically immunoreactive
with the IVA OR polypeptide or an immunogenic portion thereof are
not with other proteins, except for orthologs or polymorphic
variants and alleles of the IVA OR polypeptide. This selection may
be achieved by subtracting out antibodies that cross-react with IVA
OR molecules from other species or other IVA OR molecules.
Antibodies can also be selected that recognize only IVA OR GPCR but
not other GPCRs. A variety of immunoassay formats may be used to
select antibodies specifically immunoreactive with a particular IVA
protein. For example, solid-phase ELISA immunoassays are routinely
used to select antibodies specifically immunoreactive with a
protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory
Manual, (1988), for a description of immunoassay formats and
conditions that can be used to determine specific
immunoreactivity). Typically a specific or selective reaction will
be at least twice background signal or noise and more typically
more than 10 to 100 times background.
[0092] The phrase "selectively associates with" refers to the
ability of a nucleic acid to "selectively hybridize" with another
as defined above, or the ability of an antibody to "selectively (or
specifically) bind to a protein, as defined above.
[0093] 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, plan
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.
[0094] By "host cell" is meant a cell that contains an expression
vector and supports the replication or expression of the expression
vector. Host cells may be prokaryotic cells such as E. coli, or
eukaryotic cells such as yeast, insect, amphibian, or mammalian
cells such as CHO, HeLa, HEK-293, and the like, e.g., cultured
cells, explants, and cells in vivo.
Isolation and Expression of IVA Olfactory Receptors
[0095] Isolation and expression of the IVA ORs, or fragments or
variants thereof, of the invention can be performed as described
below. PCR primers can be used for the amplification of nucleic
acids encoding olfactory receptor ligand-binding regions and
libraries of these nucleic acids can thereby be generated.
Libraries of expression vectors can then be used to infect or
transfect host cells for the functional expression of these
libraries. These genes and vectors can be made and expressed in
vitro or in vivo. 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.
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.
[0096] 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.
[0097] Alternatively, these nucleic acids can be synthesized in
vitro by well-know chemical synthesis techniques, as described in,
e.g., Carruthers, Cold Spring Harbor Symp. Quant. Biol. 47:411-418
(1982); Adams, Am. Chem. Soc. 105:661 (1983); Belousov, Nucleic
Acids Res. 25:3440-3444 (1997); Frenkel, Free Radic. Biol. Med.
19:373-380 (1995); Blommers, Biochemistry 33:7886-7896 (1994);
Narang, Meth. Enzymol. 68:90 (1979); Brown, Meth. Enzymol. 68:109
(1979); Beaucage, Tetra. Lett. 22:1859 (1981); 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.
[0098] Techniques for the manipulation of nucleic acids, such as,
for example, for 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).
[0099] 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.
[0100] Oligonucleotide primers are used to amplify nucleic acid
encoding an olfactory receptor ligand-binding region. The nucleic
acids described herein can also be cloned or measured
quantitatively using amplification techniques. Using exemplary
degenerate primer pair sequences, (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, ed. Innis, Academic Press, Inc., N.Y.
(1995), ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560
(1989); Landegren, Science 241:1077, (1988); Barringer, Gene 89:117
(1990)); transcriptior amplification (see, e.g., Kwoh, PNAS,
86:1173 (1989)); and, self-sustained sequence replication (see,
e.g., Guatelli, PNAS, 87:1874 (1990)); Q Beta replicase
amplification (see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491
(1997)); automated Q-beta replicase amplification assay (see, e.g.,
Burg, Mol. Cell. Probes 10:257-271 (1996)) and other RNA polymerase
mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario);
see also Berger, Methods Enzymol. 152:307-316 (1987); Sambrook;
Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan,
Biotechnology 13:563-564 (1995).
[0101] Once amplified, the nucleic acids, either individually or as
libraries, may be cloned according to methods known in the art, 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 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
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 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).
[0102] The primer pairs are designed to selectively amplify
ligand-binding regions of IVA olfactory receptor proteins. These
domain regions may vary for different IVA ORs. Thus, domain regions
of different sizes comprising different domain structure may be
amplified; 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
OR.
[0103] 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 11 through VII can be generated by PCR amplification using
a primer pair. 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 E/DFILLG (SEQ
ID NO: 51). 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).
[0104] 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: 52) (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: 53))).
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).
[0105] The primer pairs are designed to selectively amplify
ligand-binding regions of IVA olfactory receptor proteins. These
domain regions may vary for different IVA ORs. Thus, domain regions
of different sizes comprising different domain structure may be
amplified; 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
OR.
[0106] 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(G/A)SHL (SEQ
ID NO:54), 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:55).
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).
[0107] 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, Nucleic Acids Res. 26:1628-1635 (1998); Singh,
Biotechniques, 24:318-19 (1998)).
[0108] 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, Nucleic Acids Res. 25:4866-4871 (1997). 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, Nat. Struct. Biol. 5:950-954 (1998)).
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, PNAS, 95:4258-63
(1998)). Exemplary degenerate primers of the invention incorporate
the nucleobase analog
5'-Dimethoxytrityl-N-benzoyl-2'-deoxy-Cytidine,3'-[(2-cyanoethyl)--
(N,N-diisopropyl)]-phosphoramidite (the term "P" in the sequences,
see above). This pyrimidine analog hydrogen bond with purines,
including A and G residues.
[0109] Exemplary primer pairs for amplification of olfactory
receptor transmembrane domains II through VII include:
TABLE-US-00003 (a) (SEQ ID NO:56)
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:57)
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/ G/T)TT(CT)
(C/T)T-3'; (b) (SEQ ID NO:58)
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:59)
5'-GGGGCTGCAGACACC(AC/G/T)ATGTA(C/T)(C/T)T(A/C/G/ T)TT(C/T)
(C/T)T-3'; (c) (SEQ ID NO:60)
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:61)
5'-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/G/ T)TT(C/T)
(C/T)T-3'; (d) (SEQ ID NO:62) primer TM7I
5'-AT(G/A)AAIGG(G/A)TTIA(G/A)GATIGG; (e) (SEQ ID NO:63) primer TM3D
5'-ATGGCITA(C/T)GA(C/T)(A/C)GOTA(C/T)G TIGC; (f) (SEQ ID NO:64)
primer 213 5'-CGGATCCGCITA(C/G)GA(C/T)GCITA(C/T)GT
IGC(A/C/G/T)AT(A/C/T)TG; (g) (SEQ ID NO:65) primer 214
5'-CCTGCAG(A/G)TA(A/G/T)AT(A/G)AAIGG(A/ G)TTIA(A/G)CAT(A/C/G/T)GG;
and (h) (SEQ ID NO:66) primer p41
5'-AA(A/G)(G/T)CITTI(C/G/T)(A/C)IACITG (C/T)G(C/G)ITCICA,
where I represents 2'-deoxyinosine, and O represents
2'-deoxycytidine phosphorothioate.
[0110] Nucleic acids that encode ligand-binding regions of IVA
olfactory receptors may be generated 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.
[0111] Isolation from olfactory receptor-expressing cells is well
known in the art (cells expressing naturally or inducibly
expressing olfactory receptors can be used in 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, PNAS, 93:9858-63 (1996)). Shirley, Eur. J. Biochem.
32:485-494 (1983: 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,
Chem. Senses 24:211-216 (1999). 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, J. Neurosci.
19:8260-8270 (1999), describes differentiated olfactory
receptor-expressing cells in culture that respond to odorants, as
measured by an influx of calcium.
[0112] In one embodiment, hybrid protein-coding sequences
comprising nucleic acids ORs fused to the translocation sequences
described herein may be constructed. Also provided are hybrid ORs
comprising the translocation motifs and 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, and 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, 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, J. Neurosci. 18:227-236 (1998)). 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.
[0113] In another embodiment, fusion proteins, either having
C-terminal or, more preferably, N-terminal translocation sequences,
may also comprise the translocation motif described herein.
However, these fusion proteins can also comprise addition elements
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.).
[0114] The inclusion of a cleavable linker sequences such as Factor
Xa (see, e.g. Ottavi, Biochimie 80:289-293 (1998)), subtilisin
protease recognition motif (see, e.g. Polyak, Protein Eng.
10:615-619 (1997)); enterokinase (invitrogen, San Diego, Calif. and
the like, between the translocation domain (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,
Biochemistry 34:1787-1797 (1995)), 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, DNA Cell. Biol. 12:441-53 (1993)).
[0115] Expression vectors, either as individual expression vectors
or as libraries expression vectors, comprising the olfactory
binding domain-encoding sequences 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, Nature
328:731 (1987); Berger supra; Schneider, Protein Expr. Purif.
6435:10 (1995); Sambrook; Tijssen; 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.
[0116] The nucleic acids 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, Gene 190:315-17 (1997); Aubrecht, J.
Pharmacol. Exp. Ther., 281:992-97 (1997)). 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.
[0117] A chimeric nucleic acid sequence may encode a ligand-binding
domain within any 7-transmembrane polypeptide. 7-transmembrane
receptors belong to a superfamily of transmembrane (TM) proteins
having seven domains that traverse 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 with 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, Protein Science 8:969-977
(1999); Rost, Protein Sci. 4:521-533 (1995). Periodicity detection
enhancement and alpha helical periodicity index can be done as by,
e.g., Donnelly, Protein Sci. 2:55-70 (1993). Other alignment and
modeling algorithms are well known in the art, see, e.g., Peitsch,
Receptors Channels 4:161-164 (1996); Cronet, Protein Eng. 6:59-64
(1993) (homology and "discover modeling");
http://bioinfo.weizmann.ac.il/.
[0118] The library sequences 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 TI 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.
[0119] Libraries of olfactory receptor ligand-binding TM domain
sequences can include a various TM domains or variations thereof,
as described 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 supra), as described above.
Using this information sequences flanking the seven domains can be
identified and used to design degenerate primers for amplification
of various combinations of TM regions and subsequences.
[0120] The present invention also includes not only the DNA and
proteins having the specified amino acid sequences corresponding to
IVA olfactory receptors, but also fragments thereof, particularly
DNA fragments of, for example, 40, 60, 80, 100, 150, 200, or 250
nucleotides, or more, as well as protein fragments of, for example,
10, 20, 30, 50, 70, 100, or 150 amino acids, or more.
[0121] Also contemplated are chimeric proteins, comprising at least
10, 20, 30, 50, 70, 100, or 150 amino acids, or more, of one of at
least one of the IVA olfactory receptors described herein, coupled
to additional amino acids representing all or part of another G
protein receptor, preferably a member of the 7.TM. superfamily.
These chimeras can be made from the instant receptors and a G
protein receptor described herein, or they can be made by combining
two or more of the present proteins. In one preferred embodiment,
one portion of the chimera corresponds to and is derived from one
or more of the domains of the seven transmembrane protein described
herein, and the remaining portion or portions come from another G
protein-coupled receptor. Chimeric receptors are well known in the
art, and the techniques for creating them and the selection and
boundaries of domains or fragments of G protein-coupled receptors
for incorporation therein are also well known. Thus, this knowledge
of those skilled in the art can readily be used to create such
chimeric receptors. The use of such chimeric receptors can provide,
for example, an olfactory selectivity characteristic of one of the
IVA olfactory receptors specifically disclosed herein, coupled with
the signal transduction characteristics of another receptor, such
as a well known receptor used in prior art assay systems.
[0122] For example, a domain such as a ligand-binding domain, an
extracellular domain, a transmembrane domain (e.g., one comprising
seven transmembrane regions and corresponding extracellular and
cytosolic loops), the transmembrane domain and a cytoplasmic
domain, an active site, a subunit association region, etc. can be
covalently linked to a heterologous protein. For instance, an
extracellular domain can be linked to a heterologous GPCR
transmembrane domain, or a heterologous GPCR extracellular domain
can be linked to a transmembrane domain. Other heterologous
proteins of choice can include, e.g., green fluorescent protein,
.beta.-gal, glutamate receptor, and the rhodopsin presequence.
[0123] Polymorphic variants, alleles, and interspecies homologs
that are substantially identical to an IVA olfactory receptor
disclosed herein can be isolated using the nucleic acid probes
described above. It is hypothesized that allelic differences in
receptors may explain why there is a difference in olfactory
sensation in different human subjects. Accordingly, the
identification of such alleles may be significant, especially with
respect to producing receptor libraries that adequately represent
the olfactory capability of the human population, i.e., which take
into account allelic differences in different individuals.
Alternatively, expression libraries can be used to clone olfactory
receptors and polymorphic variants, alleles, and interspecies
homologs thereof, by detecting expressed homologs immunologically
with antisera or purified antibodies made against an olfactory
polypeptide, which also recognize and selectively bind to the
olfactory receptor homolog.
[0124] Also within the scope of the invention are host cells for
expressing the IVA ORs, fragments, chimeras or variants of the
invention. To obtain high levels of expression of a cloned gene or
nucleic acid, such as cDNAs encoding the IVA olfactory receptors,
fragments, or variants of the invention, one of skill typically
subclones the nucleic acid sequence of interest into an expression
vector that contains a strong promoter to direct transcription, a
transcription/translation terminator, and if for a nucleic acid
encoding a protein, a ribosome binding site for translational
initiation. Suitable bacterial promoters are well known in the art
and described, e.g., in Sambrook et al., However, bacterial or
eukaryotic expression systems can be used.
[0125] Any of the well-known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, liposomes, microinjection, plasma vectors,
viral vectors and any of the other well known methods for
introducing cloned genomic DNA, cDNA, synthetic DNA or other
foreign genetic material into a host cell (see, e.g., Sambrook et
al.) It is only necessary that the particular genetic engineering
procedure used be capable of successfully introducing at lest one
gene into the host cell capable of expressing the olfactory
receptor, fragment, or variant of interest.
[0126] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of the receptor, fragment, or variant of interest, which
is then recovered from the culture using standard techniques.
Examples of such techniques are well known in the art. See, e.g.,
WO 00/06593, which is incorporated by reference in a manner
consistent with this disclosure.
Immunological Detection of IVA OR Polypeptides
[0127] In addition to the detection of IVA OR genes and gene
expression using nucleic acid hybridization technology, one can
also use immunoassays to detect IVA ORs, e.g., to identify
olfactory receptor cells, and variants of IVA OR genus members.
Immunoassays can be used to qualitatively or quantitatively analyze
the IVA ORs. A general overview of the applicable technology can be
found in Harlow & Lane, Antibodies: A Laboratory Manual
(1988).
Antibodies to IVA or Family Members
[0128] Methods of producing polyclonal and monoclonal antibodies
that react specifically with an IVA OR family member are known to
those of skill in the art (see e.g., Coligan, Current Protocols in
Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal
Antibodies: Principles and Practice (2d ed. 1986); and Kohler &
Milstein, Nature, 256:495-97 (1975)). Such techniques include
antibody preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors, as well as
preparation of polyclonal and monoclonal antibodies by immunizing
rabbits or mice (see, e.g., Huse et al., Science, 246:1275-81
(1989); Ward et al., Nature, 341:544-46 (1989)).
[0129] A number of IVA OR-comprising immunogens may be used to
produce antibodies specifically reactive with an IVA OR family
member. For example, a recombinant IVA OR protein, or an antigenic
fragment thereof, can be isolated as described herein. Suitable
antigenic regions include, e.g., the conserved motifs the are used
to identify members of the OR family. Recombinant proteins can be
expressed in eukaryotic or prokaryotic cells as described above,
and purified as generally described above. Recombinant protein is
the preferred immunogen for the production of monoclonal or
polyclonal antibodies. Alternatively, a synthetic peptide derived
from the sequences disclosed herein and conjugated to a carrier
protein can be used an immunogen. Naturally occurring protein may
also be used either in pure or impure form. The product is then
injected into an animal capable of producing antibodies. Either
monoclonal or polyclonal antibodies may be generated, for
subsequent use in immunoassays to measure the protein.
[0130] Methods of production of polyclonal antibodies are known to
those of skill in the art. For example, an inbred strain of mice
(e.g., BALB/C mice) or rabbits may be immunized with the protein
using a standard adjuvant, such as Freund's adjuvant, and a
standard immunization protocol. The animal's immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the OR. When appropriately
high titers of antibody to the immunogen are obtained, blood is
collected from the animal and antisera are prepared. Further
fractionation of the antisera to enrich for antibodies reactive to
the protein can be done if desired (see Harlow & Lane,
supra).
[0131] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen may be immortalized,
commonly by fusion with a myeloma cell (see Kohler & Milstein,
Eur. J. Immunol., 6:511-19 (1976)). Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods well known in the art.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity
for the antigen, and yield of the monoclonal antibodies produced by
such cells may be enhanced by various techniques, including
injection into the peritoneal cavity of a vertebrate host.
Alternatively, one may isolate DNA sequences which encode a
monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells according to the general protocol
outlined by Huse et al., Science, 246:1275-1281 (1989).
[0132] Monoclonal antibodies and polyclonal sera are collected and
titered again: the immunogen protein in an immunoassay, for
example, a solid phase immunoassay with the immunogen immobilized
on a solid support. Typically, polyclonal antisera with a titer of
109 or greater are selected and tested for their cross reactivity
against non-OR proteins, or other OR family members or other
related proteins from other organisms, using a competitive binding
immunoassay. Specific polyclonal antisera and monoclonal antibodies
will usually bind with a Kd of at least about 0.1 mM, more usually
at least about 1 pM, optionally at least about 0.1 pM or better,
and optionally 0.01 pM or better.
[0133] Once OR family member specific antibodies are available,
individual OR proteins can be detected by a variety of immunoassay
methods. For a review of immunological and immunoassay procedures,
see Basic and Clinical Immunology (Stites & Terr eds., 7th ed.
1991). Moreover, the immunoassays of the present invention can be
performed in any of several configurations, which are reviewed
extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow
& Lane, supra.
Immunological Binding Assays
[0134] IVA OR proteins can be detected and/or quantified using any
of a number of well recognized immunological binding assays (see,
e.g., U.S. Pat. Nos. 4,366,241 4,376,110; 4,517,288; and
4,837,168). For a review of the general immunoassays, see also
Methods in Cell Biology: Antibodies in Cell Biology, volume 37
(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr,
eds., 7th ed. 1991). Immunological binding assays (or immunoassays)
typically use an antibody that specifically binds to a protein or
antigen of choice (in this case an OR family member or an antigenic
subsequence thereof). The antibody (e.g., anti-OR) may t produced
by any of a number of means well known to those of skill in the art
and a described above.
[0135] Immunoassays also often use a labeling agent to specifically
bind to and label the complex formed by the antibody and antigen.
The labeling agent may itself be one of the moieties comprising the
antibody/antigen complex. Thus, the labeling agent may be a labeled
OR polypeptide or a labeled anti-OR antibody. Alternatively, the
labeling agent may be a third moiety, such a secondary antibody
that specifically binds to the antibody/OR complex (a secondary
antibody is typically specific to antibodies of the species from
which the first antibody is derived). Other proteins capable of
specifically binding immunoglobulin constant regions, such as
protein A or protein G may also be used as the label agent. These
proteins exhibit strong non-immunogenic reactivity with
immunoglobulin constant regions from a variety of species (see,
e.g., Kronval et al., J. Immunol., 111:1401-1406 (1973); Akerstrom
et al., J. Immunol., 135:2589-2542 (1985)). The labeling agent can
be modified with a detectable moiety, such as biotin, to which
another molecule can specifically bind, such as streptavidin. A
variety of detectable moieties are well known to those skilled in
the art.
[0136] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, optionally from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, antigen, volume of solution,
concentrations, and the like. Usually, the assays will be carried
out at ambient temperature, although they can be conducted over a
range of temperatures, such a 10.degree. C. to 40.degree. C.
Non-Competitive Assay Formats
[0137] Immunoassays for detecting an IVA OR protein in a sample may
be either competitive or noncompetitive. Noncompetitive
immunoassays are assays in which the amount of antigen is directly
measured. In one preferred "sandwich" assay, for example, the
anti-IVA OR antibodies can be bound directly to a solid substrate
on which they are immobilized. These immobilized antibodies then
capture the OR protein present in the test sample. The IVA OR
protein is thus immobilized is then bound by a labeling agent, such
as a second OR antibody bearing a label. Alternatively, the second
antibody may lack a label, but it may, in turn, be bound by a
labeled third antibody specific to antibodies of the species from
which the second antibody is derived. The second or third antibody
is typically modified with a detectable moiety, such as biotin, to
which another molecule specifically binds, e.g. streptavidin, to
provide a detectable moiety.
Competitive Assay Formats
[0138] In competitive assays, the amount of IVA OR protein present
in the sample is measured indirectly by measuring the amount of a
known, added (exogenous) IVA OR protein displaced (competed away)
from an anti-IVA OR antibody by the unknown IVA OR protein present
in a sample. In one competitive assay, a known amount of IVA OR
protein is added to a sample and the sample is then contacted with
an antibody that specifically binds to the IVA OR. The amount of
exogenous IVA OR protein bound to the antibody is inversely
proportional to the concentration of IVA OR protein present in the
sample. In a particularly preferred embodiment, the antibody is
immobilized on a solid substrate. The amount of IVA OR protein
bound to the antibody may be determined either by measuring the
amount of IVA OR protein present in an IVA OR/antibody complex, or
alternatively by measuring the amount of remaining uncomplexed
protein. The amount of IVA OR protein may be detected by providing
a labeled OR molecule.
[0139] A hapten inhibition assay is another preferred competitive
assay. In this assay the known IVA OR protein is immobilized on a
solid substrate. A known amount of anti-IVA OR antibody is added to
the sample, and the sample is then contacted with the immobilized
IVA OR. The amount of anti-IVA OR antibody bound to the known
immobilized OR protein is inversely proportional to the amount of
IVA OR protein present in the sample. Again, the amount of
immobilized antibody may be detected by detecting either the
immobilized fraction of antibody or the fraction of the antibody
that remains in solution. Detection may be direct where the
antibody is labeled or indirect by the subsequent addition of a
labeled moiety that specifically binds to the antibody as described
above.
Cross-Reactivity Determinations
[0140] Immunoassays in the competitive binding format can also be
used for cross-reactivity determinations. For example, a protein at
least partially encoded by the nucleic acid sequences disclosed
herein can be immobilized to a solid support. Proteins (e.g., IVA
OR proteins and homologs) are added to the assay that competes for
binding of the antisera to the immobilized antigen. The ability of
the added proteins to compete for binding of the antisera to the
immobilized protein is compared to the ability of the IVA OR
polypeptide encoded by the nucleic acid sequences disclosed herein
to compete with itself. The percent cross-reactivity for the above
proteins is calculated, using standard calculations. Those antisera
with less than 10% cross-reactivity with each of the added proteins
listed above are selected and pooled. The cross-reacting antibodies
are optionally removed from the pooled antisera by immunoabsorption
with the added considered proteins, e.g., distantly related
homologs. In addition, peptides comprising amino acid sequences
representing conserved motifs that are used to identify members of
the OR family can be used in cross-reactivity determinations.
[0141] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein, thought to be perhaps an allele or polymorphic
variant of a OR family member, to the immunogen protein (i.e., IVA
OR protein encoded by the nucleic acid sequences disclosed herein).
In order to make this comparison, the two proteins are each assayed
at a wide range of concentrations and the amount of each protein
required to inhibit 50% of the binding of the antisera to the
immobilized protein is determined If the amount of the second
protein required to inhibit 50% of binding is less than 10 times
the amount of the protein encoded by nucleic acid sequences
disclosed herein required to inhibit 50% of binding, then the
second protein is said to specifically bind to the polyclonal
antibodies generated to an IVA OR immunogen.
[0142] Antibodies raised against OR conserved motifs can also be
used to prepare antibodies that specifically bind only to GPCRs of
the OR family, but not to GPCRs from other families.
[0143] Polyclonal antibodies that specifically bind to a particular
member of the IVA or subgenus disclosed herein can be make by
subtracting out cross-reactive antibodies using other OR family
members. Species-specific polyclonal antibodies can be made in a
similar way. For example, antibodies specific to a particular human
IVA OR can be made by, subtracting out antibodies that are
cross-reactive with orthologous sequences, e.g., the murine
counterpart.
Other Assay Formats
[0144] Western blot (immunoblot) analysis is used to detect and
quantify the presence of IVA OR protein in the sample. The
technique generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support, (such as a
nitrocellulose filter, a nylon filter, or derivatized nylon
filter), and incubating the sample with the antibodies that
specifically bind the IVA OR protein. The anti-IVA OR polypeptide
antibodies specifically bind to the IVA OR polypeptide on the solid
support. These antibodies may be directly labeled or alternatively
may be subsequently detected using labeled antibodies (e.g.,
labeled sheep anti-mouse antibodies) that specifically bind to the
anti-IVA OR antibodies.
[0145] Other, assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see Monroe et al., Amer. Clin. Prod. Rev., 5:34-41
(1986)).
Reduction of Non-Specific Binding
[0146] One of skill in the art will appreciate that it is often
desirable to minimize non-specific binding in immunoassays.
Particularly, where the assay involves an antigen or antibody
immobilized on a solid substrate it is desirable to minimize the
amount of non-specific binding to the substrate. Means of reducing
such non-specific binding are well known to those of skill in the
art. Typically, this technique involves coating the substrate with
a proteinaceous composition. In particular, protein compositions
such as bovine serum albumin (BSA), nonfat powdered milk, and
gelatin are widely used with powdered milk being most
preferred.
Labels
[0147] The particular label or detectable group used in the assay
is not a critical aspect of the invention, as long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., DYNABEADS.TM.) (SEQ ID NO:
62), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,
rhodamine, and the like), radiolabels (e.g., .sup.3H, .sup.125I,
.sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horseradish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and calorimetric labels such as colloidal gold or colored
glass or plastic beads (e.g., polystyrene, polypropylene, latex,
etc.).
[0148] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0149] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to another molecules (e.g.,
streptavidin) molecule, which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. The ligands
and their targets can be used in any suitable combination with
antibodies that recognize a OF protein, or secondary antibodies
that recognize anti-OR.
[0150] The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidotases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds
include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
For a review of various labeling or signal producing systems that
may be used, see U.S. Pat. No. 4,391,904.
[0151] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple calorimetric labels may be
detected simply by observing the color associate with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink while various conjugated beads appear the color of the
bead.
[0152] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need be
labeled and the presence of the target antibody is detected by
simple visual inspection.
Detection of IVA Olfactory Receptor Modulators
[0153] Methods and compositions for determining whether a test
compound specifically binds to a mammalian chemosensory, and more
particularly, an IVA olfactory receptor of the invention, both in
vitro and in vivo are described below. Many aspects of cell
physiology can be monitored to assess the effect of ligand-binding
to a naturally-occurring or chimeric olfactory receptor. These
assays may be performed on intact cells expressing an olfactory
receptor, on permeabilized cells or on membrane fractions produced
by standard methods.
[0154] 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. An activated or inhibited G protein will in turn alter the
properties of target enzymes, channels, and other effector
proteins. Some examples include the activation of cGMP
phosphodiesterase by transducin in the visual system, adenylate
cyclase by the stimulatory G protein, phospholipase C by Gq and
other cognate G proteins, and modulation of diverse channels by Gi
and other G proteins. Downstream consequences can also be examined
such as generation of diacyl glycerol and IP3 by phospholipase C,
and in turn, for calcium mobilization by IP3.
[0155] Preferably, the amino acid sequence identity will be at
least 50-75% preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
Optionally, the polypeptide of the assays can comprise a domain of
an IVA OR protein, such as an extracellular domain, transmembrane
region, transmembrane domain, cytoplasmic domain, ligand-binding
domain, subunit association domain, active site, and the like.
Either the IVA OR protein or a domain thereof can be covalently
linked to a heterologous protein to create a chimeric protein used
in the assays described herein. The subgenus of IVA ORs provided
herein exhibits substantial sequence similarity at both the DNA and
protein level, but also significant dissimilarly. With respect
thereto, the OR family members possess an average percentage
sequence identity to other members of the family when determined
over the full length of the gene by about 30%. Moreover, different
members of the genes at the protein level exhibit an average on the
order of about 40% sequence identity to other members of the family
when the full length protein sequences are compared. However, while
there exist differences, there are characteristic similarities,
e.g. the consensus sequence already mentioned, which further define
members of this novel genus of receptors.
[0156] Modulators of IVA OR activity can be tested using IVA OR
polypeptides as described above, either recombinant or naturally
occurring. The protein can be isolated, expressed in a cell,
expressed in a membrane derived from a cell, expressed in tissue or
in an animal, either recombinant or naturally occurring. Modulation
can be tested using one of the in vitro or in vivo assays described
herein.
In Vitro Binding Assays
[0157] Olfactory transduction can also be examined in vitro with
soluble or solid state reactions, using a full-length IVA OR or a
chimeric molecule such as an extracellular domain or transmembrane
region, or combination thereof, of a IVA OR covalently linked to a
heterologous signal transduction domain, or a heterologous
extracellular domain and/or transmembrane region covalently linked
to the transmembrane and/or cytoplasmic domain of an IVA OR.
Furthermore, ligand-binding domains of the protein of interest can
be used in vitro in soluble or solid state reactions to assay for
ligand binding. In numerous embodiments, a chimeric receptor will
be made that comprises all or part of an IVA OR polypeptide, as
well an additional sequence that facilitates the localization of
the IVA OR to the membrane, such as a rhodopsin, e.g., an
N-terminal fragment of a rhodopsin protein, e.g. bovine or another
mammalian rhodopsin. Ligand binding to a IVA OR protein, a domain,
or chimeric protein can be tested in solution, in a bilayer
membrane, attached to a solid phase, in a lipid monolayer, or in
vesicles. Binding of a modulator can be tested using, e.g., changes
in spectroscopic characteristics (e.g., fluorescence, absorbence,
refractive index) hydrodynamic (e.g., shape), chromatographic, or
solubility properties.
[0158] Receptor-G protein interactions can also be examined. For
example, binding of the G protein to the receptor or its release
from the receptor can be examined. For example, in the absence of
GTP, an activator will lead to the formation of a tight complex of
a G protein (all three subunits) with the receptor. This complex
can be detected in a variety of ways, as noted above. Such an assay
can be modified to search for inhibitors, e.g., by adding an
activator to the receptor and G protein in the absence of GTP,
which form a tight complex, and then screen for inhibitors by
looking at dissociation of the receptor-G protein complex. In the
presence of GTP, release of the alpha subunit of the G protein from
the other two G protein subunits serves as a criterion of
activation.
[0159] Particularly preferred G proteins include the chimeric and
variant G proteins disclosed in U.S. Provisional Application No.
60/243,770, filed on Oct. 30, 2000; U.S. Ser. No. 09/984,292 filed
Oct. 29, 2001; and U.S. Ser. No. 09/989,497 filed Nov. 21, 2001.
These G proteins have been altered such that they exhibit greater
coupling efficiencies with specific olfactory or taste
receptor.
[0160] An activated or inhibited G protein will in turn alter the
properties of target enzymes, channels, and other effector
proteins. The classic examples are the activation of cGMP
phosphodiesterase by transducin in the visual system, adenylate
cyclase by the stimulatory G protein, phospholipase C by Gq and
other cognate G proteins, and modulation of diverse channels by Gi
and other G proteins. Downstream consequences can also be examined
such as generation of diacyl glycerol and IP3 by phospholipase C,
and in turn, for calcium mobilization by IP3.
[0161] In another embodiment of the invention, a GTP.gamma.S assay
may be used. As described above, upon activation of a GPCR, the
G.alpha. subunit of the G protein complex is stimulated to exchange
bound GDP for GTP. Ligand-mediated stimulation of G protein
exchange activity can be measured in a biochemical assay measuring
the binding of added radioactively-labeled GTP.gamma..sup.35S to
the G protein in the presence of a putative ligand. Typically,
membranes containing the chemosensory receptor of interest are
mixed with a complex of G proteins. Potential inhibitors and/or
activators and GTP.gamma.S are added to the assay, and binding of
GTP.gamma.S to the G protein is measured. Binding can be measured
by liquid scintillation counting or by any other means known in the
art, including scintillation proximity assays (SPA). In other
assays formats, fluorescently-labeled GTP.gamma.S can be
utilized.
Fluorescence Polarization Assays
[0162] In another embodiment, Fluorescence Polarization ("FP")
based assays may be used to detect and monitor odorant binding.
Fluorescence polarization is a versatile laboratory technique for
measuring equilibrium binding, nucleic acid hybridization, and
enzymatic activity. Fluorescence polarization assays are
homogeneous in that they do not require a separation step such as
centrifugation, filtration, chromatography, precipitation or
electrophoresis. These assays are done in real time, directly in
solution and do not require an immobilized phase. Polarization
values can be measured repeatedly and after the addition of
reagents since measuring the polarization is rapid and does not
destroy the sample. Generally, this technique can be used to
measure polarization values of fluorophores from low picomolar to
micromolar levels. This section describes how fluorescence
polarization can be used in a simple and quantitative way to
measure the binding of odorants to the olfactory receptors of the
invention.
[0163] When a fluorescently labeled molecule is excited with plane
polarized light, it emits light that has a degree of polarization
that is inversely proportional to its molecular rotation. Large
fluorescently labeled molecules remain relatively stationary during
the excited state (4 nanoseconds in the case of fluorescein) and
the polarization of the light remains relatively constant between
excitation and emission. Small fluorescently labeled molecules
rotate rapidly during the excited state and the polarization
changes significantly between excitation and emission. Therefore,
small molecules have low polarization values and large molecules
have high polarization values. For example, a single-stranded
fluorescein-labeled oligonucleotide has a relatively low
polarization value but when it is hybridized to a complementary
strand, it has a higher polarization value. When using FP to detect
and monitor odorant-binding which may activate or inhibit the
olfactory receptors of the invention, fluorescence-labeled odorants
or auto-fluorescent odorants may be used.
[0164] Fluorescence polarization (P) is defined as:
P = Int .PI. - Int .perp. Int .PI. ##EQU00001##
where .PI. is the intensity of the emission light parallel to the
excitation light plane and Int .perp. is the intensity of the
emission light perpendicular to the excitation light plane. P,
being a ratio of light intensities, is a dimensionless number. For
example, the Beacon.RTM. and Beacon 2000.TM. System may be used in
connection with these assays. Such systems typically express
polarization in millipolarization units (1 Polarization Unit=1000
mP Units).
[0165] The relationship between molecular rotation and size is
described by the Perrin equation and the reader is referred to
Jolley, M. E. (1991) in Journal of Analytical Toxicology, pp.
236-240, which gives a thorough explanation of this equation.
Summarily, the Perrin equation states that polarization is directly
proportional to the rotational relaxation time, the time that it
takes a molecule to rotate through an angle of approximately 68.50
Rotational relaxation time is related to viscosity (.eta.),
absolute temperature (T), molecular volume (V), and the gas
constant (R) by the following equation:
Rotational Relaxation Time = 3 .eta. V RT ##EQU00002##
[0166] The rotational relaxation time is small (.apprxeq.1
nanosecond) for small molecules (e.g. fluorescein) and large
(.apprxeq.100 nanoseconds) for large molecules (e.g.
immunoglobulins). If viscosity and temperature are held constant,
rotational relaxation time, and therefore polarization, is directly
related to the molecular volume. Changes in molecular volume may be
due to interactions with other molecules, dissociation,
polymerization, degradation, hybridization, or conformational
changes of the fluorescently labeled molecule. For example,
fluorescence polarization has been used to measure enzymatic
cleavage of large fluorescein labeled polymers by proteases,
DNases, and RNases. It also has been used to measure equilibrium
binding for protein/protein interactions, antibody/antigen binding,
and protein/DNA binding.
Solid State and Soluble High Throughout Assays
[0167] In yet another embodiment, the invention provides soluble
assays using molecules such as a domain such as ligand-binding
domain, an extracellular domain, a transmembrane domain (e.g., one
comprising seven transmembrane regions and cytosolic loops), the
transmembrane domain and a cytoplasmic domain, an active site, a
subunit association region, etc.; a domain that is covalently
linked to a heterologous protein to create a chimeric molecule; an
IVA OR protein; or a cell or tissue expressing an IVA OR protein,
either naturally occurring or recombinant. In another embodiment,
the invention provides solid phase based in vitro assays in a high
throughput format, where the domain, chimeric molecule, IVA OR
protein, or cell or tissue expressing the IVA OR is attached to a
solid phase substrate.
[0168] In the high throughput assays of the invention, it is
possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter
plate can be used to run a separate assay against a selected
potential modulator, or, if concentration or incubation time
effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 1000 to about 1500
different compounds. It is also possible to assay multiple
compounds in each plate well. Further, it is possible to assay
several different plates per day; assay screens for up to about
6,000-20,000 different compounds is possible using the integrated
systems of the invention. More recently, microfluidic approaches to
reagent manipulation have been developed.
[0169] The molecule of interest can be bound to the solid state
component, directly or indirectly, via covalent or non covalent
linkage, e.g., via a tag. The tag can be any of a variety of
components. In general, a molecule which binds the tag (a tag
binder) is fixed to a solid support, and the tagged molecule of
interest (e.g., the olfactory transduction molecule of interest) is
attached to the solid support by interaction of the tag and the tag
binder.
[0170] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an immunoglobulin, etc.). Antibodies to molecules with
natural binders such as biotin are also widely available and
appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.).
[0171] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody which recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs. For example, agonists and antagonists of cell
membrane receptors (e.g., cell receptor-ligand interactions such as
transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherein family, the integrin
family, the selectin family, and the like; see, e.g., Pigott &
Power, The Adhesion Molecule Facts Book I (1993)). Similarly,
toxins and venoms, viral epitopes, hormones (e.g., opiates,
steroids, etc.), intracellular receptors (e.g., which mediate the
effects of various small ligands, including steroids, thyroid
hormone, retinoids and vitamin D; peptides), drugs, lectins,
sugars, nucleic acids (both linear and cyclic polymer
configurations), oligosaccharides, proteins, phospholipids and
antibodies can all interact with various cell receptors.
[0172] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0173] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly gly sequences of between about 5 and 200 amino acids. Such
flexible linkers are known to persons of skill in the art. For
example, poly(ethelyne glycol) linkers are available from
Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally
have amide linkages, sulfhydryl linkages, or heterofunctional
linkages.
[0174] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent that fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example groups that are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature. See, e.g., Merrifield, J. Am. Chem.
Soc., 85:2149-54 (1963) (describing solid phase synthesis of, e.g.,
peptides); Geysen et al., J. Immun. Meth. 102:259-74 (1987)
(describing synthesis of solid phase components on pins); Frank
& Doring, Tetrahedron, 44:60316040 (1988) (describing synthesis
of various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-77 (1991); Sheldon et. al., Clinical Chemistry,
39(4):718-19 (1993); and Kozal et al., Nature Medicine, 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
Computer-Based Assays
[0175] Yet another assay for compounds that modulate IVA OR protein
activity involves computer assisted compound design, in which a
computer system is used to generate a three-dimensional structure
of an IVA OR protein based on the structural information encoded by
its amino acid sequence. The input amino acid sequence interacts
directly and actively with a pre-established algorithm in a
computer program to yield secondary, tertiary, and quaternary
structural models of the protein. The models of the protein
structure are then examined to identify regions of the structure
that have the ability to bind, e.g., ligands. These regions are
then used to identify ligands that bind to the protein.
[0176] The three-dimensional structural model of the protein is
generated by entering protein amino acid sequences of at least 10
amino acid residues or corresponding nucleic acid sequences
encoding an IVA OR polypeptide into the computer system. The
nucleotide sequence encoding the polypeptide, or the amino acid
sequence thereof, can be any of the IVA or polypeptides already
identified or chimeras, variants or fragments thereof.
[0177] The amino acid sequence represents the primary sequence or
subsequence of the protein, which encodes the structural
information of the protein. At least 10 residues of the amino acid
sequence (or a nucleotide sequence encoding 10 amino acids) are
entered into the computer system from computer keyboards, computer
readable substrates that include, but are not limited to,
electronic storage media (e.g., magnetic diskettes, tapes,
cartridges, and chips), optical media (e.g., CD ROM), information
distributed by internet sites, and by RAM The three-dimensional
structural model of the protein is then generated by the
interaction of the amino acid sequence and the computer system,
using software known to those of skill in the art.
[0178] The amino acid sequence represents a primary structure that
encodes the information necessary to form the secondary, tertiary
and quaternary structure of the protein of interest. The software
looks at certain parameters encoded by the primary sequence to
generate the structural model. These parameters are referred to as
"energy terms," and primarily include electrostatic potentials,
hydrophobic potentials, solvent accessible surfaces, and hydrogen
bonding. Secondary energy terms include van der Waals potentials.
Biological molecules form the structures that minimize the energy
terms in a cumulative fashion. The computer program is therefore
using these terms encoded by the primary structure or amino acid
sequence to create the secondary structural model.
[0179] The tertiary structure of the protein encoded by the
secondary structure is then formed on the basis of the energy terms
of the secondary structure. The user at this point can enter
additional variables such as whether the protein is membrane bound
or soluble, its location in the body, and its cellular location,
e.g., cytoplasmic surface, or nuclear. These variables along with
the energy terms of the secondary structure are used to form the
model of the tertiary structure. In modeling the tertiary
structure, the computer program matches hydrophobic faces of
secondary structure with like, and hydrophilic faces of secondary
structure with like.
[0180] Once the structure has been generated, potential
ligand-binding regions are identified by the computer system.
Three-dimensional structures for potential ligands are generated by
entering amino acid or nucleotide sequences or chemical formulas of
compounds, as described above. The three-dimensional structure of
the potential ligand is then compared to that of the IVA OR protein
to identify ligand that bind to the protein. Binding affinity
between the protein and ligands is determined using energy terms to
determine which ligands have an enhanced probability of binding to
the protein.
[0181] Computer systems are also used to screen for mutations,
polymorphic variants, alleles and interspecies homologs of IVA OR
genes. Such mutations can be associated with disease states or
genetic traits. As described above, GeneChip.TM. and related
technology can also be used to screen for mutations, polymorphic
variants, alleles and interspecies homologs. Once the variants are
identified, diagnostic assays can be used to identify patients
having such mutated genes. Identification of the mutated IVA OR
genes involves receiving input of a first nucleic acid or amino
acid sequence of a IVA OR gene, or conservatively modified versions
thereof. The sequence is entered into the computer system as
described above. The first nucleic acid or amino acid sequence is
then compared to a second nucleic acid or amino acid sequence that
has substantial identity to the first sequence. The second sequence
is entered into the computer system in the manner described above.
Once the first and second sequences are compared, nucleotide or
amino acid differences between the sequences are identified. Such
sequences can represent allelic differences in various IVA OR
genes, and mutation associated with disease states and genetic
traits.
Cell-Based Binding Assays
[0182] In a preferred embodiment, an IVA OR polypeptide is
expressed in a eukaryotic cell as a chimeric receptor with a
heterologous, chaperone sequence the facilitates its maturation and
targeting through the secretory pathway. For example the
heterologous sequence can comprise a rhodopsin sequence, such as an
N-terminal fragment of a rhodopsin. Such chimeric IVA OR receptors
can be expressed in any eukaryotic cell, such as HEK-293 cells.
Preferably, the cells comprise a functional G protein, e.g.,
G.alpha.15, that is capable of coupling the chimeric receptor to an
intracellular signaling pathway or to a signaling protein such as
phospholipase C or the chimeric or variant G proteins previously
identified. Activation of such chimeric receptors in such cells can
be detected using any standard method, such as by detecting changes
in intracellular calcium by detecting FURA-2 dependent fluorescence
in the cell.
[0183] Activated GPCR receptors become substrates for kinases that
phosphorylate the C-terminal tail of the receptor (and possibly
other sites as well). Thus, activators will promote the transfer of
.sup.32P from gamma-labeled GTP to the receptor, which can be
assayed with a scintillation counter. The phosphorylation of the
C-terminal tail will promote the binding of arrestin-like proteins
and will interfere with the binding of G proteins. The
kinase/arrestin pathway plays a key role in the desensitization of
many GPCR receptors. For example, compounds that modulate the
duration an olfactory receptor stays active would be useful as a
means of prolonging a desired odor or cutting off an unpleasant
one. For a general review of GPCR signal transduction and methods
of assaying signal transduction, see, e.g., Methods in Enzymology,
vols. 237 and 238 (1994) and volume 96 (1983); Bourne et. al.,
Nature, 10:349:117-27 (1991); Bourne et al., Nature, 348:125-32
(1990); Pitcher et al., Annu. Rev. Biochem., 67:653-92 (1998).
[0184] IVA OR modulation may be assayed by comparing the response
of an IVA OR polypeptide treated with a putative IVA OR modulator
to the response of an untreated control sample. Such putative IVA
OR modulators can include odorants that either inhibit or activate
IVA OR polypeptide activity. In one embodiment, control samples
(untreated with activators or inhibitors) are assigned a relative
OR activity value of 100. Inhibition of an IVA OR polypeptide is
achieved when the OR activity value relative to the control is
about 90%, optionally 50%, optionally 25-0%. Activation of an OR
polypeptide is achieved when the OR activity value relative to the
control is 110%, optionally 150%, 200-500%, or 1000-2000%.
[0185] Preferably molecules will be identified that inhibit IVA OR
activity, as these molecules should be useful as anti-odorants for
blocking the odor of IVA and structurally related compounds.
[0186] Changes in ion flux may be assessed by determining changes
in polarization (i.e., electrical potential) of the cell or
membrane expressing a OR protein. One means to determine changes in
cellular polarization is by measuring changes in current (thereby
measuring changes in polarization) with voltage-clamp and
patch-clamp techniques, e.g., the "cell-attached" mode, the
"inside-out" mode, and the "whole cell" mode (see, e.g., Ackerman
et al., New Engl. J. Med., 336:1575-1595 (1997)). Whole cell
currents are conveniently determined using the standard. Other
known assays include: radiolabeled ion flux assays and fluorescence
assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind
et al., J. Membrane Biol., 88:67-75 (1988); Gonzales & Tsien,
Chem. Biol., 4:269277 (1997); Daniel et al., J. Pharmacol. Meth.,
25:185-193 (1991); Holevinsky et al., J. Membrane Biology,
137:59-70 (1994)). Generally, the compounds to be tested are
present in the range from 1 pM to 100 mM.
[0187] The effects of the test compounds upon the function of the
polypeptides can be measured by examining any of the parameters
described above. Any suitable physiological change that affects
GPCR activity can be used to assess the influence of a test
compound on the polypeptides of this invention. When the functional
consequences are determined using intact cells or animals, one can
also measure a variety of effects such as transmitter release,
hormone release, transcriptional changes to both known and
uncharacterized genetic markers (e.g., northern blots), changes in
cell metabolism such as cell growth or pH changes, and changes in
intracellular second messengers such as Ca.sup.2+, IP3, cGMP, or
cAMP.
[0188] Preferred assays for GPCRs include cells that are loaded
with ion or voltage sensitive dyes to report receptor activity.
Assays for determining activity of such receptors can also use
known agonists and antagonists for other G protein coupled
receptors as negative or positive controls to assess activity of
tested compounds. In assays for identifying modulatory compounds
(e.g., agonists, antagonists), changes in the level of ions in the
cytoplasm or membrane voltage will be monitored using an ion
sensitive or membrane voltage fluorescent indicator, respectively.
Among the ion-sensitive indicators and voltage probes that may be
employed are those disclosed in the Molecular Probes 1997 Catalog.
For G protein coupled receptors, promiscuous G proteins such as
G.alpha.15 and G.alpha.16 can be used in the assay of choice
(Wilkie et al., PNAS, 88:10049-53 (1991)). Such promiscuous G
proteins allow coupling of a wide range of receptors.
Alternatively, the chimeric and variant G proteins disclosed in the
patent applications incorporated by reference supra may be
utilized. In case of mammalian olfactory receptors, such G proteins
as Golf and Gs may also be utilized to couple these receptors to
cyclic nucleotide-regulated signaling pathways.
[0189] Receptor activation typically initiates subsequent
intracellular events, e.g., increases in second messengers such as
IP3, which releases intracellular stores of calcium ions.
Activation of some G protein coupled receptors stimulates the
formation of inositol triphosphate (IP3) through phospholipase
C-mediated hydrolysis of phosphatidylinositol (Berridge &
Irvine, Nature, 312:315-21 (1984)). IP3 in turn stimulates the
release of intracellular calcium ion stores. Thus, a change in
cytoplasmic calcium ion levels, or a change in second messenger
levels such as IP3 can be used to assess G protein coupled receptor
function. Cells expressing such G protein coupled receptors may
exhibit increased cytoplasmic calcium levels as a result of
contribution from both intracellular stores and via activation of
ion channels, in which case it may be desirable although not
necessary to conduct such assays in calcium-free buffer, optionally
supplemented with a chelating agent such as EGTA, to distinguish
fluorescence response resulting from calcium release from internal
stores.
[0190] Other assays can involve determining the activity of
receptors which, when activated, result in a change in the level of
intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating
or inhibiting enzymes such as adenylate cyclase. There are cyclic
nucleotide-gated ion channels, e.g., rod photoreceptor cell
channels and olfactory neuron channels that are permeable to
cations upon activation by binding of cAMP or cGMP (see, e.g.,
Altenhofen et al., PNAS, 88:9868-72 (1991) and Dhallan et al.,
Nature, 347:184-187 (1990)). In cases where activation of the
receptor results in a decrease in cyclic nucleotide levels, it may
be preferable to expose the cells to agents that increase
intracellular cyclic nucleotide levels, e.g., forskolin, prior to
adding a receptor-activating compound to the cells in the assay.
Cells for this type of assay can be made by co-transfection of a
host cell with DNA encoding a cyclic nucleotide-crated ion channel,
GPCR phosphatase and DNA encoding a receptor (e.g., certain
glutamate receptors, muscarinic acetylcholine receptors, dopamine
receptors, serotonin receptors, and the like), which, when
activated, causes a change in cyclic nucleotide levels in the
cytoplasm
[0191] In a preferred embodiment, IVA OR protein activity is
measured by expressing an IVA OR gene in a heterologous cell with a
promiscuous G protein the links the receptor to a phospholipase C
signal transduction pathway (see Offermanns & Simon, J. Biol.
Chem., 270:15175-15180 (1995)). Optionally the cell line is HEK-293
(which does not naturally express OR genes) and the promiscuous G
protein is G.alpha.15/G.alpha.16 (Offermanns & Simon, supra).
Modulation of olfactory transduction is assayed by measuring
changes in intracellular Ca.sup.2+ levels, which change in response
to modulation of the OR signal transduction pathway via
administration of a molecule that associates with a OR protein.
Changes in Ca.sup.2+ levels are optionally measured using
fluorescent Ca.sup.2+ indicator dyes and fluorometric imaging.
[0192] In one embodiment, the changes in intracellular cAMP or cGMP
can be measured using immunoassays. The method described in
Offermanns & Simon, J. Bio. Chem., 270:15175-15180 (1995), may
be used to determine the level of cAMP. Also, the method described
in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol.,
11:159-164 (1994), may be used to determine the level of cGMP.
Further, an assay kit for measuring cAMP and/or cGMP is described
in U.S. Pat. No. 4,115,538, herein incorporated by reference.
[0193] In another embodiment, phosphatidyl inositol (PI) hydrolysis
can be analyzed according to U.S. Pat. No. 5,436,128, herein
incorporated by reference. Briefly, the assay involves labeling of
cells with 3H-myoinositol for 48 or more hrs. The labeled cells are
treated with a test compound for one hour. The treated cells are
lysed and extracted in chloroform-methanol-water after which the
inositol phosphates were separated by ion exchange chromatography
and quantified by scintillation counting. Fold stimulation is
determined by calculating the ratio of cpm in the presence of
agonist, to cpm in the presence of buffer control. Likewise, fold
inhibition is determined by calculating the ratio of cpm in the
presence of antagonist to cpm in the presence of buffer control
(which may or may not contain an agonist).
[0194] In another embodiment, transcription levels can be measured
to assess th effects of a test compound on signal transduction. A
host cell containing an OR protein of interest is contacted with a
test compound for a sufficient time to effect any interactions, and
then the level of gene expression is measured. The amount time to
effect such interactions may be empirically determined, such as by
running time course and measuring the level of transcription as a
function of time. The amount of transcription may be measured by
using any method known to those of skill in the art to be suitable.
For example, mRNA expression of the protein of interest may be
detected using northern blots or their polypeptide products may be
identified using immunoassays. Alternatively, transcription based
assays using reporter gene may be used as described in U.S. Pat.
No. 5,436,128, herein incorporated by reference. The reporter genes
can be, e.g., chloramphenicol acetyltransferase, luciferase,
'3-galactosidase and alkaline phosphatase. Furthermore, the protein
of interest can be used as an indirect reporter via attachment to a
second reporter such as green fluorescent protein (see, e.g.,
Mistili & Spector, Nature Biotechnology, 15:961-64 (1997)).
[0195] The amount of transcription is then compared to the amount
of transcription in either the same cell in the absence of the test
compound, or it may be compared with the amount of transcription in
a substantially identical cell that lacks the IVA OR protein of
interest. A substantially identical cell may be derived from the
same cells from which the recombinant cell was prepared but which
had not been modified by introduction of heterologous DNA. Any
difference in the amount of transcription indicates that the test
compound has in some manner altered the activity of the IVA OR
protein of interest.
Transgenic Non-Human Animals Expressing IVA Olfactory Receptors
[0196] Non-human animals expressing one or more IVA olfactory
receptor sequences of the invention, particularly human IVA
olfactory receptor sequences, can also be used for receptor assays.
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 transfected with a nucleic acid encoding an olfactory
receptor c ligand-binding region thereof with a test compound and
determining whether the animal reacts to the test compound by
specifically binding to the receptor polypeptide.
[0197] Use of the translocation domains of the invention in the
fusion polypeptide generates a cell expressing high levels of
olfactory receptor. Animals transfected C 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 ca 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.
[0198] Means to infect/express the nucleic acids and vectors,
either individually c as libraries, are well known in the art. 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 Brain Res. Protoc. 1:287-291 (1997)). 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, J. Neurophysiol.
77:1950-1962 (1997); Scott, J. Neurophysiol. 75:2036-2049 (1996);
Ezeh, J. Neurophysiol. 73:2207-2220 (1995). In other systems,
fluorescence changes in nasal epithelium can be measured using the
dye di4-ANEPPS, which is applied on the rat's nasal septum and
medial surface of the turbinates (see, e.g., Youngentob, J.
Neurophysiol. 73:387-398 (1995)). 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, Brain Res. 539:1-5
(1991)).
[0199] The IVA OR sequences of the invention can be for example
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, PNAS, 96:4040-45 (1999).
[0200] 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.
[0201] 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 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, Transgenic Res 6:97-106
(1997)). The insertion of the exogenous 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, Hum. Mol.
Genet. 7:53-62 (1998); Moreadith, J. Mol. Med. 75:208-216 (1997);
Tojo, Cytotechnology 19:161-165 (1995); Mudgett, Methods Mol. Biol.
48:167-184 (1995); Longo, Transgenic Res. 6:321-328 (1997); U.S.
Pat. Nos. 5,616,491; 5,464,764; 5,631,153; 5,487,992; 5,627,059;
5,272,071; WO 91/09955; WO 93/09222; WO 96/29411; WO 95/31560; WO
91/12650.
[0202] The nucleic acid libraries of the invention can also be used
as reagents to produce "knockout" human cells and their progeny.
Likewise, the nucleic acids of the invention can also be used as
reagents to produce "knock-ins" in mice. The human or rat IVA OR
gene sequences can replace the orthologous IVA ORs in the mouse
genome. In this way, a mouse expressing a human or rat IVA OR can
be produced. This mouse can then be used to analyze the function of
human or rat IVA ORs, and to identify ligands for such IVA ORs.
Modulators
[0203] The compounds tested as modulators of an IVA OR subgenus
member can be any small chemical compound, or a biological entity,
such as a protein, sugar, nucleic acid or lipid. Alternatively,
modulators can be genetically altered versions of an IVA OR gene.
Typically, test compounds will be small chemical molecules and
peptides. Essentially any chemical compound can be used as a
potential modulator or ligand in the assays of the invention,
although most often compounds can be dissolved in aqueous or
organic (especially DMSO-based) solutions are used. The assays are
designed to screen large chemical libraries by automating the assay
steps and providing compounds from any convenient source to assays,
which are typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland) and the like.
[0204] The IVA OR modulating compounds can be used in any number of
consumer products, including, but not limited to, perfumes,
fragrance compositions, deodorants, air fresheners, foods, drugs,
etc., or ingredients thereof, to thereby modulate the odor of the
product, composition, or ingredient in a desired manner. As one of
skill in the art will recognize, that IVA OR modulating compounds
preferably will be used to block malodors, i.e. attributable to IVA
and structurally related carboxylic acids.
[0205] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial chemical or peptide
library containing a large number of potential anti-odorant
compounds (potential modulator or ligand compounds). Such
"combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual anti-odorant
compositions.
[0206] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0207] Preparation and screening of combinatorial chemical
libraries is well know to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res., 37:487-93 (1991) and Houghton et al., Nature,
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091),
benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., PNAS,
90:6909-13 (1993)), vinylogous polypeptides (Hagihara et al., J.
Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimetics
with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.,
114:9217-18 (1992)), analogous organic syntheses of small compound
libraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)),
oligocarbamates (Cho et al., Science, 261:1303 (1993)), peptidyl
phosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)),
nucleic acid libraries (Ausubel, Berger and Sambrook, all supra),
peptide nucleic acid libraries (U.S. Pat. No. 5,539,083), antibody
libraries (Vaughn et al., Nature Biotechnology, 14(3):309-14 (1996)
and PCT/US96/10287), carbohydrate libraries (Liang et al., Science,
274:1520-22 (1996) and U.S. Pat. No. 5,593,853), small organic
molecule libraries (benzodiazepines, Baum, C&EN, January 18,
page 33 (1993); thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pynrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
5,288,514, and the like).
[0208] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS (Advanced Chem
Tech, Louisville Ky.), Symphony (Rainin, Woburn, Mass.), 433A
(Applied Biosystems, Foster City, Calif.), 905 Plus (Millipore,
Bedford, Mass.)). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J.; Tripos, Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.;
Martek Biosciences; Columbia Md.; etc.).
Kits
[0209] IVA OR genes and their homologs are useful tools for
identifying IVA olfactory receptor expressing cells, for forensics
and paternity determinations, and for examining olfactory
transduction. IVA OR subgenus member-specific reagents that
specifically hybridize to IVA OR nucleic acids, and IVA OR subgenus
member-specific reagents that specifically bind to an IVA OR
protein, e.g., anti-IVA OR antibodies are used to examine olfactory
cell expression and olfactory transduction regulation.
[0210] Nucleic acid assays for the presence of DNA and RNA for an
OR family member in a sample include numerous techniques are known
to those skilled in the art, such as southern analysis, northern
analysis, dot blots, RNase protection, S1 analysis, amplification
techniques such as PCR, and in situ hybridization. In in situ
hybridization, for example, the target nucleic acid is liberated
from its cellular surroundings in such a form so as to be available
for hybridization within the cell, while preserving the cellular
morphology for subsequent interpretation and analysis The following
articles provide an overview of the art of in situ hybridization:
Singer E al., Biotechniques, 4:230-50 (1986); Haase et al., Methods
in Virology, vol. VII, pp., 189-226 (1984); and Nucleic Acid
Hybridization: A Practical Approach (Names et al. eds. 1987). In
addition, an OR protein can be detected with the various
immunoassay techniques described above. The test sample is
typically compared to both a positive control (e.g., a sample
expressing a recombinant OR protein) and a negative control.
[0211] The present invention also provides for kits for screening
for modulators of IVA OR subgenus members. Such kits can be
prepared from readily available materials and reagents. For
example, such kits can comprise any one or more of the following
materials: one or more IVA OR nucleic acids or proteins, reaction
tubes, and instructions for testing IVA OR activity. Optionally,
the kit contains a biologically active IVA OR receptor. A wide
variety of kits and components can be prepared according to the
present invention, depending upon the intended user of the kit and
the particular needs of the user.
[0212] The present invention also embraces compositions containing
compounds which modulate the activity of at least one IVA OR
receptor according to the invention. These compounds based on their
activity should inhibit malodor attributable to isovaleric acid and
structurally related carboxylic acids such as those identified
infra that are comprised in animal and human axillary secretions
(sweat).
[0213] Compositions wherein such compounds should be useful include
by the way of example deodorants, pet deodorizers, carpet
fresheners, air deodorizers, fabric deodorizers, and other
compositions that are intended to inhibit such odors.
EXAMPLES
Example 1
Isolation and Imaging of Individual Murine Olfactory Neurons
[0214] In this example, the details of how the individual murine
olfactory neurons were isolated from murine olfactory epithelium
and subjected to Ca.sup.2+ imaging are described.
[0215] Olfactory neuroepithelium was dissected from Balb/C mice and
placed in a small Petri dish with phosphate-buffered saline (PBS)
without Ca.sup.2+ and Mg.sup.2+. The tissue was fragmented under
the dissecting microscope into pieces as small as possible. The
buffer was removed and replaced by 2 ml of fresh PBS without
Ca.sup.2+ and Mg.sup.2+ supplemented with 0.025% trypsin, 0.75 mM
EDTA for 15 min. After dissociation, the tissue was transferred
into 5 ml PBS without Ca.sup.2+ and Mg.sup.2+ and the tissue was
triturated very gently by pipetting up and down 4-5 times with
plastic disposable, progress of cell dissociation was monitored
under inverted microscope. After dissociation, the tissue was
transferred into 10 ml of Dulbecco's modified Eagle medium (DMEM)
supplemented with 10% Calf serum, centrifuged 10 min at 2000 g.
Supernatant was removed, 5 ml of cold PBS without Ca.sup.2+ and
Mg.sup.2+ were added and the tissue suspension (kept on ice) was
triturated very gently by pipetting up and down 4-5 times with
pipettes and pipetman tips of gradually decreasing diameters: 2 ml
plastic pipette, 1 ml plastic pipette, then 1 ml followed by 200
.mu.l-tip Pipetman. The isolated cells were then incubated with 10
.mu.M Fura-2 AM for 45 min Isolated olfactory sensory neurons were
identified in an inverted microscope field either by their
morphology alone (for random control neurons) or by the morphology
and ability to respond to IVA as detected by Ca.sup.2+-imaging. An
example of IVA response of an isolated murine olfactory sensory
neuron is represented In FIG. 3. Fluorescence measurements were
conducted at room temperature on an inverted microscope (Zeiss
Axiovert 100, Carl Zeiss). Fura-2 loaded neurons were illuminated
at excitation wavelengths (360 and 380 nm). Emitted fluorescence
passed through the objective, the dichroic mirror (DCPL405) and an
emission filter (510.+-.40 nm) and was collected by a cooled
charge-coupled device (CCD) camera (Quantix, Photomerics) mounted
on the microscopy. Metafluoro imaging software (Universal Imaging
Inc.) were used to acquire the images at 10 seconds intervals. The
responsive neurons were then picked with a glass microcapillary
pipette.
[0216] The pipette contents (.about.0.5 .mu.l or less) were then
ejected by positive pressure (with syringe) into a PCR tube with 4
.mu.l of the lysis buffer (0.5% Nonidet NP-40, 10 .mu.M of each
dNTP, 5 units/.mu.l of Prime RNAse inhibitor (3'-5', Inc.), 324
units/ml RNAguard (Pharmacia), 1.times.MMLV reverse transcriptase
buffer (GIBCO-BRL), 0.005 OD.sub.260/ml pd(T) 19-24 primer in
DEPC-treated water). The collection tubes were briefly centrifuged
immediately to make sure the cell is indeed in the lysis buffer
Sample tubes were stored on ice for up to 2 hours before starting
the next step.
[0217] FIG. 3 shows response of a single olfactory neuron to IVA
indicated by changes in fura-2 fluorescence intensity ratios
(340/380 nm). Either high K.sup.+ solution or IVA at either 100
.mu.M or 10 .mu.M were applied. The neuron was washed continuously
between applications of high K.sup.+ and IVA.
Example 2
Single-Cell cDNA Synthesis and PCR Amplification of or cDNA
Fragments
[0218] The single neurons collected in lysis buffer were heated to
65.degree. C. in a water for 1 min to lyse the cells, then kept at
room temperature for 1-2 min to allow the oli primer to anneal to
the RNA and put back on ice. The tube was centrifuged briefly .mu.l
of a 1:1 mix of AMV- and MMLV-reverse transcriptases (Gibco-BRL)
were added followed by incubation for 15 min at 37.degree. C. For
control experiments, the reaction mi was divided into two equal
aliquots--one for reverse transcriptase reaction (cDNA synthesis)
and another for a negative control without reverse transcriptase.
After incubation, the reverse transcriptases were inactivated by
incubation for 10 min at 6 the tubes were put back on ice, then
briefly centrifuged (2 min at 4.degree. C.). 4.5 .mu.l of 2.times.
buffer (stock tailing buffer: 800 .mu.l of 5.times. Gibco-BRL
terminal transferase buffer, 30 .mu.l 100 mM dATP, 1.17 ml
H.sub.2O) containing 10 units of terminal transferase were added
reaction mix was incubated at 37.degree. C. for 15 min, inactivated
for 10 min at 65.degree. C., put ice and centrifuged briefly. PCR
amplification reactions were done in Perkin Elmer Thermal Cycler
480. Freshly made ice cold PCR buffer (90 .mu.l) was added to the
re mixture described above. Composition of the PCR buffer (all
reagents and PCR m kept on ice to avoid primer dimers): 10 .mu.l of
10.times.PCR buffer 11 (Perkin Elmer), 10 .mu.l 25 mM MgCl.sub.2,
0.5 .mu.l of 20 mg/ml BSA, 1 .mu.l of each 100 mM dNTP, 1 .mu.l of
5% Triton) 5 .mu.g of AL1 oligonucleotide primer
dATTGGATCCAGGCCGCTTGGACAAAAATGAA TC(T).sub.24, 90 .mu.l H.sub.2O, 2
.mu.l of AmpliTaq polymerase (Perkin Elmer), 1-2 drops of minera
(Sigma M5904). Twenty five cycles of amplification were performed
in the following conditions: 94.degree. C. for 1 min, 42.degree. C.
for 2 min, 72.degree. C. for 6 min with 10 sec extension time each
cycle. When these 25 cycles were finished, 1 .mu.l of AmpliTaq
polymerase were directly to each tube and 25 more cycles were
performed with the same program as but without extension time. One
.mu.l aliquots of the first PCR reactions supplied with 0 of each
dNTP, 2 .mu.M of each degenerate OR primer in a pair used for a
given reaction (primers p41+214, 213+214, TM3D+TM71) and 2.5 U of
AmpliTaq polymerase in 1.times. buffer II (Perkin Elmer) were
amplified using the following program: 94.degree. C. for 5 min,
cycles of PCR (1 min 94.degree. C., 3 min 40.degree. C., 3 min
72.degree. C.), final extension at 72.degree. C. for 7 The PCR
reaction mixtures were analyzed by 1% agarose gel electrophoresis.
DNA fragments from the successful reactions (as judged by the
expected size of a fragm absence of DNA contamination confirmed by
negative control reactions) were TA-cl pCRII-TOPO vector
(invitrogen) and fully sequenced.
[0219] While the foregoing detailed description has described
several embodiments of the present invention, it is to be
understood that the above description is illustrative only and not
limiting of the disclosed invention. The invention is to be limited
only by the claims which follow.
Sequence CWU 1
1
861936DNAMus musculus 1atgggaaaag aaaatcacac agaactatca caattcctgc
tactgggtct ctcagatgat 60cctaaattgc agcctattct tttcgggata ttcttattta
tgtacctggt cacagtgctt 120ggtaacctgc tcatcatcct ggctgtcagt
tctgattccc atctccacaa ccccatgtac 180ttcttcctct ccaacctctc
atttgtagac atgtgtttca cttctaccac tgtcccaaag 240atgctggtga
acatccagac aaagaacaaa aatatctcct acatgcagtg cctcactcaa
300gtctattttt ttatggtgtt tgctggaatg gataatttct tactgactgt
aatggccttt 360gaccgctttg tggctatttg tcacccctta aactacacag
tcatcatgaa ccctcacttc 420tgttgcttcc ttgtgctaat gtgctggatt
atcattttat cagtctccct gtttcatagt 480ctattaatga agcaattaac
tttttccatg ggtactgaaa tcccacattt cttctgtgag 540ttggctcaaa
ttctcagagt agcaagctct gatattctca tcaataatat cgcattatat
600gtggctactg ccctgttatg tgtgtttcct gtcactggaa ttctcttctc
ttactcgcag 660attgtctcct ccttattgaa tatgtcttca gtagtcagca
agtatagagc cttttccacc 720tgtggatctc acctctgtgt ggtctgtttg
ttttatggta cagcactggg ggtttacctc 780agttcagctg ggactgatgt
ttctcaagga agcactatag cctcagtgat gtatactgtg 840gtcactccta
tgctcaaccc attcatctac agcctgagga ataaagatgt gaagggggct
900ctggtaagaa tccttaaagt atattcttgt ccctga 9362311PRTMus musculus
2Met Gly Lys Glu Asn His Thr Glu Leu Ser Gln Phe Leu Leu Leu Gly1 5
10 15Leu Ser Asp Asp Pro Lys Leu Gln Pro Ile Leu Phe Gly Ile Phe
Leu20 25 30Phe Met Tyr Leu Val Thr Val Leu Gly Asn Leu Leu Ile Ile
Leu Ala35 40 45Val Ser Ser Asp Ser His Leu His Asn Pro Met Tyr Phe
Phe Leu Ser50 55 60Asn Leu Ser Phe Val Asp Met Cys Phe Thr Ser Thr
Thr Val Pro Lys65 70 75 80Met Leu Val Asn Ile Gln Thr Lys Asn Lys
Asn Ile Ser Tyr Met Gln85 90 95Cys Leu Thr Gln Val Tyr Phe Phe Met
Val Phe Ala Gly Met Asp Asn100 105 110Phe Leu Leu Thr Val Met Ala
Phe Asp Arg Phe Val Ala Ile Cys His115 120 125Pro Leu Asn Tyr Thr
Val Ile Met Asn Pro His Phe Cys Cys Phe Leu130 135 140Val Leu Met
Cys Trp Ile Ile Ile Leu Ser Val Ser Leu Phe His Ser145 150 155
160Leu Leu Met Lys Gln Leu Thr Phe Ser Met Gly Thr Glu Ile Pro
His165 170 175Phe Phe Cys Glu Leu Ala Gln Ile Leu Arg Val Ala Ser
Ser Asp Ile180 185 190Leu Ile Asn Asn Ile Ala Leu Tyr Val Ala Thr
Ala Leu Leu Cys Val195 200 205Phe Pro Val Thr Gly Ile Leu Phe Ser
Tyr Ser Gln Ile Val Ser Ser210 215 220Leu Leu Asn Met Ser Ser Val
Val Ser Lys Tyr Arg Ala Phe Ser Thr225 230 235 240Cys Gly Ser His
Leu Cys Val Val Cys Leu Phe Tyr Gly Thr Ala Leu245 250 255Gly Val
Tyr Leu Ser Ser Ala Gly Thr Asp Val Ser Gln Gly Ser Thr260 265
270Ile Ala Ser Val Met Tyr Thr Val Val Thr Pro Met Leu Asn Pro
Phe275 280 285Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Gly Ala Leu
Val Arg Ile290 295 300Leu Lys Val Tyr Ser Cys Pro305 3103942DNAMus
musculus 3atggaagaac acaatcttac attaatgact gaattcatcc taatgggtat
cagtgaccac 60tctgaattgc aggccccatt atttgggctg atccttgcca tatacatgac
ctcaatggta 120ggtaatatgg gaatcattgt tttaatcact ttggactcac
gcctgctaac acccatgtac 180ttctttataa aacacctggc tattacagat
cttggatatt ctacagctgt gggacccaaa 240atgttggaaa attttgttgt
agatcaaaat acaatttcat ttaatctttg tgccacacaa 300ctagctttct
ttcttgtatt cattggtagt gagctattca ttctctctgc gatgtcctat
360gaccgctatg tggccatctg taagcctctg ctctacactg tcctcatgtc
ccaaaaacta 420tgttgggttc ttatgtcaat gccttatctc tactgcacat
ttgtgtctct tctcatcaca 480gtgaagattt ttacttcatc cttctgtggc
tacaatgtca ttaaccattt ctactgtgac 540tgtatcccct tgctgtctct
actctgttca catgcagagg aaatcgcatt tattgttatg 600atctttgcag
cttttgattt gattgtgtct cttcttattg ttctggtatc ctacatgttt
660atcctcatag cagttctcag gatgaactct gcagagggca ggtacaaggc
tttctccaca 720tgtgggtccc acctgacagt ggtgacagtg ttctatggta
ctttaatatt tatgtatgta 780caacctcagt ccagtcattc tgatgacaat
gataaggtgt cttcaatttt ttacaccctc 840gttataccca tgctgaatcc
tttgatctat agtttgagga acaaggatgt aaaatttgcc 900ctacatagga
cttggagaaa tatttgtaag atcttccctt ag 9424313PRTMus musculus 4Met Glu
Glu His Asn Leu Thr Leu Met Thr Glu Phe Ile Leu Met Gly1 5 10 15Ile
Ser Asp His Ser Glu Leu Gln Ala Pro Leu Phe Gly Leu Ile Leu20 25
30Ala Ile Tyr Met Thr Ser Met Val Gly Asn Met Gly Ile Ile Val Leu35
40 45Ile Thr Leu Asp Ser Arg Leu Leu Thr Pro Met Tyr Phe Phe Ile
Lys50 55 60His Leu Ala Ile Thr Asp Leu Gly Tyr Ser Thr Ala Val Gly
Pro Lys65 70 75 80Met Leu Glu Asn Phe Val Val Asp Gln Asn Thr Ile
Ser Phe Asn Leu85 90 95Cys Ala Thr Gln Leu Ala Phe Phe Leu Val Phe
Ile Gly Ser Glu Leu100 105 110Phe Ile Leu Ser Ala Met Ser Tyr Asp
Arg Tyr Val Ala Ile Cys Lys115 120 125Pro Leu Leu Tyr Thr Val Leu
Met Ser Gln Lys Leu Cys Trp Val Leu130 135 140Met Ser Met Pro Tyr
Leu Tyr Cys Thr Phe Val Ser Leu Leu Ile Thr145 150 155 160Val Lys
Ile Phe Thr Ser Ser Phe Cys Gly Tyr Asn Val Ile Asn His165 170
175Phe Tyr Cys Asp Cys Ile Pro Leu Leu Ser Leu Leu Cys Ser His
Ala180 185 190Glu Glu Ile Ala Phe Ile Val Met Ile Phe Ala Ala Phe
Asp Leu Ile195 200 205Val Ser Leu Leu Ile Val Leu Val Ser Tyr Met
Phe Ile Leu Ile Ala210 215 220Val Leu Arg Met Asn Ser Ala Glu Gly
Arg Tyr Lys Ala Phe Ser Thr225 230 235 240Cys Gly Ser His Leu Thr
Val Val Thr Val Phe Tyr Gly Thr Leu Ile245 250 255Phe Met Tyr Val
Gln Pro Gln Ser Ser His Ser Asp Asp Asn Asp Lys260 265 270Val Ser
Ser Ile Phe Tyr Thr Leu Val Ile Pro Met Leu Asn Pro Leu275 280
285Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Phe Ala Leu His Arg
Thr290 295 300Trp Arg Asn Ile Cys Lys Ile Phe Pro305 3105930DNAMus
musculus 5atgactgagg acaactactc cttgacaaca gagttcatcc tcataggatt
ctcagaccac 60ccagacttaa agatacttct attcctggtg ttatctacca tctatctggt
caccatggtg 120gggaatcttg ggctggtggc cttgatctac atggagcctc
gtctccacac acccatgtac 180atctttctgg gcaacctggc tctcatggat
tcctgttgct cctgtgccat cactcctaag 240atgctagaga actttttttc
tgtgaacaga aggatttctc tctatgaatg catggcacag 300ttctattttc
tctgtcttgc tgaaactgca gactgcttcc ttctggcagc catggcctat
360gaccgctatg tggccatatg caaccctctg cagtaccaca ccatgatgtc
caagaagctc 420tgccttcaaa tgaccacagg agcctacata gcaggaaacc
tgcattccat gattcacata 480gggttcttgt tcaggttaat tttctgcagg
tctcatgtga tcaagcactt cttttgtgat 540gtcctccccc tatacagact
ctcatgtgtt gacccttata tcaatgaact gatgatactc 600atcttttctg
gttcagttca aaccttttcc attattatag tcttgatttc ttatttctgc
660atccttttta ctatattcac aatgaagtcc agagagggaa gaagcaaagc
cttatctact 720tgtgcatccc actttctgtc tgtgtcaata ttctatgggt
ctcttctcta cacatatatt 780cgaccaagtt cacttaatga agggtataaa
gacatacctg ttgctatatt ttatactcta 840gtaattcctt tattaaaccc
gtttatttat agtctgagaa ataaagaagt aattaatgtg 900atgaaaagag
caatgaagaa aagattataa 9306309PRTMus musculus 6Met Thr Glu Asp Asn
Tyr Ser Leu Thr Thr Glu Phe Ile Leu Ile Gly1 5 10 15Phe Ser Asp His
Pro Asp Leu Lys Ile Leu Leu Phe Leu Val Leu Ser20 25 30Thr Ile Tyr
Leu Val Thr Met Val Gly Asn Leu Gly Leu Val Ala Leu35 40 45Ile Tyr
Met Glu Pro Arg Leu His Thr Pro Met Tyr Ile Phe Leu Gly50 55 60Asn
Leu Ala Leu Met Asp Ser Cys Cys Ser Cys Ala Ile Thr Pro Lys65 70 75
80Met Leu Glu Asn Phe Phe Ser Val Asn Arg Arg Ile Ser Leu Tyr Glu85
90 95Cys Met Ala Gln Phe Tyr Phe Leu Cys Leu Ala Glu Thr Ala Asp
Cys100 105 110Phe Leu Leu Ala Ala Met Ala Tyr Asp Arg Tyr Val Ala
Ile Cys Asn115 120 125Pro Leu Gln Tyr His Thr Met Met Ser Lys Lys
Leu Cys Leu Gln Met130 135 140Thr Thr Gly Ala Tyr Ile Ala Gly Asn
Leu His Ser Met Ile His Ile145 150 155 160Gly Phe Leu Phe Arg Leu
Ile Phe Cys Arg Ser His Val Ile Lys His165 170 175Phe Phe Cys Asp
Val Leu Pro Leu Tyr Arg Leu Ser Cys Val Asp Pro180 185 190Tyr Ile
Asn Glu Leu Met Ile Leu Ile Phe Ser Gly Ser Val Gln Thr195 200
205Phe Ser Ile Ile Ile Val Leu Ile Ser Tyr Phe Cys Ile Leu Phe
Thr210 215 220Ile Phe Thr Met Lys Ser Arg Glu Gly Arg Ser Lys Ala
Leu Ser Thr225 230 235 240Cys Ala Ser His Phe Leu Ser Val Ser Ile
Phe Tyr Gly Ser Leu Leu245 250 255Tyr Thr Tyr Ile Arg Pro Ser Ser
Leu Asn Glu Gly Tyr Lys Asp Ile260 265 270Pro Val Ala Ile Phe Tyr
Thr Leu Val Ile Pro Leu Leu Asn Pro Phe275 280 285Ile Tyr Ser Leu
Arg Asn Lys Glu Val Ile Asn Val Met Lys Arg Ala290 295 300Met Lys
Lys Arg Leu3057933DNAMus musculus 7atgggcaaat taaaccacac ttatctgacg
gagttcatct tgctgggcct ctcttcagat 60catcagactc agatcctgct gtttgtggta
tttctcatca tctacctgat cactgtgttt 120gggaacctgc tcatcatact
cctcattcat gttgactccc gacttcatac accaatgtac 180ttctttctaa
aaatcctgtc attcaatgat ctctgtttct ctacaacaat tgttccaaag
240atgctagtcc actttctagg tgtcagaaag accatttcat ttgctgggtg
ctcagtgcaa 300atgttttctt tcctcataat ggggtgtaca gaaagctctc
ttctggcagt catgtcatat 360gaccgctaca tagctgtctg caaacccctg
cactactcca ccatcatgac acataaggtt 420tgtgttctgc tagttgtagg
atcctggact agtggaatat ttgtgtctgt agtagatacc 480tcatttactt
tatgcttgac gtaccgggga ccaaatataa tcaatcatta cttttgtgag
540cctcctgcac tcttaaagct ggcttcagaa gaaacctaca cagctgaaat
ggtcatattt 600gcaatgggta taataattct cttaggtcct gtctctctta
tccttttctc ctattggaat 660attatctcca ctgtggttca aatacaatca
ggtgagggga ggctcaaggt tttctctacc 720tgcagttccc attttattgt
tgttatcttc ttctatggct caacaatatt tacctacatg 780cagccaaact
caaagaaaat gaatgaaaag gataaggtaa tctcggtatt ctactcaata
840gtaacatcca tgatgaaccc attcatttat agcctaagga acaaagatgt
gaaaggggca 900ttaaagaaag tacttaaaag agagataaga taa 9338310PRTMus
musculus 8Met Gly Lys Leu Asn His Thr Tyr Leu Thr Glu Phe Ile Leu
Leu Gly1 5 10 15Leu Ser Ser Asp His Gln Thr Gln Ile Leu Leu Phe Val
Val Phe Leu20 25 30Ile Ile Tyr Leu Ile Thr Val Phe Gly Asn Leu Leu
Ile Ile Leu Leu35 40 45Ile His Val Asp Ser Arg Leu His Thr Pro Met
Tyr Phe Phe Leu Lys50 55 60Ile Leu Ser Phe Asn Asp Leu Cys Phe Ser
Thr Thr Ile Val Pro Lys65 70 75 80Met Leu Val His Phe Leu Gly Val
Arg Lys Thr Ile Ser Phe Ala Gly85 90 95Cys Ser Val Gln Met Phe Ser
Phe Leu Ile Met Gly Cys Thr Glu Ser100 105 110Ser Leu Leu Ala Val
Met Ser Tyr Asp Arg Tyr Ile Ala Val Cys Lys115 120 125Pro Leu His
Tyr Ser Thr Ile Met Thr His Lys Val Cys Val Leu Leu130 135 140Val
Val Gly Ser Trp Thr Ser Gly Ile Phe Val Ser Val Val Asp Thr145 150
155 160Ser Phe Thr Leu Cys Leu Thr Tyr Arg Gly Pro Asn Ile Ile Asn
His165 170 175Tyr Phe Cys Glu Pro Pro Ala Leu Leu Lys Leu Ala Ser
Glu Glu Thr180 185 190Tyr Thr Ala Glu Met Val Ile Phe Ala Met Gly
Ile Ile Ile Leu Leu195 200 205Gly Pro Val Ser Leu Ile Leu Phe Ser
Tyr Trp Asn Ile Ile Ser Thr210 215 220Val Val Gln Ile Gln Ser Gly
Glu Gly Arg Leu Lys Val Phe Ser Thr225 230 235 240Cys Ser Ser His
Phe Ile Val Val Ile Phe Phe Tyr Gly Ser Thr Ile245 250 255Phe Thr
Tyr Met Gln Pro Asn Ser Lys Lys Met Asn Glu Lys Asp Lys260 265
270Val Ile Ser Val Phe Tyr Ser Ile Val Thr Ser Met Met Asn Pro
Phe275 280 285Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Gly Ala Leu
Lys Lys Val290 295 300Leu Lys Arg Glu Ile Arg305 3109960DNAMus
musculus 9atggaaacag gaaatgacac tcagctttca gaattctttc ttctgggatt
ttcagagaat 60caacctcaaa ttcagcctgt catatttgga ctgttcctct tcatgtatat
attgactttc 120actggaaacc tactcatcat catggccatc attgttgact
cccacctgca cacacccatg 180tacctcttcc tctctaatct gtcctttgtg
gacatctgct tcacttccac cactgttcca 240cagatgctgg taaacattca
cacacaaagc aaggccatca cctatgcagg ctgcatcatc 300cagatgtact
tcttactgct tttttcaggg ttagacatct ttctgctgac tgtgatggcc
360tatgaccgct atgtggccat ctgtcacccc ctgcattaca tgatcatcat
gagcacaaga 420cgctgtggat tgatgattct ggcatgctgg attataggtg
ttataaattc cctgttacac 480acctttttgg tgttacggct gtcattctgc
acaaacttgg aaatccccca ttttttctgt 540gaacttaatc aagttgtaca
ccaggcctgt tctgacacct ttcttaatga tatggtaatt 600tacattacag
ctatgctact ggctgttggc cccttctctg gtatccttta ctcttactct
660aggatagtat cctccatttg tgcaatctcc tcagtgcagg ggaagtacaa
agcattttcc 720acctgtgcat ctcacctctc agttgtctcc ttattttatt
gcaccctcct gggagtgtac 780ctcagctctg ctgtgaccca aaactcacat
gctactgcaa cagcttcatt gatgtacact 840gtggtcaccc ccatgctgaa
tcccttcatc tacagtctga ggaacaaaga cataaagaca 900gctctgaaaa
tcctgttagg gagtgtaact agaagcagat caatggattc accttcataa
96010318PRTMus musculus 10Met Glu Thr Gly Asn Asp Thr Gln Leu Ser
Glu Phe Phe Leu Leu Gly1 5 10 15Phe Ser Glu Asn Gln Pro Gln Ile Gln
Pro Val Ile Phe Gly Leu Phe20 25 30Leu Phe Met Tyr Ile Leu Thr Phe
Thr Gly Asn Leu Leu Ile Ile Met35 40 45Ala Ile Ile Val Asp Ser His
Leu His Thr Pro Met Tyr Leu Phe Leu50 55 60Ser Asn Leu Ser Phe Val
Asp Ile Cys Phe Thr Ser Thr Thr Val Pro65 70 75 80Gln Met Leu Val
Asn Ile His Thr Gln Ser Lys Ala Ile Thr Tyr Ala85 90 95Gly Cys Ile
Ile Gln Met Tyr Phe Leu Leu Leu Phe Ser Gly Leu Asp100 105 110Ile
Phe Leu Leu Thr Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys115 120
125His Pro Leu His Tyr Met Ile Ile Met Ser Thr Arg Arg Cys Gly
Leu130 135 140Met Ile Leu Ala Cys Trp Ile Ile Gly Val Ile Asn Ser
Leu Leu His145 150 155 160Thr Phe Leu Val Leu Arg Leu Ser Phe Cys
Thr Asn Leu Glu Ile Pro165 170 175His Phe Phe Cys Glu Leu Asn Gln
Val Val His Gln Ala Cys Ser Asp180 185 190Thr Phe Leu Asn Asp Met
Val Ile Tyr Ile Thr Ala Met Leu Leu Ala195 200 205Val Gly Pro Phe
Ser Gly Ile Leu Tyr Ser Tyr Ser Arg Ile Val Ser210 215 220Ser Ile
Cys Ala Ile Ser Ser Val Gln Gly Lys Tyr Lys Ala Phe Ser225 230 235
240Thr Cys Ala Ser His Leu Ser Val Val Ser Leu Phe Tyr Cys Thr
Leu245 250 255Leu Gly Val Tyr Leu Ser Ser Ala Val Thr Gln Asn Ser
His Ala Thr260 265 270Ala Thr Ala Ser Leu Met Tyr Thr Val Val Thr
Pro Met Leu Asn Pro275 280 285Phe Ile Tyr Ser Leu Arg Asn Lys Asp
Ile Lys Thr Ala Leu Lys Ile290 295 300Leu Leu Gly Ser Val Thr Arg
Ser Arg Ser Met Asp Ser Pro305 310 31511942DNAMus musculus
11atgagtgtgg ccaatgagag catctcacgg gagttcattc tcttagggtt ttcagatcgg
60ccatggctgg agctgccgct ctttgtggtg tttctagtgt cctatattct gaccatcttt
120ggaaatatga tgatcattct tgtgtcccgc ctggattcca aactccacac
ccccatgtac 180tttttcctca ctaacctgtc cttgctggac ctgtgctaca
ccacaagcac ggtcccacag 240atgctcatca acatctgcag cacccggaag
gtgatcagct atggtggctg tgtggcccag 300cttttcattt tcctggcctt
gggttccaca gaatgctttc tgctgggcgt catgtccttt 360gacaggtttg
tagccatctg tcggcctctc cactactcag tcatcatgca ccagaggcgc
420tgcctccagt tggcggctgc atgttggatc agtggcttca gcaactcagt
attacagtct 480acgtggaccc ttcagatgcc actgtgtgga cacaaggaag
tggaccattt cttttgcgaa 540gtccctgccc tgctcaagtt gtcctgtgtg
gatacgacag ctaatgaagc agagctgttc 600ttcatcagtg tgctgtttct
tttaataccc gtgaccctca tcctcatatc atatgctttt 660attgtccagg
cagtgttgag aataagatca gctgaaggtc ggcgaaaggc atttgggaca
720tgtggctccc acctcatcgt ggtggtcctt ttctatggca ctgccatcta
catgtatctg 780cagccaccat cccctacttc caaggaccgg gggaaaatgg
tgtctctctt ttatgggatc 840atcacaccca tgctgaaccc cctcatctac
acactcagga acaaagaggt aaagggagcg 900ttcaagaggt tggtgacaag
gatcatcctg agtagaaaat aa 94212313PRTMus musculus 12Met Ser Val Ala
Asn Glu Ser Ile Ser Arg Glu Phe Ile Leu Leu Gly1 5 10 15Phe Ser Asp
Arg Pro Trp Leu Glu Leu Pro Leu Phe Val Val Phe Leu20 25 30Val Ser
Tyr Ile Leu Thr Ile Phe Gly Asn Met Met Ile Ile Leu Val35
40 45Ser Arg Leu Asp Ser Lys Leu His Thr Pro Met Tyr Phe Phe Leu
Thr50 55 60Asn Leu Ser Leu Leu Asp Leu Cys Tyr Thr Thr Ser Thr Val
Pro Gln65 70 75 80Met Leu Ile Asn Ile Cys Ser Thr Arg Lys Val Ile
Ser Tyr Gly Gly85 90 95Cys Val Ala Gln Leu Phe Ile Phe Leu Ala Leu
Gly Ser Thr Glu Cys100 105 110Phe Leu Leu Gly Val Met Ser Phe Asp
Arg Phe Val Ala Ile Cys Arg115 120 125Pro Leu His Tyr Ser Val Ile
Met His Gln Arg Arg Cys Leu Gln Leu130 135 140Ala Ala Ala Cys Trp
Ile Ser Gly Phe Ser Asn Ser Val Leu Gln Ser145 150 155 160Thr Trp
Thr Leu Gln Met Pro Leu Cys Gly His Lys Glu Val Asp His165 170
175Phe Phe Cys Glu Val Pro Ala Leu Leu Lys Leu Ser Cys Val Asp
Thr180 185 190Thr Ala Asn Glu Ala Glu Leu Phe Phe Ile Ser Val Leu
Phe Leu Leu195 200 205Ile Pro Val Thr Leu Ile Leu Ile Ser Tyr Ala
Phe Ile Val Gln Ala210 215 220Val Leu Arg Ile Arg Ser Ala Glu Gly
Arg Arg Lys Ala Phe Gly Thr225 230 235 240Cys Gly Ser His Leu Ile
Val Val Val Leu Phe Tyr Gly Thr Ala Ile245 250 255Tyr Met Tyr Leu
Gln Pro Pro Ser Pro Thr Ser Lys Asp Arg Gly Lys260 265 270Met Val
Ser Leu Phe Tyr Gly Ile Ile Thr Pro Met Leu Asn Pro Leu275 280
285Ile Tyr Thr Leu Arg Asn Lys Glu Val Lys Gly Ala Phe Lys Arg
Leu290 295 300Val Thr Arg Ile Ile Leu Ser Arg Lys305 31013945DNAMus
musculus 13atgttccaag gaaatctttc cggagtaact gagttcaatc ttgctggttt
aacagacaaa 60ccagggctgc agctgcccct cttcctcctg ttcctaggaa tctatgtggt
cacagtggtg 120gggaatctca gcatgatcac cctgatacta ttcagttctc
aactacacac acccatgtat 180tattttctca gcagtctgtc cttcattgac
ctctgccagt ccattgtcat tattcccaaa 240atgttggtga actttgtgac
agtgcagaac atcatctcct accctgaatg tatgacacag 300ttttgctttt
tgctactttt tactattgca gagtgtcaca tgttagctgt aatggcatat
360gaccgctatg ttgccatttg taagcccttg ctttacaatg ctgtaatgtc
ctatcaagtt 420tgttcctgga tgatatttgg agtatatatt atggcttttg
ttggtgccac aactcaaaca 480gtcttcatgt taaaagtgca tttttgtaag
gccaatgtaa taaatcatta cttctgtgat 540ctttccccac tcctggaact
ctcttgttct gatactttta ttaatgaagt attagctttg 600tgcttcagtg
ttttcaatat ctttattcca actctgacaa ttctaagctc ttacatcttc
660atcatagcca gcatcctccg gattaaatcc actgaaggca ggtccaaagc
cttcagcact 720tgcagctcac acatatcagc agttgctata ttctttggat
cccttgcatt catgtacctg 780cagccatcat caatcaactc catggaccaa
aggaaagtgt cctctgtatt ttataccatt 840gtcgtgccca tgctgaatcc
tttgatctac agcctgagga ataaggatgt caaagttgct 900ctaaataagt
tccttgaaag aattttttct tgtgaacaaa actaa 94514314PRTMus musculus
14Met Phe Gln Gly Asn Leu Ser Gly Val Thr Glu Phe Asn Leu Ala Gly1
5 10 15Leu Thr Asp Lys Pro Gly Leu Gln Leu Pro Leu Phe Leu Leu Phe
Leu20 25 30Gly Ile Tyr Val Val Thr Val Val Gly Asn Leu Ser Met Ile
Thr Leu35 40 45Ile Leu Phe Ser Ser Gln Leu His Thr Pro Met Tyr Tyr
Phe Leu Ser50 55 60Ser Leu Ser Phe Ile Asp Leu Cys Gln Ser Ile Val
Ile Ile Pro Lys65 70 75 80Met Leu Val Asn Phe Val Thr Val Gln Asn
Ile Ile Ser Tyr Pro Glu85 90 95Cys Met Thr Gln Phe Cys Phe Leu Leu
Leu Phe Thr Ile Ala Glu Cys100 105 110His Met Leu Ala Val Met Ala
Tyr Asp Arg Tyr Val Ala Ile Cys Lys115 120 125Pro Leu Leu Tyr Asn
Ala Val Met Ser Tyr Gln Val Cys Ser Trp Met130 135 140Ile Phe Gly
Val Tyr Ile Met Ala Phe Val Gly Ala Thr Thr Gln Thr145 150 155
160Val Phe Met Leu Lys Val His Phe Cys Lys Ala Asn Val Ile Asn
His165 170 175Tyr Phe Cys Asp Leu Ser Pro Leu Leu Glu Leu Ser Cys
Ser Asp Thr180 185 190Phe Ile Asn Glu Val Leu Ala Leu Cys Phe Ser
Val Phe Asn Ile Phe195 200 205Ile Pro Thr Leu Thr Ile Leu Ser Ser
Tyr Ile Phe Ile Ile Ala Ser210 215 220Ile Leu Arg Ile Lys Ser Thr
Glu Gly Arg Ser Lys Ala Phe Ser Thr225 230 235 240Cys Ser Ser His
Ile Ser Ala Val Ala Ile Phe Phe Gly Ser Leu Ala245 250 255Phe Met
Tyr Leu Gln Pro Ser Ser Ile Asn Ser Met Asp Gln Arg Lys260 265
270Val Ser Ser Val Phe Tyr Thr Ile Val Val Pro Met Leu Asn Pro
Leu275 280 285Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Val Ala Leu
Asn Lys Phe290 295 300Leu Glu Arg Ile Phe Ser Cys Glu Gln Asn305
31015912DNAMus musculus 15tctgagttta tcctcttaga gctccccatt
cagccagagg atcaagctgt gtactttgcc 60ctgttcctgg ccatgtacct gacaactgtg
ctggggaacc tgctcatcat tcttctcatt 120aggctggact ctcacctcca
cacccccatg tacttcttcc tcagtcactt ggccttcacg 180gacatctctt
tctcatctgt cacagctcca aagatgctca tgaatatgct gacacatagc
240caatccatct cacatgctgg gtgtgtttcc caaatatatt ttttcttatt
gtttgggtgt 300attgacaact tccttctgac ttccatggcc tatgacaggt
atgtggccat ctgccaccct 360ctgcattata ccactatcat gagtcaaagc
ctctgtgttc tgctagtgat ggtgtcctgg 420gcattttcct cttctaatgg
ccttgtgcat actcttctct ttgctcgtct ctctcttttt 480agagacaaca
ctgtccacca ttttttctgt gatctctctg ctttgctgaa gctgtccagc
540tcagacacta ctatcaatga actagtaatc ctcactttag cagtggtggt
catcactgta 600ccattcatat gcatcctggt ttcttatggc cacatggggg
ccactatcct aagaactcca 660tccatcaagg gtatctgcaa agccttgtcc
acatgtggtt ctcatctctg tgtagtttct 720ttatattatg gagccattat
tgggttatat tttttcccct cctccaataa tactaatgat 780aaagatgtca
tagtagctgt gttgtacact gtggttacac ccatgctgaa tccctttatc
840tatagtctga ggaatcggga tataaatgga gcattgagaa agacactcag
caggagactg 900tgttcacact ga 91216303PRTMus musculus 16Ser Glu Phe
Ile Leu Leu Glu Leu Pro Ile Gln Pro Glu Asp Gln Ala1 5 10 15Val Tyr
Phe Ala Leu Phe Leu Ala Met Tyr Leu Thr Thr Val Leu Gly20 25 30Asn
Leu Leu Ile Ile Leu Leu Ile Arg Leu Asp Ser His Leu His Thr35 40
45Pro Met Tyr Phe Phe Leu Ser His Leu Ala Phe Thr Asp Ile Ser Phe50
55 60Ser Ser Val Thr Ala Pro Lys Met Leu Met Asn Met Leu Thr His
Ser65 70 75 80Gln Ser Ile Ser His Ala Gly Cys Val Ser Gln Ile Tyr
Phe Phe Leu85 90 95Leu Phe Gly Cys Ile Asp Asn Phe Leu Leu Thr Ser
Met Ala Tyr Asp100 105 110Arg Tyr Val Ala Ile Cys His Pro Leu His
Tyr Thr Thr Ile Met Ser115 120 125Gln Ser Leu Cys Val Leu Leu Val
Met Val Ser Trp Ala Phe Ser Ser130 135 140Ser Asn Gly Leu Val His
Thr Leu Leu Phe Ala Arg Leu Ser Leu Phe145 150 155 160Arg Asp Asn
Thr Val His His Phe Phe Cys Asp Leu Ser Ala Leu Leu165 170 175Lys
Leu Ser Ser Ser Asp Thr Thr Ile Asn Glu Leu Val Ile Leu Thr180 185
190Leu Ala Val Val Val Ile Thr Val Pro Phe Ile Cys Ile Leu Val
Ser195 200 205Tyr Gly His Met Gly Ala Thr Ile Leu Arg Thr Pro Ser
Ile Lys Gly210 215 220Ile Cys Lys Ala Leu Ser Thr Cys Gly Ser His
Leu Cys Val Val Ser225 230 235 240Leu Tyr Tyr Gly Ala Ile Ile Gly
Leu Tyr Phe Phe Pro Ser Ser Asn245 250 255Asn Thr Asn Asp Lys Asp
Val Ile Val Ala Val Leu Tyr Thr Val Val260 265 270Thr Pro Met Leu
Asn Pro Phe Ile Tyr Ser Leu Arg Asn Arg Asp Ile275 280 285Asn Gly
Ala Leu Arg Lys Thr Leu Ser Arg Arg Leu Cys Ser His290 295
30017939DNAMus musculus 17atgcctagaa acaacaacca gactaccatc
tctcagttcc tcctcctggg tctgcccatc 60ccccaagagt ttcagcatct gttctatgcc
ctgttcctgg ccatgtacct caccactgtc 120ttggggaacc tcatcatcat
catactcatt cgactggact cccatctcca cacacccatg 180tacttgtttc
tcagcaactt gtccttcact gacctctaat tttcctctgt cacaatgccc
240aagttgctgc agaacatgca gagccaagtt ccttcaatcc cctatgcagg
ctgcctgaca 300caaatgtact tccttttgtt ttttggagat cttgagagct
tcctccttgt ggccatggcc 360tatgaccgct atgtagccat ctgcttccct
cttcattaca ccagcatcat gagccccagg 420ctctgtgtga gtcttgtgct
gctgtcctgg ttgctgacca tgtcccattc catgctgcac 480actttgctct
taactaggtt gtctttctgt gaaaacaatg tgatccccca ttttttctgt
540gatctgtctg ctctgctgaa gctggcctgc tctgatattc acattaatga
attggtgata 600ttgatcatag gagggcttgt tgttatactt ccatttctac
tcatcacagt gtcttatgca 660cgcatcatct cctccattct caaggtccct
tcaactcaag gcatccacaa ggtcttctcc 720acttgtggtt ctcacctgtc
tgtggtgtca ctgttctatg ggacaattat tggcctctac 780ttatgtccat
ctgctaataa ctctactcta aaggacactg tcatgtctat gatgtacacc
840gtggtaactc ccatgctgaa ccccttcatc tacagcctga ggaacagaga
catgaaggaa 900gccctaaaaa gagtgcttca aaagaaaact atcttttga
93918312PRTMus musculusVARIANT(73)Variable amino acid 18Met Pro Arg
Asn Asn Asn Gln Thr Thr Ile Ser Gln Phe Leu Leu Leu1 5 10 15Gly Leu
Pro Ile Pro Gln Glu Phe Gln His Leu Phe Tyr Ala Leu Phe20 25 30Leu
Ala Met Tyr Leu Thr Thr Val Leu Gly Asn Leu Ile Ile Ile Ile35 40
45Leu Ile Arg Leu Asp Ser His Leu His Thr Pro Met Tyr Leu Phe Leu50
55 60Ser Asn Leu Ser Phe Thr Asp Leu Xaa Phe Ser Ser Val Thr Met
Pro65 70 75 80Lys Leu Leu Gln Asn Met Gln Ser Gln Val Pro Ser Ile
Pro Tyr Ala85 90 95Gly Cys Leu Thr Gln Met Tyr Phe Leu Leu Phe Phe
Gly Asp Leu Glu100 105 110Ser Phe Leu Leu Val Ala Met Ala Tyr Asp
Arg Tyr Val Ala Ile Cys115 120 125Phe Pro Leu His Tyr Thr Ser Ile
Met Ser Pro Arg Leu Cys Val Ser130 135 140Leu Val Leu Leu Ser Trp
Leu Leu Thr Met Ser His Ser Met Leu His145 150 155 160Thr Leu Leu
Leu Thr Arg Leu Ser Phe Cys Glu Asn Asn Val Ile Pro165 170 175His
Phe Phe Cys Asp Leu Ser Ala Leu Leu Lys Leu Ala Cys Ser Asp180 185
190Ile His Ile Asn Glu Leu Val Ile Leu Ile Ile Gly Gly Leu Val
Val195 200 205Ile Leu Pro Phe Leu Leu Ile Thr Val Ser Tyr Ala Arg
Ile Ile Ser210 215 220Ser Ile Leu Lys Val Pro Ser Thr Gln Gly Ile
His Lys Val Phe Ser225 230 235 240Thr Cys Gly Ser His Leu Ser Val
Val Ser Leu Phe Tyr Gly Thr Ile245 250 255Ile Gly Leu Tyr Leu Cys
Pro Ser Ala Asn Asn Ser Thr Leu Lys Asp260 265 270Thr Val Met Ser
Met Met Tyr Thr Val Val Thr Pro Met Leu Asn Pro275 280 285Phe Ile
Tyr Ser Leu Arg Asn Arg Asp Met Lys Glu Ala Leu Lys Arg290 295
300Val Leu Gln Lys Lys Thr Ile Phe305 31019936DNAMus musculus
19atgcaaaacc agagctttgt cactgagttc atactcttgg ggctttccca gaacccaaaa
60gttgagaaaa tactgtttgt tgtattttta ttggtctata ttgcaactat tgggggaaac
120atgataattg tggtgaccat catctatagc cctgcactgt tgagttcccc
catgtacttc 180ttcttaatat ttctgtcttt cctggatgct tgcacttcct
ctactgtcac ccccaagatg 240attgtagact tcttctatga gaggaagacc
atctcctttg aatgttgcat cacacaactg 300tttactagcc acttctttgc
aggagttgag gtgattatct tgacatctat ggcctatgac 360cgctatgtgg
ccatctgcaa gcctcttcac tactcttcca tcatgaccag gaggctctgt
420ggcactctcg taatggtggc ctggacagga ggattcttac attctatcac
acaagttatc 480ttcacgttgc agctaccctt ctgtgggccc aattttattg
atcatttcat atgtgacttg 540ttcccattac tgcagcttgc ctgcactgac
acacacattt ttgtcatttt ggtgtttgct 600aatagtgggt ctttctgcat
cattatcttc tccttgttga ttgtttccta tggtgtcatc 660ctcttctctc
taagaggtca cagctcagaa ggacgaagga aagctctctc aacctgtgga
720tcccatatta ctgttatgat attattcttt gtcccatgta tgctaatata
tgcacggcct 780tcatctgcct tttcctttga gaaaaacaca cttatatttg
cctctgtcct gacaccattg 840ttcaatccta tggtttacac tttcagaaat
aaagaaatga agaatgccat caggaaaatg 900tgtaggaaaa tgttagtaga
ttctgataac ttttaa 93620311PRTMus musculus 20Met Gln Asn Gln Ser Phe
Val Thr Glu Phe Ile Leu Leu Gly Leu Ser1 5 10 15Gln Asn Pro Lys Val
Glu Lys Ile Leu Phe Val Val Phe Leu Leu Val20 25 30Tyr Ile Ala Thr
Ile Gly Gly Asn Met Ile Ile Val Val Thr Ile Ile35 40 45Tyr Ser Pro
Ala Leu Leu Ser Ser Pro Met Tyr Phe Phe Leu Ile Phe50 55 60Leu Ser
Phe Leu Asp Ala Cys Thr Ser Ser Thr Val Thr Pro Lys Met65 70 75
80Ile Val Asp Phe Phe Tyr Glu Arg Lys Thr Ile Ser Phe Glu Cys Cys85
90 95Ile Thr Gln Leu Phe Thr Ser His Phe Phe Ala Gly Val Glu Val
Ile100 105 110Ile Leu Thr Ser Met Ala Tyr Asp Arg Tyr Val Ala Ile
Cys Lys Pro115 120 125Leu His Tyr Ser Ser Ile Met Thr Arg Arg Leu
Cys Gly Thr Leu Val130 135 140Met Val Ala Trp Thr Gly Gly Phe Leu
His Ser Ile Thr Gln Val Ile145 150 155 160Phe Thr Leu Gln Leu Pro
Phe Cys Gly Pro Asn Phe Ile Asp His Phe165 170 175Ile Cys Asp Leu
Phe Pro Leu Leu Gln Leu Ala Cys Thr Asp Thr His180 185 190Ile Phe
Val Ile Leu Val Phe Ala Asn Ser Gly Ser Phe Cys Ile Ile195 200
205Ile Phe Ser Leu Leu Ile Val Ser Tyr Gly Val Ile Leu Phe Ser
Leu210 215 220Arg Gly His Ser Ser Glu Gly Arg Arg Lys Ala Leu Ser
Thr Cys Gly225 230 235 240Ser His Ile Thr Val Met Ile Leu Phe Phe
Val Pro Cys Met Leu Ile245 250 255Tyr Ala Arg Pro Ser Ser Ala Phe
Ser Phe Glu Lys Asn Thr Leu Ile260 265 270Phe Ala Ser Val Leu Thr
Pro Leu Phe Asn Pro Met Val Tyr Thr Phe275 280 285Arg Asn Lys Glu
Met Lys Asn Ala Ile Arg Lys Met Cys Arg Lys Met290 295 300Leu Val
Asp Ser Asp Asn Phe305 31021921DNAMus musculus 21atgggacaga
accacaatgt cacagaattc atttttgtgg gtcttagtca agatcctgct 60gggcaaaaag
tattatttgt cttgttttca ctgacttaca ttgtgacaat gttcggaaac
120ctgctcattg cacttacagt gattgccagc ccctccttaa actccccaat
gtacttcttc 180cttgcctgtc tgtcagtcct ggatgctctt tattgcaata
caatctcacc aaatttgatt 240atagacttgt tatataataa aaagaatatc
tccttcagag cttgcatgct ccagctgttt 300gtagagcact tatttggagg
tgttgaggtc ttccttctgg tattcatggc ctatgatcgc 360tatgtggcca
tctgtaagcc actgcactat ttgaccatca tgaaccagag ggtgtgcatt
420cttctattgc tgatagctgg agttggaggc atcttacact cactgattca
agttctgact 480gtgtataaac ttcctttttg tggtcccaat gtcattgatc
acttcatgtg tgacatgaat 540caattactcg ggcttgcatg cactgacacc
tacttccttg gcatcactgt catggccaat 600ggtggagtaa tctgtgtggg
aattttcacc tttctcttag tctcctatgg aatcattcta 660aactctctta
agacccacag tcgggaagga agacataaag ctctgtttac ctgcagttct
720cacatcatgg ttgttgtctg cttttttgct ccctgtagtt ttatatatgc
tagacctgtc 780tccaactttc cagtggataa atatattgct gtgttttata
cagttgttag tcccatgctg 840aatccattga tatatacctt gagaaattca
gagatgaaaa actctattaa aaagctctgg 900tgtaaaactc taacaacata a
92122240PRTMus musculus 22Met Gly Gln Asn His Asn Val Thr Glu Phe
Ile Phe Val Gly Leu Ser1 5 10 15Gln Asp Pro Ala Gly Gln Lys Val Leu
Phe Val Leu Phe Ser Leu Thr20 25 30Tyr Ile Val Thr Met Phe Gly Asn
Leu Leu Ile Ala Leu Thr Val Ile35 40 45Ala Ser Pro Ser Leu Asn Ser
Pro Met Tyr Phe Phe Leu Ala Cys Leu50 55 60Ser Val Leu Asp Ala Leu
Tyr Cys Asn Thr Ile Ser Pro Asn Leu Ile65 70 75 80Ile Asp Leu Leu
Tyr Asn Lys Lys Asn Ile Ser Phe Arg Ala Cys Met85 90 95Leu Gln Leu
Phe Val Glu His Leu Phe Gly Gly Val Glu Val Phe Leu100 105 110Leu
Val Phe Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Lys Pro Leu115 120
125His Tyr Leu Thr Ile Met Asn Gln Arg Val Cys Ile Leu Leu Leu
Leu130 135 140Ile Ala Gly Val Gly Gly Ile Leu His Ser Leu Ile Gln
Val Leu Thr145 150 155 160Val Tyr Lys Leu Pro Phe Cys Gly Pro Asn
Val Ile Asp His Phe Met165 170 175Cys Asp Met Asn Gln Leu Leu Gly
Leu Ala Cys Thr Asp Thr Tyr Phe180 185 190Leu Gly Ile Thr Val Met
Ala Asn Gly Gly Val Ile Cys Val Gly Ile195 200 205Phe Thr Phe Leu
Leu Val Ser Tyr Gly Ile Ile Leu Asn Ser Leu Lys210 215 220Thr His
Ser Arg Glu Gly Arg His Lys Ala Leu Phe Thr Cys Ser Ser225 230 235
24023993DNAHomo sapiens 23atgtgttctt ttttcttgtg ccaaacaggt
aaacaggcaa aaatatcaat gggagaagaa 60aaccaaacct ttgtgtccaa gtttatcttc
ctgggtcttt cacaggactt gcagacccag 120atcctgctat ttatcctttt
cctcatcatt
tatctgctga ccgtgcttgg aaaccagctc 180atcatcattc tcatcttcct
ggattctcgc cttcacactc ccatgtattt ttttcttaga 240aatctctcct
ttgcagatct ctgtttctct actagcattg tccctcaagt gttggttcac
300ttcttggtaa agaggaaaac catttctttt tatgggtgta tgacacagat
aattgtcttt 360cttctggttg ggtgtacaga gtgtgcgctg ctggcagtga
tgtcctatga ccggtatgtg 420gctgtctgca agcccctgta ctactctacc
atcatgacac aacgggtgtg tctctggctg 480tccttcaggt cctgggccag
tggggcacta gtgtctttag tagataccag ctttactttc 540catcttccct
actggggaca gaatataatc aatcactact tttgtgaacc tcctgccctc
600ctgaagctgg cttccataga cacttacagc acagaaatgg ccatcttttc
aatgggcgtg 660gtaatcctcc tggcccctgt ctccctgatt cttggttctt
attggaatat tatctccact 720gttatccaga tgcagtctgg ggaagggaga
ctcaaggctt tttccacctg tggctcccat 780cttattgttg ttgtcctctt
ctatgggtca ggaatattca cctacatgcg accaaactcc 840aagactacaa
aagaactgga taaaatgata tctgtgttct atacagcggt gactccaatg
900ttgaacccca taatttatag cttgaggaac aaagatgtca aaggggctct
caggaaacta 960gttgggagaa agtgcttctc tcataggcag tga 99324330PRTHomo
sapiens 24Met Cys Ser Phe Phe Leu Cys Gln Thr Gly Lys Gln Ala Lys
Ile Ser1 5 10 15Met Gly Glu Glu Asn Gln Thr Phe Val Ser Lys Phe Ile
Phe Leu Gly20 25 30Leu Ser Gln Asp Leu Gln Thr Gln Ile Leu Leu Phe
Ile Leu Phe Leu35 40 45Ile Ile Tyr Leu Leu Thr Val Leu Gly Asn Gln
Leu Ile Ile Ile Leu50 55 60Ile Phe Leu Asp Ser Arg Leu His Thr Pro
Met Tyr Phe Phe Leu Arg65 70 75 80Asn Leu Ser Phe Ala Asp Leu Cys
Phe Ser Thr Ser Ile Val Pro Gln85 90 95Val Leu Val His Phe Leu Val
Lys Arg Lys Thr Ile Ser Phe Tyr Gly100 105 110Cys Met Thr Gln Ile
Ile Val Phe Leu Leu Val Gly Cys Thr Glu Cys115 120 125Ala Leu Leu
Ala Val Met Ser Tyr Asp Arg Tyr Val Ala Val Cys Lys130 135 140Pro
Leu Tyr Tyr Ser Thr Ile Met Thr Gln Arg Val Cys Leu Trp Leu145 150
155 160Ser Phe Arg Ser Trp Ala Ser Gly Ala Leu Val Ser Leu Val Asp
Thr165 170 175Ser Phe Thr Phe His Leu Pro Tyr Trp Gly Gln Asn Ile
Ile Asn His180 185 190Tyr Phe Cys Glu Pro Pro Ala Leu Leu Lys Leu
Ala Ser Ile Asp Thr195 200 205Tyr Ser Thr Glu Met Ala Ile Phe Ser
Met Gly Val Val Ile Leu Leu210 215 220Ala Pro Val Ser Leu Ile Leu
Gly Ser Tyr Trp Asn Ile Ile Ser Thr225 230 235 240Val Ile Gln Met
Gln Ser Gly Glu Gly Arg Leu Lys Ala Phe Ser Thr245 250 255Cys Gly
Ser His Leu Ile Val Val Val Leu Phe Tyr Gly Ser Gly Ile260 265
270Phe Thr Tyr Met Arg Pro Asn Ser Lys Thr Thr Lys Glu Leu Asp
Lys275 280 285Met Ile Ser Val Phe Tyr Thr Ala Val Thr Pro Met Leu
Asn Pro Ile290 295 300Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Gly
Ala Leu Arg Lys Leu305 310 315 320Val Gly Arg Lys Cys Phe Ser His
Arg Gln325 33025951DNAHomo sapiens 25atggagctct ggaacttcac
cttgggaagt ggcttcattt tggtggggat tctgaatgac 60agtgggtctc ctgaactgct
ctgtgctaca attacaatcc tatacttgtt ggccctgatc 120agcaatggcc
tactgctcct ggctatcacc atggaagccc ggctccacat gcccatgtac
180ctcctgcttg ggcagctctc tctcatggac ctcctgttca catctgttgt
cactcccaag 240gcccttgcgg actttctgcg cagagaaaac accatctcct
ttggaggctg tgcccttcag 300atgttcctgg cactgacaat gggtggtgct
gaggacctcc tactggcctt catggcctat 360gacaggtatg tggccatttg
tcatcctctg acatacatga ccctcatgag ctcaagagcc 420tgctggctca
tggtggccac gtcctggatc ctggcatccc taagtgccct aatatatacc
480gtgtatacca tgcactatcc cttctgcagg gcccaggaga tcaggcatct
tctctgtgag 540atcccacact tgctgaaggt ggcctgtgct gatacctcca
gatatgagct catggtatat 600gtgatgggtg tgaccttcct gattccctct
cttgctgcta tactggcctc ctatacacaa 660attctactca ctgtgctcca
tatgccatca aatgagggga ggaagaaagc ccttgtcacc 720tgctcttccc
acctgactgt ggttgggatg ttctatggag ctgccacatt catgtatgtc
780ttgcccagtt ccttccacag caccagacaa gacaacatca tctctgtttt
ctacacaatt 840gtcactccag ccctgaatcc actcatctac agcctgagga
ataaggaggt catgcgggcc 900ttgaggaggg tcctgggaaa atacatgctg
ccagcacact ccacgctcta g 95126316PRTHomo sapiens 26Met Glu Leu Trp
Asn Phe Thr Leu Gly Ser Gly Phe Ile Leu Val Gly1 5 10 15Ile Leu Asn
Asp Ser Gly Ser Pro Glu Leu Leu Cys Ala Thr Ile Thr20 25 30Ile Leu
Tyr Leu Leu Ala Leu Ile Ser Asn Gly Leu Leu Leu Leu Ala35 40 45Ile
Thr Met Glu Ala Arg Leu His Met Pro Met Tyr Leu Leu Leu Gly50 55
60Gln Leu Ser Leu Met Asp Leu Leu Phe Thr Ser Val Val Thr Pro Lys65
70 75 80Ala Leu Ala Asp Phe Leu Arg Arg Glu Asn Thr Ile Ser Phe Gly
Gly85 90 95Cys Ala Leu Gln Met Phe Leu Ala Leu Thr Met Gly Gly Ala
Glu Asp100 105 110Leu Leu Leu Ala Phe Met Ala Tyr Asp Arg Tyr Val
Ala Ile Cys His115 120 125Pro Leu Thr Tyr Met Thr Leu Met Ser Ser
Arg Ala Cys Trp Leu Met130 135 140Val Ala Thr Ser Trp Ile Leu Ala
Ser Leu Ser Ala Leu Ile Tyr Thr145 150 155 160Val Tyr Thr Met His
Tyr Pro Phe Cys Arg Ala Gln Glu Ile Arg His165 170 175Leu Leu Cys
Glu Ile Pro His Leu Leu Lys Val Ala Cys Ala Asp Thr180 185 190Ser
Arg Tyr Glu Leu Met Val Tyr Val Met Gly Val Thr Phe Leu Ile195 200
205Pro Ser Leu Ala Ala Ile Leu Ala Ser Tyr Thr Gln Ile Leu Leu
Thr210 215 220Val Leu His Met Pro Ser Asn Glu Gly Arg Lys Lys Ala
Leu Val Thr225 230 235 240Cys Ser Ser His Leu Thr Val Val Gly Met
Phe Tyr Gly Ala Ala Thr245 250 255Phe Met Tyr Val Leu Pro Ser Ser
Phe His Ser Thr Arg Gln Asp Asn260 265 270Ile Ile Ser Val Phe Tyr
Thr Ile Val Thr Pro Ala Leu Asn Pro Leu275 280 285Ile Tyr Ser Leu
Arg Asn Lys Glu Val Met Arg Ala Leu Arg Arg Val290 295 300Leu Gly
Lys Tyr Met Leu Pro Ala His Ser Thr Leu305 310 31527948DNAHomo
sapiensvariation(9)..(12)a, t, c, g, other or unknown 27atgagacann
nnaacaatat nacagaattt gtcctcctgg gcttttctca ggatcctggt 60gtgnnnaaag
cattatttgt catgttttta ctcacatacn nnnnnacagt ggtggggaac
120ctgctcattg tngtggatat tattgccagc ccttnnttgg gttccccaat
gtatttcttc 180cttgcctgcc tgtcatttat agatgctgca tattccacta
ccatttctcc caagttaatt 240gtaggcttat tctgtgataa aaagactatt
tccttccaag gttgcatggg ccagctattt 300atagaccatt tctttggtgg
ggctgaggtc ttccttctgg tggtgatggc ctgtgatcgc 360tatgtggcca
tctgtaagcc actgcactat ttgaccatca tgaatcgaca ggtttgcttc
420cttctgttgg tnntnnccat gattggaggt tttgtacatt ctgcgtttca
aattgttgtg 480tacagtctcc ctttctgtgg tcccnatgtc attgttcatt
tcagttgtga catgcaccca 540ttactggaac tggcatgcac tgacacctac
tttataggcc tcactgttgt tgtcaatagt 600ggagcaatct gtatggtcat
tttcaacctt ctgttaatct cctatggagt catcctaagc 660tcccttaaaa
cttacagtca ggaaaagagg ggtaaagcct tgtctacctg cagctccggc
720agtaccgttg ttgtcctctt ttttgtaccc tgtattttca tatatgttag
acctgtttca 780aactttccta ctgataagtt catgactgtg ttttatacca
ttatcacaca catgctgagt 840cctttaatat atacgttgag aaattcagag
atgagaaatg ctatagaaaa actcttgggt 900aaaaagttaa ctatatttat
tataggagga gtgtccgtcc tcatgtag 94828315PRTHomo
sapiensVARIANT(3)..(4)Varible amino acid 28Met Arg Xaa Xaa Asn Asn
Xaa Thr Glu Phe Val Leu Leu Gly Phe Ser1 5 10 15Gln Asp Pro Gly Val
Xaa Lys Ala Leu Phe Val Met Phe Leu Leu Thr20 25 30Tyr Xaa Xaa Thr
Val Val Gly Asn Leu Leu Ile Val Val Asp Ile Ile35 40 45Ala Ser Pro
Xaa Leu Gly Ser Pro Met Tyr Phe Phe Leu Ala Cys Leu50 55 60Ser Phe
Ile Asp Ala Ala Tyr Ser Thr Thr Ile Ser Pro Lys Leu Ile65 70 75
80Val Gly Leu Phe Cys Asp Lys Lys Thr Ile Ser Phe Gln Gly Cys Met85
90 95Gly Gln Leu Phe Ile Asp His Phe Phe Gly Gly Ala Glu Val Phe
Leu100 105 110Leu Val Val Met Ala Cys Asp Arg Tyr Val Ala Ile Cys
Lys Pro Leu115 120 125His Tyr Leu Thr Ile Met Asn Arg Gln Val Cys
Phe Leu Leu Leu Val130 135 140Xaa Xaa Met Ile Gly Gly Phe Val His
Ser Ala Phe Gln Ile Val Val145 150 155 160Tyr Ser Leu Pro Phe Cys
Gly Pro Xaa Val Ile Val His Phe Ser Cys165 170 175Asp Met His Pro
Leu Leu Glu Leu Ala Cys Thr Asp Thr Tyr Phe Ile180 185 190Gly Leu
Thr Val Val Val Asn Ser Gly Ala Ile Cys Met Val Ile Phe195 200
205Asn Leu Leu Leu Ile Ser Tyr Gly Val Ile Leu Ser Ser Leu Lys
Thr210 215 220Tyr Ser Gln Glu Lys Arg Gly Lys Ala Leu Ser Thr Cys
Ser Ser Gly225 230 235 240Ser Thr Val Val Val Leu Phe Phe Val Pro
Cys Ile Phe Ile Tyr Val245 250 255Arg Pro Val Ser Asn Phe Pro Thr
Asp Lys Phe Met Thr Val Phe Tyr260 265 270Thr Ile Ile Thr His Met
Leu Ser Pro Leu Ile Tyr Thr Leu Arg Asn275 280 285Ser Glu Met Arg
Asn Ala Ile Glu Lys Leu Leu Gly Lys Lys Leu Thr290 295 300Ile Phe
Ile Ile Gly Gly Val Ser Val Leu Met305 310 31529939DNAHomo sapiens
29atggaacaac acaatctaac aacggtgaat gaattcattc ttacgggaat cacagatatc
60gctgagctgc aggcaccatt atttgcattg ttcctcatga tctatgtgat ctcagtgatg
120ggcaatttgg gcatgattgt cctcaccaag ttggactcca ggttgcaaac
ccctatgtac 180ttttttctca gacatctggc tttcatggat cttggttatt
caacaactgt gggacccaaa 240atgttagtaa attttgttgt ggataagaat
ataatttctt attatttttg tgcaacacag 300ctagctttct ttcttgtgtt
cattggtagt gaacttttta ttctctcagc catgtcctac 360gacctctatg
tggccatctg taaccctctg ctatacacag taatcatgtc acgaagggta
420tgtcaggtgc tggtagcaat cccttacctc tattgcacat tcatttctct
tctagtcacc 480ataaagattt ttactttatc cttctgtggc tacaacgtca
ttagtcattt ctactgtgac 540agtctccctt tgttaccttt gctttgttca
aatacacatg aaattgaatt gataattctg 600atctttgcag ctattgattt
gatttcatct cttctgatag ttcttttatc ttacctgctc 660atccttgtag
ccattctcag gatgaattct gctggcagac aaaaggcttt ttctacctgt
720ggagcccacc tgacagtggt catagtgttc tatgggactt tgcttttcat
gtacgtgcag 780cccaagtcca gtcattcctt tgacactgat aaagtggctt
ccatatttta caccctggtt 840atccccatgt tgaatccctt gatctatagt
ttacgaaaca aagatgtaaa atatgcccta 900cgaaggacat ggaataactt
atgtaatatt tttgtttaa 93930312PRTHomo sapiens 30Met Glu Gln His Asn
Leu Thr Thr Val Asn Glu Phe Ile Leu Thr Gly1 5 10 15Ile Thr Asp Ile
Ala Glu Leu Gln Ala Pro Leu Phe Ala Leu Phe Leu20 25 30Met Ile Tyr
Val Ile Ser Val Met Gly Asn Leu Gly Met Ile Val Leu35 40 45Thr Lys
Leu Asp Ser Arg Leu Gln Thr Pro Met Tyr Phe Phe Leu Arg50 55 60His
Leu Ala Phe Met Asp Leu Gly Tyr Ser Thr Thr Val Gly Pro Lys65 70 75
80Met Leu Val Asn Phe Val Val Asp Lys Asn Ile Ile Ser Tyr Tyr Phe85
90 95Cys Ala Thr Gln Leu Ala Phe Phe Leu Val Phe Ile Gly Ser Glu
Leu100 105 110Phe Ile Leu Ser Ala Met Ser Tyr Asp Leu Tyr Val Ala
Ile Cys Asn115 120 125Pro Leu Leu Tyr Thr Val Ile Met Ser Arg Arg
Val Cys Gln Val Leu130 135 140Val Ala Ile Pro Tyr Leu Tyr Cys Thr
Phe Ile Ser Leu Leu Val Thr145 150 155 160Ile Lys Ile Phe Thr Leu
Ser Phe Cys Gly Tyr Asn Val Ile Ser His165 170 175Phe Tyr Cys Asp
Ser Leu Pro Leu Leu Pro Leu Leu Cys Ser Asn Thr180 185 190His Glu
Ile Glu Leu Ile Ile Leu Ile Phe Ala Ala Ile Asp Leu Ile195 200
205Ser Ser Leu Leu Ile Val Leu Leu Ser Tyr Leu Leu Ile Leu Val
Ala210 215 220Ile Leu Arg Met Asn Ser Ala Gly Arg Gln Lys Ala Phe
Ser Thr Cys225 230 235 240Gly Ala His Leu Thr Val Val Ile Val Phe
Tyr Gly Thr Leu Leu Phe245 250 255Met Tyr Val Gln Pro Lys Ser Ser
His Ser Phe Asp Thr Asp Lys Val260 265 270Ala Ser Ile Phe Tyr Thr
Leu Val Ile Pro Met Leu Asn Pro Leu Ile275 280 285Tyr Ser Leu Arg
Asn Lys Asp Val Lys Tyr Ala Leu Arg Arg Thr Trp290 295 300Asn Asn
Leu Cys Asn Ile Phe Val305 31031312PRTHomo sapiens 31Met Glu Ala
Glu Asn Leu Thr Glu Leu Ser Lys Phe Leu Leu Leu Gly1 5 10 15Leu Ser
Asp Asp Pro Glu Leu Gln Pro Val Leu Phe Gly Leu Phe Leu20 25 30Ser
Met Tyr Leu Val Thr Val Leu Gly Asn Leu Leu Ile Ile Leu Ala35 40
45Val Ser Ser Asp Ser His Leu His Thr Pro Met Tyr Phe Phe Leu Ser50
55 60Asn Leu Ser Phe Val Asp Ile Cys Phe Ile Ser Thr Thr Val Pro
Lys65 70 75 80Met Leu Val Ser Ile Gln Ala Arg Ser Lys Asp Ile Ser
Tyr Met Gly85 90 95Cys Leu Thr Gln Val Tyr Phe Leu Met Met Phe Ala
Gly Met Asp Thr100 105 110Phe Leu Leu Ala Val Met Ala Tyr Asp Arg
Phe Val Ala Ile Cys His115 120 125Pro Leu His Tyr Thr Val Ile Met
Asn Pro Cys Leu Cys Gly Leu Leu130 135 140Val Leu Ala Ser Trp Phe
Ile Ile Phe Trp Phe Ser Leu Val His Ile145 150 155 160Leu Leu Met
Lys Arg Leu Thr Phe Ser Thr Gly Thr Glu Ile Pro His165 170 175Phe
Phe Cys Glu Pro Ala Gln Val Leu Lys Val Ala Cys Ser Asn Thr180 185
190Leu Leu Asn Asn Ile Val Leu Tyr Val Ala Thr Ala Leu Leu Gly
Val195 200 205Phe Pro Val Ala Gly Ile Leu Phe Ser Tyr Ser Gln Ile
Val Ser Ser210 215 220Leu Met Gly Met Ser Ser Thr Lys Gly Lys Tyr
Lys Ala Phe Ser Thr225 230 235 240Cys Gly Ser His Leu Cys Val Val
Ser Leu Phe Tyr Gly Thr Gly Leu245 250 255Gly Val Tyr Leu Ser Ser
Ala Val Thr His Ser Ser Gln Ser Ser Ser260 265 270Thr Ala Ser Val
Met Tyr Ala Met Val Thr Pro Met Leu Asn Pro Phe275 280 285Ile Tyr
Ser Leu Arg Asn Lys Asp Val Lys Gly Ala Leu Glu Arg Leu290 295
300Leu Ser Arg Ala Asp Ser Cys Pro305 31032314PRTHomo sapiens 32Met
Gly Glu Glu Asn Gln Thr Phe Val Ser Lys Phe Ile Phe Leu Gly1 5 10
15Leu Ser Gln Asp Leu Gln Thr Gln Ile Leu Leu Phe Ile Leu Phe Leu20
25 30Ile Ile Tyr Leu Leu Thr Val Leu Gly Asn Gln Leu Ile Ile Ile
Leu35 40 45Ile Phe Leu Asp Ser Arg Leu His Thr Pro Met Tyr Phe Phe
Leu Arg50 55 60Asn Leu Ser Phe Ala Asp Leu Cys Phe Ser Thr Ser Ile
Val Pro Gln65 70 75 80Val Leu Val His Phe Leu Val Lys Arg Lys Thr
Ile Ser Phe Tyr Gly85 90 95Cys Met Thr Gln Ile Ile Val Phe Leu Leu
Val Gly Cys Thr Glu Cys100 105 110Ala Leu Leu Ala Val Met Ser Tyr
Asp Arg Tyr Val Ala Val Cys Lys115 120 125Pro Leu Tyr Tyr Ser Thr
Ile Met Thr Gln Arg Val Cys Leu Trp Leu130 135 140Ser Phe Arg Ser
Trp Ala Ser Gly Ala Leu Val Ser Leu Val Asp Thr145 150 155 160Ser
Phe Thr Phe His Leu Pro Tyr Trp Gly Gln Asn Ile Ile Asn His165 170
175Tyr Phe Cys Glu Pro Pro Ala Leu Leu Lys Leu Ala Ser Ile Asp
Thr180 185 190Tyr Ser Thr Glu Met Ala Ile Phe Ser Met Gly Val Val
Ile Leu Leu195 200 205Ala Pro Val Ser Leu Ile Leu Gly Ser Tyr Trp
Asn Ile Ile Ser Thr210 215 220Val Ile Gln Met Gln Ser Gly Glu Gly
Arg Leu Lys Ala Phe Ser Thr225 230 235 240Cys Gly Ser His Leu Ile
Val Val Val Leu Phe Tyr Gly Ser Gly Ile245 250 255Phe Thr Tyr Met
Arg Pro Asn Ser Lys Thr Thr Lys Glu Leu Asp Lys260 265 270Met Ile
Ser Val Phe Tyr Thr Ala Val Thr Pro Met Leu Asn Pro Ile275 280
285Ile Tyr Ser Leu Arg Asn Lys Asp Val Lys Gly Ala Leu Arg Lys
Leu290 295 300Val Gly Arg Lys Cys Phe Ser His Arg Gln305
31033318PRTMus musculus 33Met Glu Thr Gly Asn Asp Thr Gln Leu Ser
Glu Phe Phe Leu Leu Gly1 5 10 15Phe Ser Glu Asn Gln Pro Gln Ile Gln
Pro Val Ile Phe Gly Leu Phe20 25 30Leu Phe Met Tyr Ile Leu Thr Phe
Thr Gly Asn Leu Leu Ile Ile Met35 40 45Ala Ile Ile Val Asp Ser His
Leu His
Thr Pro Met Tyr Leu Phe Phe50 55 60Ser Asn Leu Ser Phe Val Asp Ile
Cys Phe Thr Ser Thr Thr Val Pro65 70 75 80Gln Met Leu Val Asn Ile
His Thr Gln Ser Lys Ala Ile Thr Tyr Ala85 90 95Gly Cys Ile Ile Gln
Met Tyr Phe Leu Leu Leu Phe Ser Gly Leu Asp100 105 110Ile Phe Leu
Leu Thr Val Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys115 120 125His
Pro Leu His Tyr Met Ile Met Ser Thr Arg Arg Cys Gly Leu Met130 135
140Ile Leu Ala Cys Trp Ile Ile Gly Val Ile Asn Ser Leu Leu His
Thr145 150 155 160Phe Leu Val Leu Arg Leu Ser Phe Cys Thr Asn Leu
Glu Ile Pro His165 170 175Phe Phe Cys Glu Leu Asn Gln Val Val His
Gln Ala Cys Ser Asp Thr180 185 190Phe Leu Asn Asp Met Val Ile Tyr
Ile Thr Ala Met Leu Leu Ala Val195 200 205Gly Pro Phe Ser Gly Ile
Leu Tyr Ser Tyr Ser Arg Ile Val Ser Ser210 215 220Ile Cys Ala Ile
Ser Ser Val Gln Gly Lys Tyr Lys Ala Phe Ser Thr225 230 235 240Cys
Ala Ser His Leu Ser Val Val Ser Leu Phe Tyr Cys Thr Leu Leu245 250
255Gly Val Tyr Leu Ser Ser Ala Val Thr Gln Asn Ser His Ala Thr
Ala260 265 270Thr Ala Ser Leu Met Tyr Thr Val Val Thr Pro Met Leu
Asn Pro Phe275 280 285Ile Tyr Ser Leu Arg Asn Lys Asp Ile Lys Thr
Ala Leu Lys Ile Leu290 295 300Leu Gly Ser Val Thr Arg Ser Arg Ser
Met Asp Ser Pro Ser305 310 31534310PRTHomo sapiens 34Met Asn Trp
Val Asn Lys Ser Val Pro Gln Glu Phe Ile Leu Leu Val1 5 10 15Phe Ser
Asp Gln Pro Trp Leu Glu Ile Pro Pro Phe Val Met Phe Leu20 25 30Phe
Ser Tyr Ile Leu Thr Ile Phe Gly Asn Leu Thr Ile Ile Leu Val35 40
45Ser His Val Asp Phe Lys Leu His Thr Pro Met Tyr Phe Phe Leu Ser50
55 60Asn Leu Ser Leu Leu Asp Leu Cys Tyr Thr Thr Ser Thr Val Pro
Gln65 70 75 80Met Leu Val Asn Ile Cys Asn Thr Arg Lys Val Ile Ser
Tyr Gly Gly85 90 95Cys Val Ala Gln Leu Phe Ile Phe Leu Ala Leu Gly
Ser Thr Glu Cys100 105 110Leu Leu Leu Ala Val Met Cys Phe Asp Arg
Phe Val Ala Ile Cys Arg115 120 125Pro Leu His Tyr Ser Ile Ile Met
His Gln Arg Leu Cys Phe Gln Leu130 135 140Ala Ala Ala Ser Trp Ile
Ser Gly Phe Ser Asn Ser Val Leu Gln Ser145 150 155 160Thr Trp Thr
Leu Lys Met Pro Leu Cys Gly His Lys Glu Val Asp His165 170 175Phe
Phe Cys Glu Val Pro Ala Leu Leu Lys Leu Ser Cys Val Asp Thr180 185
190Thr Ala Asn Glu Ala Glu Leu Phe Phe Ile Ser Val Leu Phe Leu
Leu195 200 205Ile Pro Val Thr Leu Ile Leu Ile Ser Tyr Ala Phe Ile
Val Gln Ala210 215 220Val Leu Arg Ile Gln Ser Ala Glu Gly Gln Arg
Lys Ala Phe Gly Thr225 230 235 240Cys Gly Ser His Leu Ile Val Val
Ser Leu Phe Tyr Gly Thr Ala Ile245 250 255Ser Met Tyr Leu Gln Pro
Pro Ser Pro Ser Ser Lys Asp Arg Gly Lys260 265 270Met Val Ser Leu
Phe Cys Gly Ile Ile Ala Pro Met Leu Asn Pro Leu275 280 285Ile Tyr
Thr Leu Arg Asn Lys Glu Val Lys Glu Ala Phe Lys Arg Leu290 295
300Val Ala Lys Ser Leu Leu305 31035309PRTHomo sapiens 35Met Lys Ser
Trp Asn Asn Thr Ile Ile Leu Glu Phe Leu Leu Leu Gly1 5 10 15Ile Ser
Glu Glu Pro Glu Leu Gln Ala Phe Leu Phe Gly Leu Phe Leu20 25 30Ser
Met Tyr Leu Val Thr Val Leu Gly Asn Leu Leu Ile Ile Leu Ala35 40
45Thr Ile Ser Asp Ser His Leu His Thr Pro Met Tyr Phe Phe Leu Ser50
55 60Asn Leu Ser Phe Val Asp Ile Cys Phe Val Ser Thr Thr Val Pro
Lys65 70 75 80Met Leu Val Asn Ile Gln Thr His Asn Lys Val Ile Thr
Tyr Ala Gly85 90 95Cys Ile Thr Gln Met Cys Phe Phe Leu Leu Phe Val
Gly Leu Asp Asn100 105 110Phe Leu Leu Thr Val Met Ala Tyr Asp Arg
Phe Val Ala Ile Cys His115 120 125Pro Leu His Tyr Met Val Ile Met
Asn Pro Gln Leu Cys Gly Leu Leu130 135 140Val Leu Ala Ser Trp Ile
Met Ser Val Leu Asn Ser Met Leu Gln Ser145 150 155 160Leu Met Val
Leu Pro Leu Pro Phe Cys Thr His Met Glu Ile Pro His165 170 175Phe
Phe Cys Glu Ile Asn Gln Val Val His Leu Ala Cys Ser Asp Thr180 185
190Phe Leu Asn Asp Ile Val Met Tyr Phe Ala Val Ala Leu Leu Gly
Gly195 200 205Gly Pro Leu Thr Gly Ile Leu Tyr Ser Tyr Ser Lys Ile
Val Ser Ser210 215 220Ile Arg Ala Ile Ser Ser Ala Gln Gly Lys Tyr
Lys Ala Phe Ser Thr225 230 235 240Cys Ala Ser His Leu Ser Val Val
Ser Leu Phe Tyr Gly Thr Cys Leu245 250 255Gly Val Tyr Leu Ser Ser
Ala Ala Thr His Asn Ser His Thr Gly Ala260 265 270Ala Ala Ser Val
Met Tyr Thr Val Val Thr Pro Met Leu Asn Pro Phe275 280 285Ile Tyr
Ser Leu Arg Asn Lys His Ile Lys Gly Ala Met Lys Thr Phe290 295
300Phe Arg Gly Lys Gln30536313PRTHomo sapiens 36Met Lys Arg Glu Asn
Gln Ser Ser Val Ser Glu Phe Leu Leu Leu Asp1 5 10 15Leu Pro Ile Trp
Pro Glu Gln Gln Ala Val Phe Phe Thr Leu Phe Leu20 25 30Gly Met Tyr
Leu Ile Thr Val Leu Gly Asn Leu Leu Ile Ile Leu Leu35 40 45Ile Arg
Leu Asp Ser His Leu His Thr Pro Met Phe Phe Phe Leu Ser50 55 60His
Leu Ala Leu Thr Asp Ile Ser Leu Ser Ser Val Thr Val Pro Lys65 70 75
80Met Leu Leu Ser Met Gln Thr Gln Asp Gln Ser Ile Leu Tyr Ala Gly85
90 95Cys Val Thr Gln Met Tyr Phe Phe Ile Phe Phe Thr Asp Leu Asp
Asn100 105 110Phe Leu Leu Thr Ser Met Ala Tyr Asp Arg Tyr Val Ala
Ile Cys His115 120 125Pro Leu Arg Tyr Thr Thr Ile Met Lys Glu Gly
Leu Cys Asn Leu Leu130 135 140Val Thr Val Ser Trp Ile Leu Ser Cys
Thr Asn Ala Leu Ser His Thr145 150 155 160Leu Leu Leu Ala Gln Leu
Ser Phe Cys Ala Asp Asn Thr Ile Pro His165 170 175Phe Phe Cys Asp
Leu Val Ala Leu Leu Lys Leu Ser Cys Ser Asp Ile180 185 190Ser Leu
Asn Glu Leu Val Ile Phe Thr Val Gly Gln Ala Val Ile Thr195 200
205Leu Pro Leu Ile Cys Ile Leu Ile Ser Tyr Gly His Ile Gly Val
Thr210 215 220Ile Leu Lys Ala Pro Ser Thr Lys Gly Ile Phe Lys Ala
Leu Ser Thr225 230 235 240Cys Gly Ser His Leu Ser Val Val Ser Leu
Tyr Tyr Gly Thr Ile Ile245 250 255Gly Leu Tyr Phe Leu Pro Ser Ser
Ser Ala Ser Ser Asp Lys Asp Val260 265 270Ile Ala Ser Val Met Tyr
Thr Val Ile Thr Pro Leu Leu Asn Pro Phe275 280 285Ile Tyr Ser Leu
Arg Asn Arg Asp Ile Lys Gly Ala Leu Glu Arg Leu290 295 300Phe Asn
Arg Ala Thr Val Leu Ser Gln305 31037314PRTHomo sapiens 37Met Met
Gly Gln Asn Gln Ile Ser Ile Ser Asp Phe Leu Leu Leu Gly1 5 10 15Leu
Pro Ile Gln Pro Glu Gln Gln Asn Leu Cys Tyr Ala Leu Phe Leu20 25
30Ala Met Tyr Leu Thr Thr Leu Leu Gly Asn Leu Leu Ile Ile Val Leu35
40 45Ile Arg Leu Asp Ser His Leu His Thr Pro Met Tyr Leu Phe Leu
Ser50 55 60Asn Leu Ser Phe Ser Asp Leu Cys Phe Ser Ser Val Thr Ile
Pro Lys65 70 75 80Leu Leu Gln Asn Met Gln Asn Gln Asp Pro Ser Ile
Pro Tyr Ala Asp85 90 95Cys Leu Thr Gln Met Tyr Phe Phe Leu Leu Phe
Gly Asp Leu Glu Ser100 105 110Phe Leu Leu Val Ala Met Ala Tyr Asp
Arg Tyr Val Ala Ile Cys Phe115 120 125Pro Leu His Tyr Thr Ala Ile
Met Ser Pro Met Leu Cys Leu Ala Leu130 135 140Val Ala Leu Ser Trp
Val Leu Thr Thr Phe His Ala Met Leu His Thr145 150 155 160Leu Leu
Met Ala Arg Leu Cys Phe Cys Ala Asp Asn Val Ile Pro His165 170
175Phe Phe Cys Asp Met Ser Ala Leu Leu Lys Leu Ala Phe Ser Asp
Thr180 185 190Arg Val Asn Glu Trp Val Ile Phe Ile Met Gly Gly Leu
Ile Leu Val195 200 205Ile Pro Phe Leu Leu Ile Leu Gly Ser Tyr Ala
Arg Thr Val Ser Ser210 215 220Ile Leu Lys Val Pro Ser Ser Lys Gly
Ile Cys Lys Ala Phe Ser Thr225 230 235 240Cys Gly Ser His Leu Ser
Val Val Ser Leu Phe Tyr Gly Thr Val Ile245 250 255Gly Leu Tyr Leu
Cys Ser Ser Ala Asn Ser Ser Thr Leu Lys Asp Thr260 265 270Val Met
Ala Met Met Tyr Thr Val Val Thr Pro Met Leu Asn Pro Phe275 280
285Ile Tyr Ser Leu Arg Asn Arg Asp Met Lys Gly Ala Leu Ser Arg
Val290 295 300Ile His Gln Lys Lys Thr Phe Phe Ser Leu305
31038316PRTHomo sapiens 38Met Gln Asn Gln Ser Phe Val Thr Glu Phe
Val Leu Leu Gly Leu Ser1 5 10 15Gln Asn Pro Asn Val Gln Glu Ile Val
Phe Val Val Phe Leu Phe Val20 25 30Tyr Ile Ala Thr Val Gly Gly Asn
Met Leu Ile Val Val Thr Ile Leu35 40 45Ser Ser Pro Ala Leu Leu Val
Ser Pro Met Tyr Phe Phe Leu Gly Phe50 55 60Leu Ser Phe Leu Asp Ala
Cys Phe Ser Ser Val Ile Thr Pro Lys Met65 70 75 80Ile Val Asp Ser
Leu Tyr Val Thr Lys Thr Ile Ser Phe Glu Gly Cys85 90 95Met Val Gln
Leu Phe Ala Glu His Phe Phe Ala Gly Val Glu Val Ile100 105 110Val
Leu Thr Ala Met Ala Tyr Asp Arg Tyr Val Ala Ile Cys Lys Pro115 120
125Leu His Tyr Ser Ser Ile Met Asn Arg Arg Leu Cys Gly Ile Leu
Met130 135 140Gly Val Ala Trp Thr Gly Gly Leu Leu His Ser Met Ile
Gln Ile Leu145 150 155 160Phe Thr Phe Gln Leu Pro Phe Cys Gly Pro
Asn Val Ile Asn His Phe165 170 175Met Cys Asp Leu Tyr Pro Leu Leu
Glu Leu Ala Cys Thr Asp Thr His180 185 190Ile Phe Gly Leu Met Val
Val Ile Asn Ser Gly Phe Ile Cys Ile Ile195 200 205Asn Phe Ser Leu
Leu Leu Val Ser Tyr Ala Val Ile Leu Leu Ser Leu210 215 220Arg Thr
His Ser Ser Glu Gly Arg Trp Lys Ala Leu Ser Thr Cys Gly225 230 235
240Ser His Ile Ala Val Val Ile Leu Phe Phe Val Pro Cys Ile Phe
Val245 250 255Tyr Thr Arg Pro Pro Ser Ala Phe Ser Leu Asp Lys Met
Ala Ala Ile260 265 270Phe Tyr Ile Ile Leu Asn Pro Leu Leu Asn Pro
Leu Ile Tyr Thr Phe275 280 285Arg Asn Lys Glu Val Lys Gln Ala Met
Arg Arg Ile Trp Asn Arg Leu290 295 300Met Val Val Ser Asp Glu Lys
Glu Asn Ile Lys Leu305 310 31539315PRTHomo sapiens 39Met Arg Gln
Asn Asn Asn Ile Thr Phe Phe Val Leu Leu Gly Phe Ser1 5 10 15Gln Asp
Pro Gly Val Gln Lys Ala Leu Phe Val Met Phe Leu Leu Thr20 25 30Tyr
Leu Val Thr Val Val Gly Asn Leu Leu Ile Val Val Asp Ile Ile35 40
45Ala Ser Pro Ser Leu Gly Ser Pro Met Tyr Phe Phe Leu Ala Cys Leu50
55 60Ser Phe Ile Asp Ala Ala Tyr Ser Thr Thr Ile Ser Pro Lys Leu
Ile65 70 75 80Val Gly Leu Phe Cys Asp Lys Lys Thr Ile Ser Phe Gln
Gly Cys Met85 90 95Gly Gln Leu Phe Ile Asp His Phe Phe Gly Gly Ala
Glu Val Phe Leu100 105 110Leu Val Val Met Ala Cys Asp Arg Tyr Val
Ala Ile Cys Lys Pro Leu115 120 125His Tyr Leu Thr Ile Met Asn Arg
Gln Val Cys Phe Leu Leu Leu Val130 135 140Val Ala Met Ile Gly Gly
Phe Val His Ser Ala Phe Gln Ile Val Val145 150 155 160Tyr Ser Leu
Pro Phe Cys Gly Pro Asn Val Ile Val His Phe Ser Cys165 170 175Asp
Met His Pro Leu Leu Glu Leu Ala Cys Thr Asp Thr Tyr Phe Ile180 185
190Gly Leu Thr Val Val Val Asn Ser Gly Ala Ile Cys Met Val Ile
Phe195 200 205Asn Leu Leu Leu Ile Ser Tyr Gly Val Ile Leu Ser Ser
Leu Lys Thr210 215 220Tyr Ser Gln Glu Lys Arg Gly Lys Ala Leu Ser
Thr Cys Ser Ser Gly225 230 235 240Ser Thr Val Val Val Leu Phe Phe
Val Pro Cys Ile Phe Ile Tyr Val245 250 255Arg Pro Val Ser Asn Phe
Pro Thr Asp Lys Phe Met Thr Val Phe Tyr260 265 270Thr Ile Ile Thr
His Met Leu Ser Pro Leu Ile Tyr Thr Leu Arg Asn275 280 285Ser Glu
Met Arg Asn Ala Ile Glu Lys Leu Leu Gly Lys Lys Leu Thr290 295
300Ile Phe Ile Ile Gly Gly Val Ser Val Leu Met305 310 3154017PRTMus
musculus 40Val Met Ala Gly Asn Met Ile Val Phe His Leu Leu Ala Thr
Leu Cys1 5 10 15Phe4117PRTHomo sapiens 41Val Met Ala Gly Thr Ala
Ile Phe Val His Leu Leu Ala Thr Leu Gly1 5 10 15Phe42311PRTHomo
sapiens 42Met Ala Ala Glu Asn His Ser Phe Val Thr Lys Phe Ile Leu
Val Gly1 5 10 15Leu Thr Glu Lys Ser Glu Leu Gln Leu Pro Leu Phe Leu
Val Phe Leu20 25 30Gly Ile Tyr Val Val Thr Val Leu Gly Asn Leu Gly
Met Ile Thr Leu35 40 45Ile Gly Leu Ser Ser His Leu His Thr Pro Met
Tyr Cys Phe Leu Ser50 55 60Ser Leu Ser Phe Ile Asp Phe Cys His Ser
Thr Val Ile Thr Pro Lys65 70 75 80Met Leu Val Asn Phe Val Thr Glu
Lys Asn Ile Ile Ser Tyr Pro Glu85 90 95Cys Met Thr Gln Leu Tyr Phe
Phe Leu Val Phe Ala Ile Ala Glu Cys100 105 110His Met Leu Ala Ala
Met Ala Tyr Asp Gly Tyr Val Ala Ile Cys Ser115 120 125Pro Leu Leu
Tyr Ser Ile Ile Ile Ser Asn Lys Ala Cys Phe Ser Leu130 135 140Ile
Leu Val Val Tyr Val Ile Gly Leu Ile Cys Ala Ser Ala His Ile145 150
155 160Gly Cys Met Phe Arg Val Gln Phe Cys Lys Phe Asp Val Ile Asn
His165 170 175Tyr Phe Cys Asp Leu Ile Ser Ile Leu Lys Leu Ser Cys
Ser Ser Thr180 185 190Tyr Ile Asn Glu Leu Leu Ile Leu Ile Phe Ser
Gly Ile Asn Ile Leu195 200 205Val Pro Ser Leu Thr Ile Leu Ser Ser
Tyr Ile Phe Ile Ile Ala Ser210 215 220Ile Leu Arg Ile Arg Tyr Thr
Glu Gly Arg Ser Lys Ala Phe Ser Thr225 230 235 240Cys Ser Ser His
Ile Ser Ala Val Ser Val Phe Phe Gly Ser Ala Ala245 250 255Phe Met
Tyr Leu Gln Pro Ser Ser Val Ser Ser Met Asp Gln Gly Lys260 265
270Val Ser Ser Val Phe Tyr Thr Ile Val Val Pro Met Leu Asn Pro
Leu275 280 285Ile Tyr Ser Leu Arg Asn Lys Asp Val His Val Ala Leu
Lys Lys Thr290 295 300Leu Gly Lys Arg Thr Phe Leu305 3104317PRTMus
musculus 43Leu Leu Ile Gly Leu Met Tyr Val Leu Ile Lys Val Phe Ala
Asp Leu1 5 10 15Val44316PRTHomo sapiens 44Met Ala Glu Glu Asn His
Thr Met Lys Asn Glu Phe Ile Leu Thr Gly1 5 10 15Phe Thr Asp His Pro
Glu Leu Lys Thr Leu Leu Phe Val Val Phe Phe20 25 30Ala Ile Tyr Leu
Ile Thr Val Val Gly Asn Ile Ser Leu Val Ala Leu35 40 45Ile Phe Thr
His Arg Arg Leu His Thr Pro Met Tyr Ile Phe Leu Gly50 55 60Asn Leu
Ala Leu Val Asp Ser Cys Cys Ala Cys Ala Ile Thr Pro Lys65 70 75
80Met Leu Glu Asn Phe Phe Ser Glu Asn Lys Arg Ile Ser Leu Tyr Glu85
90 95Cys Ala Val Gln Phe Tyr Phe Leu Cys Thr Val Glu Thr Ala Asp
Cys100 105 110Phe Leu Leu Ala Ala Met Ala Tyr Asp Arg Tyr Val Ala
Ile Cys Asn115 120
125Pro Leu Gln Tyr His Thr Met Met Ser Lys Lys Leu Cys Ile Gln
Met130 135 140Thr Thr Gly Ala Phe Ile Ala Gly Asn Leu His Ser Met
Ile His Val145 150 155 160Gly Leu Val Phe Arg Leu Val Phe Cys Gly
Ser Asn His Ile Asn His165 170 175Phe Tyr Cys Asp Ile Leu Pro Leu
Tyr Arg Leu Ser Cys Val Asp Pro180 185 190Tyr Ile Asn Glu Leu Val
Leu Phe Ile Phe Ser Gly Ser Val Gln Val195 200 205Phe Thr Ile Gly
Ser Val Leu Ile Ser Tyr Leu Tyr Ile Leu Leu Thr210 215 220Ile Phe
Lys Met Lys Ser Lys Glu Gly Arg Ala Lys Ala Phe Ser Thr225 230 235
240Cys Ala Ser His Phe Leu Ser Val Ser Leu Phe Tyr Gly Ser Leu
Phe245 250 255Phe Met Tyr Val Arg Pro Asn Leu Leu Glu Glu Gly Asp
Lys Asp Ile260 265 270Pro Ala Ala Ile Leu Phe Thr Ile Val Val Pro
Leu Leu Asn Pro Phe275 280 285Ile Tyr Ser Leu Arg Asn Arg Glu Val
Ile Ser Val Leu Arg Lys Ile290 295 300Leu Met Lys Glu Ile Ile Ser
Arg Arg Trp Lys Gln305 310 315455PRTArtificial SequenceDescription
of Artificial Sequence Synthetic consensus amino acid motif 45Glu
Phe Ile Leu Leu1 5466PRTArtificial SequenceDescription of
Artificial Sequence Synthetic consensus amino acid motif 46Leu His
Thr Pro Met Tyr1 54710PRTArtificial SequenceDescription of
Artificial Sequence Synthetic consensus amino acid motif 47Met Ala
Tyr Asp Arg Tyr Val Ala Ile Cys1 5 10487PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
amino acid motif 48Phe Ser Thr Cys Ser Ser His1 5496PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
amino acid motif 49Pro Met Leu Asn Pro Phe1 55020PRTArtificial
SequenceDescription of Artificial Sequence Synthetic translocation
domain sequence 50Met Asn Gly Thr Glu Gly Pro Asn Phe Tyr Val Pro
Phe Ser Asn Lys1 5 10 15Thr Gly Val Val20516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 51Xaa
Phe Ile Leu Leu Gly1 5529PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 52Met Xaa Xaa Asp Arg Tyr Val
Ala Ile1 55320DNAArtificial SequenceDescription of Artificial
Sequence Primer 53atggsctwtg accghtwygt 20546PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Thr
Cys Xaa Ser His Leu1 55517DNAArtificial SequenceDescription of
Artificial Sequence Primer 55agrtgnswns crcangt 175625DNAArtificial
SequenceDescription of Artificial Sequence Primer 56ggggtccgga
grsrtadatv avvgg 255730DNAArtificial SequenceDescription of
Artificial Sequence Primer 57ggggctgcag acaccnatgt ayytnttyyt
305825DNAArtificial SequenceDescription of Artificial Sequence
Primer 58ggggtccgga grstradatv avvgg 255930DNAArtificial
SequenceDescription of Artificial Sequence Primer 59ggggctgcag
acaccnatgt ayytnttyyt 306025DNAArtificial SequenceDescription of
Artificial Sequence Primer 60ggggtccgga grstradatn anngg
256130DNAArtificial SequenceDescription of Artificial Sequence
Primer 61ggggctgcag acaccnatgt ayytnttyyt 306220DNAArtificial
SequenceDescription of Artificial Sequence Primer 62atraanggrt
tnarcatngg 206323DNAArtificial SequenceDescription of Artificial
Sequence Primer 63atggcntayg aymgntaygt ngc 236433DNAArtificial
SequenceDescription of Artificial Sequence Primer 64cggatccgcn
tasgaygcnt aygtngcnat htg 336531DNAArtificial
Sequencemodified_base(17)2'-deoxyinosine 65cctgcagrta datraanggr
ttnarcatng g 316626DNAArtificial SequenceDescription of Artificial
Sequence Primer 66aarkcnttnb mnacntgygs ntcnca 266717PRTHomo
sapiens 67Leu Leu Ile Gly Leu Ile Tyr Ile Leu Val Lys Ile Phe Ala
Asp Leu1 5 10 15Ser6817PRTMus musculus 68Phe Cys Glu Thr Cys Gly
Ala His Ile His Phe Ile Phe Ser Val Gln1 5 10 15Phe6917PRTHomo
sapiens 69Phe Cys Glu Thr Cys Gly Ala His Ile His Leu Leu Phe Ser
Val Gln1 5 10 15Phe7017PRTMus musculus 70Met Leu Gly Cys Ser Gly
Ser Val Val Asp Phe Ile Met Gly Ile Leu1 5 10 15Gly7117PRTHomo
sapiens 71Ile Leu Gly Cys Cys Arg Ser Val Val Asp Phe Ile Met Gly
Ile Leu1 5 10 15Ala7217PRTMus musculus 72Met Leu Ser Gly Ile Ala
Ile Asn Leu His Leu Ile Thr Ala Leu Ala1 5 10 15Gly7317PRTHomo
sapiens 73Met Leu Val Gly Asn Ala Met Asn Leu Gln Met Met Ala Val
Leu Gly1 5 10 15Gly7417PRTMus musculus 74Leu Leu Gly Ser Cys Ala
Ser Asn Leu Gln Trp Leu Ile Ser Phe Leu1 5 10 15Ile7517PRTHomo
sapiens 75Leu Leu Gly Ser Cys Ala Ser Asn Leu Gln Trp Leu Ile Ser
Phe Leu1 5 10 15Ile7617PRTMus musculus 76Phe Leu Thr Ile Cys Gly
Met Gly Thr Gln Phe Ala Phe Ser Asn Ile1 5 10 15Ile7717PRTHomo
sapiens 77Leu Leu Ala Ile Cys Val Ile Cys Ala His Cys Ile Phe Ser
Asn Ile1 5 10 15Val7817PRTMus musculus 78Ile Leu Gly Cys Asn Val
Phe Asn Val His Leu Ile Leu Ala Val Ile1 5 10 15Val7917PRTHomo
sapiens 79Met Ile Thr Asp Asn Val Leu Asn Ser His Leu Ile Val Gly
Val Ile1 5 10 15Leu8017PRTMus musculus 80Met Leu Gly Asp Ser Leu
Leu His Leu His Leu Ile Ile Gly Val Val1 5 10 15Leu8117PRTHomo
sapiens 81Met Leu Gly Asp Ser Leu Leu His Leu His Leu Ile Met Gly
Ile Leu1 5 10 15Ile8217PRTMus musculus 82Leu His Ala Gly Val Val
Gly His Thr Gln Phe Val Asn Ser Phe Cys1 5 10 15Ile8317PRTHomo
sapiens 83Leu His Ala Gly Val Val Gly His Ile Gln Phe Val Asn Ser
Ile Cys1 5 10 15Ile8417PRTMus musculus 84Leu His Gly Gly Val Ile
Gly His Ile Gln Thr Val Asn Gly Ile Cys1 5 10 15Gly8517PRTHomo
sapiens 85Leu His Gly Gly Val Val Gly His Phe Gln Val Val Asn Ser
Ile Cys1 5 10 15Val8657DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 86attggatcca
ggccgcttgg acaaaaatga atcttttttt tttttttttt ttttttt 57
* * * * *
References