WO1995018140A1 - Olfactory genes and receptors - Google Patents

Olfactory genes and receptors Download PDF

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Publication number
WO1995018140A1
WO1995018140A1 PCT/US1994/014554 US9414554W WO9518140A1 WO 1995018140 A1 WO1995018140 A1 WO 1995018140A1 US 9414554 W US9414554 W US 9414554W WO 9518140 A1 WO9518140 A1 WO 9518140A1
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protein
polypeptide
dna sequence
dna
sequence
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PCT/US1994/014554
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French (fr)
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Nissim Ben-Arie
Doron Lancet
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Yeda Research & Development Co., Ltd.
Rycus, Avigail
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Publication of WO1995018140A1 publication Critical patent/WO1995018140A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention is generally in the field of human genetics and concerns genes of the human olfactory system.
  • the genetic basis for the olfactory system has been subject to extensive research. An understanding of the genetic basis of olfaction is important, for example, for the fragrance industry, to enable a more systematic and accurate way of fragrance design. To date, research in this field has been centered primarily in non-human mammals, although some information on the genetic basis of the olfactory system in humans has also been found.
  • ORs olfactory receptors
  • OR gene clusters human OR genes form continuous clusters in the genome.
  • OR gene clusters One such OR gene cluster was found on human chromosome 17. This OR gene cluster was found to be about 0.35 Mb long with an average inter-genic separation between the genes of about 15 kb.
  • the present invention thus provides, by a first of its aspects, a DNA molecule selected from the group consisting of: (a) an isolated cluster of olfactory receptor genes;
  • a specific olfactory receptor gene cluster in accordance with the invention is that located on human chromosome 17 or that located on human chromosome 11.
  • a DNA molecule having a coding sequence being a member selected from the group consisting of: (a) a DNA sequence depicted in Fig. 4 with the proviso that the sequences designated 17-2 and 17-4 be excluded;
  • the above DNA molecules can be used in transforming a host cell to express the protein or polypeptide encoded by said DNA molecule.
  • the DNA molecule is incorporated into an expression vector which is introduced into suitable host cells, which may be prokaryotic or eukaryotic.
  • Eukaryotic host cells are generally preferred since the post- translational modification in prokaryotic and eukaryotic host cells is different, and thus expression in a eukaryotic host cell will bring to an expression product which more closely resembles that encoded by the olfactory genomic DNA in humans.
  • a particular example of a vector and a host cell is the Baculovirus vector and an insect host cell, respectively.
  • the present invention thus provides a method of production of a protein or a polypeptide in a host cell comprising transforming a host cell by a DNA molecule selected from those defined above, growing the host cells under conditions allowing for expression of the protein or polypeptide encoded by said DNA molecule and harvesting the expressed product.
  • a protein or polypeptide encoded by the above coding sequence and being a member selected from the group consisting of: (a) a protein or polypeptide having an amino acid sequence depicted in Fig. 3;
  • the present invention also provides, by a further of its aspects, uses of the above DNA molecules, proteins or polypeptides.
  • Another use of the DNA molecule is in the construction of probes for use in studying olfactory polymorphism in a population or of probes for Southern blot hybridization and restriction fragment length polymorphism for detecting interindividual differences in olfactory receptors or olfactory clusters in humans.
  • Useful for this purpose are characterizing fragments of an OR gene cluster or DNA sequence which hybridize thereto.
  • the integrity of gene clusters is usually preserved over generations, i.e. gene clusters are usually inherited as a single unit. This is so since the recombination between pairs of chromosomes has a much higher probability of occurring outside such clusters.
  • a fragrance preparation can be produced which will create a positive response in most individuals.
  • the proteins or polypeptides of the invention may be used in probing for the presence of a certain fragrance, aroma or flavor in a medium. Another use of said polypeptides or proteins is in the development of fragrances for human use.
  • said protein or polypeptide When used in probing or in the development of fragrances, said protein or polypeptide may be immobilized onto a solid support or may be embedded into a suitable membraneous system, e.g. membrane of a host cell transformed with a DNA encoding for said protein or polypeptide displaying same on its surface, a liposome wherein said protein or polypeptide is embedded, and the like.
  • a further use of the above protein and polypeptide is in raising antibodies.
  • Such antibodies which also form an aspect of the invention, may be used to detect and label ORs in vivo as well as in in vitro prepara ⁇ tions.
  • the antibodies may carry a detectable marker.
  • Another use of the antibodies is in assays for the detection of the presence of fragrances in a medium.
  • antibodies which recognize the fragrance binding site are brought into contact with said proteins or polypeptides in the presence of a tested medium, the level of bound or free antibodies being directly proportional to the level of fragrance in the tested medium.
  • the DNA molecules and proteins or polypeptides of the invention may have various other uses, all being within the scope of the present invention.
  • Fig. 1 shows fluorescence in situ hybridization (FISH) of cosmid 7 to metaphase spreads of human chromosomes.
  • FISH fluorescence in situ hybridization
  • Fig. 2 shows the mapping of the OR cluster in human 17pl3.3.
  • Thirty-six representative OR-positive cosmids from the human chromosome 17 library were assembled into 3 contigs utilizing a partial digest mapping technique.
  • the Hindl ⁇ l (H) and EcoRl (E) sites are shown on the top lines.
  • the numbered cosmids are shown as horizontal line, showing their positions in the contig.
  • Gap lengths between the 3 contigs were estimated to be 10-20 kb by FISH to free chromatin, and are shown to scale.
  • OR coding regions mapped to within ⁇ 4kb are shown as dark boxes, while those for which only a cosmid assignment is known are shown as open boxes.
  • Fig. 3 shows the deduced amino acid sequences of olfactory receptor clones from human chromosome 17 and their alignment.
  • the deduced protein sequences of the PCR-generated clones encoding putative human ORs were aligned using the GCG package pileup program (Genetic Computer Group, Madison, Wisconsin, U.S.A.).
  • Rat OR I14 1 is also included for comparison and positioning of the sequences in the full length open reading frame. The numbering system used is based on the 114 sequence. Transmembrane domains were determined by an averaged hydropathy plot procedure combined with a hydrophobic moment analysis and are marked by lines with roman numerals. Dark boxes represent amino acids identity, while different shades of gray stand for two different degrees of conservative substitutions.
  • ORs 17-23 and 17-90 have a deletion of one nucleotide compared to all other sequences, which leads to an immediate stop codon to be in frame. Therefore, amino acid 166 in these sequences was marked as *, and the remainder of the protein is the translation of the phase deduced by homology to the other OR sequences;
  • Fig. 4 shows the DNA sequences of the olfactory receptors from the human chromosome 17 cluster shown in Fig. 3;
  • Fig. 5 shows the expression of OR17-93 in human olfactory epitheli ⁇ um.
  • PCR analysis was performed using OR17-93 specific primers on the following templates; OR17-93 and OR17-2 clones as positive controls (OR), cDNA of olfactory epithelium (OR), cDNA of respiratory epithelium (RE), cDNA from nasal inverted papilloma (PA).
  • OR OR
  • RE cDNA of respiratory epithelium
  • PA nasal inverted papilloma
  • Arrows are pointing at the PCR products of OR17-93 and OR17-2 clones (260 and 230 bp, respectively).
  • M 100 bp ladder markers (BRL, Gaithersburg, MD).
  • Genomic DNA was extracted from male Wistar rats and from blood of a human individual.
  • the rat probe (ORI) was obtained by PCR using oligonucleotides OR5B (TM2; CCC ATG TA(T/C) TT(G/CVT) TT(C/T) CTC (A/G/T)(G/C) (C/T) AA(OT)(T/C)T(G/A) TC C) and OR3B (TM7; OR3B AG(A/G) C(A/T)(A/G) TAI ATG AAI GG(A/G) TTC AIC AT).
  • the human probe, ORIII was amplified by nested PCR, using first OR5B/OR3B and then reamplified by OR5A (GGC (QT)TA TGA CCG IT(A/T) (T/C)(G/C)T (G/A)GC (OT)AT ITG) and OR3B.
  • PCR amplifica ⁇ tion was carried out in a buffer containing 50mM KC1, lOmM Tris pH 8.3, 0.01% gelatine, 0.2 mM each deoxyribonucleotide, l M each primer and 2.5 U of Taq DNA polymerase (Cetus, Branchburg, NJ) lOO ⁇ l as follows: 30 cycles of denaturation at 94°C, annealing at 55°C and extension at 72°C, each step for 1 min. The first step of denaturation and the last step of extension were 4 min. long. Both PCR-generated probes were purified by Geneclean II (BIO 101, La Jolla, CA).
  • Chromosome 17 cosmid library was used, prepared previously from flow-sorted chromosome 17 DNA, isolated from human cell line LCL127 12 .
  • DNA probes were radiolabelled to a specificity activity of 10 s -10 9 cpm/ ⁇ g by random hexamer priming using [ -32P] dCTP (Amersham, Buckinghamshire, UK).
  • Hybridization to filters was carried out overnight in 50% formamide, 50 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA, 10X Denhardt's, 4X SSC, 1% SDS, 10% dextran sulphate, 50 ⁇ g/ml sonicated salmon sperm DNA, and 100 ⁇ g/ml sonicated human placental DNA at 42°C.
  • Cosmid and genomic filters were washed in IX SSC, 0.1% SDS at room temperature for 60 minutes and exposed to Kodak XAR 5 X-ray film at -80°C. The positive cosmids isolated were numbered sequentially from 1 to 76. The isolation of 70 OR-positive cosmids (average insert length 35 kb) in a region of 350 kb suggests a 7- fold coverage of the library, over this region, in agreement with the previously estimated coverage.
  • Cosmid DNA was purified using a Qiagen column (Diagen, Chatsworth, CA) according to the manufacturer's recommendations.
  • a Qiagen column Diagen, Chatsworth, CA
  • cosmid DNA was linearised by cleavage with lambda terminase (lU/mg DNA) for 2 hours at room temperature and 2 ⁇ g partially digested with EcoRl or Hind ⁇ Jl in a total volume of 30 ⁇ l at 37°C (0.2 U/ g DNA, removing 5 ⁇ l aliquots every 5 minutes).
  • Partially digested cosmid DNA were hybridized in solution with radiolabelled oligonucleotides complemen ⁇ tary to the protruding 12bp single-stranded sequences at the cleaved cos site and the samples fractionated on a 0.5% agarose gel.
  • PCR amplification using OR5B and OR3B as primers was performed on individual cosmids.
  • the PCR products were digested with a frequent cutter restriction enzymes (e.g. Hinfl, Hael ⁇ l, Hhal).
  • a frequent cutter restriction enzymes e.g. Hinfl, Hael ⁇ l, Hhal.
  • the lengths of the fragments were summed and divided by the length of the undigested product, as described 1>2 -
  • the value obtained is the Minimal Complexity Index (MCI), indicating the minimal number of putative receptors encoded by the cosmid.
  • MCI Minimal Complexity Index
  • Cosmid DNA was extracted by the miniprep alkaline lysis procedure. PCR was performed separately on each, using OR5B/OR3B primers. An equimolar pool of PCR products from all cosmids, as well as products amplified from individual cosmids were ligated to pCRlOOO or pCRII plasmids (Invitrogene, San Diego, CA). Recombinant clones were identified by Southern blot hybridization and PCR.
  • Sequencing was performed using Taq DNA polymerase and dye-terminators on an ABI DNA sequencer model 373 A, with universal vector primers and HOR1, an internal primer (GA/(C/T) G(A/G)(A/C) T(A/T)(CVT) (G/QTG GCC ATG TGC).
  • FISH Fluorescence in situ hybridization
  • Hybridization and detection were carried out according to a modification of the technique described by Pinkel et al. u .
  • 1 ⁇ g of cosmid DNA was biotinylated using a Bionick kit (BRL, Gaithersburg, MD).
  • 80 ng of labelled DNA was mixed with 4 ⁇ g Cot-1 DNA (BRL, Gaithersburg, MD), dried and resuspended in hybridiza ⁇ tion buffer (50% formamide, 2xSSC, 10% dextran sulphate, 1% Tween 20).
  • the sample was denatured by heating to 80°C for 3.5 minutes and incubated at 37°C for 60 minutes.
  • Chromosome slides were baked for two hours at 65°C and then denatured in 70% formamide, 2xSSC for 3 minutes at 75°C. Slides were dehydrated by washing with 70%, 95% and absolute ethanol. Hybridization took place overnight at 37°C under sealed coverslip. Post hybridization washes were with 2xSSC. 50% formamide (3 x 5 min.) and 2xSSC (3 x 5 min.) both at 42°C.
  • the slides were then washed in 4xSSC, 0.05% Tween 20, pH 7 (SSCT) for 3 minutes at 37°C and preincubated in SSCT containing 5% low fat dried milk (Marvel) (SSCTM) prior to the addition of FITC-conjugated avidin DCS (Vector labs) at 5 g/ml in SSCT.
  • SSCTM low fat dried milk
  • the signal was amplified by addition of biotinylated anti-avidin (5 ⁇ g/ml) and a second application of FITC-avidin (DCS (5 ⁇ g/ml).
  • DCS FITC-avidin
  • Chromatin was released from routinely fixed nuclei according to a recently developed method 15 .
  • two color FISH one probe was labelled with biotin and one with digoxygenin, as above. Detection was by incubation with mouse anti-digoxygenin monoclonal antibody (Boehringer Manheim, Manheim, Germany) at 0.8 ⁇ g/ml plus avidin Texas red at 2 ⁇ g/ml, both in SSCTM. This was followed by anti-mouse IgG conjugated to FITC at 30 ⁇ g/ml. The biotinated anti-avidin was used alone and finally another round of avidin Texas red was performed. Post hybridization washes were as described.
  • Somatic cell hybrids BR8 and AY1 were constructed by fusion of lymphoblastoid cell lines of Miller-Dieker syndrome patients MDS-9 and MDS-12, respectively, with TK' rodent cells followed by selection in HAT media. Hybrid clones containing a single, detected chromosome 17 were identified by RLFP analysis of polymorphic loci in distal 17p and confirmed by cytogenetic analysis.
  • RNA expression Human tissues were surgically removed from a female patient having inverted papilloma and were immediately frozen in liquid nitrogen. Total RNA was extracted using TRI-REAGENT (NRC Inc., Cincinnati, OH) following the manufacturer's procedure. Yields were 3.3, 0.9 and 1.6 mg RNA/g tissue for papilloma, olfactory epithelium and respiratory epithelium, respectively. Total RNA (2 ⁇ g) was treated with 0.2 units reverse transcriptase (BRL, Gaithersburg, MD) for 20 mins.
  • OR17-93A ATT GCT GCA GAA CAT GCA GAA
  • OR17-93B ACA TGG CAT GGA AGG TGG TC.
  • a control experiment was conducted to verify that the PCR signals are RNA-dependent, and not due to a genomic DNA contamination. Since Taq DNA polymerase is known to have a significant reverse transcriptase activity, the omission of reverse transcription was considered less desirable. Instead, control PCR reactions were performed using primers for 3-actin spanning intron 2.
  • oligonucleotide primers OR5B and OR3B, respectively. These were utilized to amplify a complex set of OR-related sequences by polymerase chain reaction (PCR), using rat and human genomic DNA as templates (probes ORI and ORIII, respectively). The resulting PCR products were radiolabelled and used to screen cosmid libraries prepared from flow-sorted human chromosomes 17, 21, 22 and X 12 .
  • Chromosomal mapping of the OR gene cluster The approximate size of the genomic OR locus was determined by hybridizing the 5B/3A PCR products from cosmids, 1,2,4 and 7 to a Southern blot containing human genomic DNA digested with a variety of infrequently cutting restriction enzymes (BssHII, SacII, Mlul, Nrul and Not ⁇ ). All four PCR products identified a ⁇ 400 kb .BssHII restriction fragment, giving an estimate for the maximal size of the region. This indicated that the clustered OR genes do not have CpG islands at their 5' flanking ends.
  • Hind ⁇ il and EcoRl restriction maps were constructed for 40 cosmids using a partial digest mapping protocol 17 , and 35 of them arranged into three contigs with a total length of 347 kb (Fig. 2). All the other cosmids were judged by full restriction digest patterns with Hindlll to be overlapping with the mapped subset. Two small gaps were seen, which probably arise due to the large distance between neighboring OR genes in these regions. The gap sizes between cosmids 53-45, 53-68 and 28-65 were estimated at 10-20 kb, and the contigs oriented relative to each other, by two-color FISH 15 to free chromatin using pairwise combination of cosmids from the ends of the three established contigs (data not shown). This produced clear linear signals from cosmids hybridized to chromatin fibers, enabling the relationship between adjacent or overlapping probes to be determined. A more accurate localization and an absolute orientation of the
  • OR cluster within the chromosomal band 17pl3.3 was obtained using DNA from somatic cell hybrids containing the deleted human chromosome 17 homolog of two Miller-Dieker Syndrome (MDS) patients 16 .
  • DNA from hybrid BR8 was positive with the ORI 7-93 PCR primers but negative with PCR marker HP5 (near OR17-210) as well as with STS marker 506 16 which is found only on cosmid 73 (Fig. 2). Southern hybridization with cosmids 7, 44 and 59 further confirmed the location of the BR8 breakpoint at the centromeric end of the OR cluster, within cosmid 7.
  • DNA from hybrid AYl was positive with all three PCR markers, consistent with its breakpoint being telomeric to STS marker 506.
  • the OR cluster is localized between the BR8 breakpoint and STS 506, immediately centromeric to the MDS critical region.
  • OR17-2 and OR17-31 For the first pair (OR17-2 and OR17-31), the difference was one silent mutation (at amino acid 218).
  • the second pair For the second pair (ORI 7-23 and OR17-90, both pseudogenes with the same single base deletion at amino acid 166) the difference was a trinucleotide deletion in OR17-90, which affected two coded amino acids (positions 243-4).
  • These two OR gene pairs may be alleles, occurring at respectively identical loci on cosmids derived from the parental and maternal chromosomes. Alternative ⁇ ly, they could be the result of a recent tandem duplication.
  • OR genes described here were isolated from cloned genomic DNA fragments. To examine whether any of them are actually expressed in human olfactory epithelium, samples of olfactory and non-sensory nasal tissues from a human subject were obtained. Total RNA was extracted, and examined for OR expression by a reverse transcriptase - polymerase chain reaction (RT-PCR) assay (Fig. 5). A control PCR with -actin primers spanning an intron was used to demonstrate that signals are not significantly generated from a genomic DNA contamination. Since OR genes are rather similar to each other, it was necessary to verify that a specific OR of the presently described cluster is the one actually amplified. The verification made use of the unusual 27 nucleotide insertion in OR17-93 and this, together with the design of a specific primer pair allowed to ascertain that only one gene product was amplified.

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Abstract

Olfactory genes appear as gene clusters. In the human genome such clusters appear on chromosomes 11 and 17. Such gene clusters are disclosed herein for the first time. A variety of DNA sequences encoding olfactory receptors or parts thereof have been found and are disclosed herein.

Description

OLFACTORY GENES AND RECEPTORS
FIELD OF THE INVENTION
The present invention is generally in the field of human genetics and concerns genes of the human olfactory system.
PRIOR ART
The following is a list of prior art referred to in the present specification:
1. Buck, L. and Axel, R., 1991, Cell 65:175-187. 2. PCT Application WO 92/17585.
3. Levy, N.S., et al, 1991, /. Steroid Biochem. Molec. BioL, 39:633- 637.
4. Buck, L.B., 1992, Current Opinion Genet. Devel, 2:467-473.
5. Parmentier, M., et al, 1992, Nature, 355:453-455. 6. Nef, P., et al, 1992, Proc. Natl. Acad. Sci. USA, 89:8948-8952.
7. Selbie, L.A., et al, 1992, Mol Brain Res., 13:159-163.
8. Raming, K., et al, 1993, Nature, 361:353-356. 9. Ressler, K.J, et al, 1993, Cell, 73: 597-609.
10. Ngai, J., et al, 1993, Cell, 72:657-666.
11. Schurmans, S., et al, 1993, Cytigenet Cell Genet, 63:200-204. 12. Nizetic, D., et al, 1991, Proc. Natl Acad. Sci. USA, 88:3233-3237.
13. Kwiatkowski, D.J., et al, 1990, Am. J. Hum. Genet, 46:559-567.
14. Pinkel, D., et al, 1986, Proc. Natl. Acad. Sci. USA, 83:2934-2939.
15. Fidlerova, H., et al, 1993, Submitted.
16. Ledbetter, D.H., et al, 1989, Proc. Natl. Acad. Sci. USA, 86:5136- 5140.
17. Rackwitz, H.R., et al, 1985, Gene, 40:259-266.
The acknowledgement herein of the above publications is given to allow an appreciation of the prior art but should not, however, be construed as an indication that this art is in any way relevant to the patentability of the invention as defined in the appended claims.
The above publications will be acknowledged herein by indicating their number from the above list.
BACKGROUND OF THE INVENTION
The genetic basis for the olfactory system has been subject to extensive research. An understanding of the genetic basis of olfaction is important, for example, for the fragrance industry, to enable a more systematic and accurate way of fragrance design. To date, research in this field has been centered primarily in non-human mammals, although some information on the genetic basis of the olfactory system in humans has also been found.
On the basis of growing body of evidence, it became clear that the olfactory receptors (ORs) are encoded by a superfamily consisting of a plurality of genes1"9. Common characteristics of all receptors identified to date is that they belong to the superfamily of G-protein coupled receptors and have seven putative trans-membrane helices.
Genomic blot analyses have shown that the rat genome may have several hundred OR genes. It is anticipated that large OR gene repertoires exist also in other mammals. In addition to the study of OR genes in ratsι,3,S } members of the OR gene family have also been cloned from fish10, mouse6,9, dog5 and human5,7'11.
It is an object of the present invention to provide novel DNA molecules encoding human olfactory receptors.
It is more specifically an object of the invention to provide a human olfactory subgenome, i.e. a cluster of OR genes.
It is another specific object of the present invention to provide novel proteins or polypeptides encoded by the DNA sequences of the invention.
The remaining objects of the invention will be realized in the following description and claims.
GENERAL DESCRIPTION OF THE INVENTION In accordance with the present invention it has been found that contrary to the prior art prevailing belief that known OR genes are randomly distributed throughout the genome, human OR genes form continuous clusters in the genome (hereinafter "OR gene clusters"). One such OR gene cluster was found on human chromosome 17. This OR gene cluster was found to be about 0.35 Mb long with an average inter-genic separation between the genes of about 15 kb.
Furthermore, in accordance with the present invention novel DNA coding sequences from OR genes belonging to the OR cluster on chromosome 17 have been identified and cloned. These sequences are depicted in Fig. 4 which shows 16 sequences of which those designated 17-2 and 17-4 are known and the rest are novel.
The present invention thus provides, by a first of its aspects, a DNA molecule selected from the group consisting of: (a) an isolated cluster of olfactory receptor genes;
(b) an isolated non-coding fragment of the DNA sequence of (a);
(c) a DNA sequence included in (a) or (b) and included within a sequence coding for an olfactory receptor with the proviso that the sequences designated 17-2 and 17-4 in Fig. 4 are excluded; (d) a DNA sequence different than the DNA sequence of (c) and encoding the same protein or polypeptide;
(e) a DNA sequence of (c) or (d) in which one or more coding triplets from the open reading frame have been added, deleted or replaced, the protein or polypeptide encoded by said sequence having similar properties to that encoded by the DNA sequence of (c) or (d); and
(f) a DNA sequence capable of hybridizing under conditions of stringency to the DNA sequence of (a), (b), (c), (d) or (e).
A specific olfactory receptor gene cluster in accordance with the invention is that located on human chromosome 17 or that located on human chromosome 11.
By a further aspect of the invention there is provided a DNA molecule having a coding sequence being a member selected from the group consisting of: (a) a DNA sequence depicted in Fig. 4 with the proviso that the sequences designated 17-2 and 17-4 be excluded;
(b) a DNA sequence encoding a functional polypeptide being a fragment of the polypeptide encoded by the DNA sequence of
(a); (c) a DNA sequence comprising the DNA sequence of (a) or (b);
(d) a DNA sequence of (a), (b) or (c) wherein one or more triplets in the open reading frame have been added, replaced or deleted, the protein or polypeptide encoded by the sequence having essentially the same properties as the protein encoded by the respective sequence of (a), (b) or (c);
(e) a DNA sequence of (a), (b), (c) and (d) comprising also non- coding stretches of nucleic acid residues; and
(f) a DNA sequence capable of hybridization under conditions of stringency with the DNA sequence of (a), (b), (c) or (d).
The above DNA molecules can be used in transforming a host cell to express the protein or polypeptide encoded by said DNA molecule. For this purpose, the DNA molecule is incorporated into an expression vector which is introduced into suitable host cells, which may be prokaryotic or eukaryotic. Eukaryotic host cells are generally preferred since the post- translational modification in prokaryotic and eukaryotic host cells is different, and thus expression in a eukaryotic host cell will bring to an expression product which more closely resembles that encoded by the olfactory genomic DNA in humans. A particular example of a vector and a host cell is the Baculovirus vector and an insect host cell, respectively.
The present invention thus provides a method of production of a protein or a polypeptide in a host cell comprising transforming a host cell by a DNA molecule selected from those defined above, growing the host cells under conditions allowing for expression of the protein or polypeptide encoded by said DNA molecule and harvesting the expressed product.
In accordance with yet another aspect of the invention, there is provided a protein or polypeptide encoded by the above coding sequence and being a member selected from the group consisting of: (a) a protein or polypeptide having an amino acid sequence depicted in Fig. 3;
(b) a protein or polypeptide wherein one or more amino acid residues has been added, deleted or chemically modified, the protein or polypeptide having essentially the same biological properties as the respective protein or polypeptide under (a); and
(c) a protein comprising a polypeptide having a sequence of (a) or (b).
The present invention also provides, by a further of its aspects, uses of the above DNA molecules, proteins or polypeptides.
Another use of the DNA molecule is in the construction of probes for use in studying olfactory polymorphism in a population or of probes for Southern blot hybridization and restriction fragment length polymorphism for detecting interindividual differences in olfactory receptors or olfactory clusters in humans. Useful for this purpose are characterizing fragments of an OR gene cluster or DNA sequence which hybridize thereto. The integrity of gene clusters is usually preserved over generations, i.e. gene clusters are usually inherited as a single unit. This is so since the recombination between pairs of chromosomes has a much higher probability of occurring outside such clusters. In such clusters, unique non-coding sequences of a length of a few tens of nucleic acid basis can usually be found, which can be used to identify the entire cluster. By the use of such fragments or nucleic acid sequences which can hybridize thereto, the distribution of OR gene cluster in a population and hence OR polymorphism, can be assayed. Probes to assay for OR polymorphism are very important in the fragrance industry since on the basis of knowledge of such polymorphism, fragrances for use by specific populations may be used. Based on the type of olfactory receptors, different people may be responsive to different fragrances. At times, a homogeneous population may have many common ORs and thus fragrance preparations specific for this population may be produced. Alternatively, by the knowledge of polymorphism in a popula¬ tion, a fragrance preparation can be produced which will create a positive response in most individuals. The proteins or polypeptides of the invention may be used in probing for the presence of a certain fragrance, aroma or flavor in a medium. Another use of said polypeptides or proteins is in the development of fragrances for human use. When used in probing or in the development of fragrances, said protein or polypeptide may be immobilized onto a solid support or may be embedded into a suitable membraneous system, e.g. membrane of a host cell transformed with a DNA encoding for said protein or polypeptide displaying same on its surface, a liposome wherein said protein or polypeptide is embedded, and the like. In such use, binding of a fragrance, aroma or a flavor to said protein or polypeptide will give rise to a detectable signal. The manner of use of a receptor protein so that binding of a ligand thereto will give rise to a detectable signal is generally known per se and the description of such manner is outside the scope of the present writing.
A further use of the above protein and polypeptide is in raising antibodies. Such antibodies, which also form an aspect of the invention, may be used to detect and label ORs in vivo as well as in in vitro prepara¬ tions. For such use, the antibodies may carry a detectable marker. Another use of the antibodies is in assays for the detection of the presence of fragrances in a medium. In such use, antibodies which recognize the fragrance binding site are brought into contact with said proteins or polypeptides in the presence of a tested medium, the level of bound or free antibodies being directly proportional to the level of fragrance in the tested medium. As will no doubt be appreciated by the artisan, the DNA molecules and proteins or polypeptides of the invention may have various other uses, all being within the scope of the present invention.
The invention will now be illustrated in the following description of specific embodiments with occasional reference to the annexed drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows fluorescence in situ hybridization (FISH) of cosmid 7 to metaphase spreads of human chromosomes. The arrows in the figure indicate labelling at the telomeric end of chromosome 17 on the two sister chromatids of both chromosomes.
Fig. 2 shows the mapping of the OR cluster in human 17pl3.3. Thirty-six representative OR-positive cosmids from the human chromosome 17 library were assembled into 3 contigs utilizing a partial digest mapping technique. The Hindlϊl (H) and EcoRl (E) sites are shown on the top lines. The numbered cosmids are shown as horizontal line, showing their positions in the contig. Gap lengths between the 3 contigs were estimated to be 10-20 kb by FISH to free chromatin, and are shown to scale. OR coding regions mapped to within ±4kb are shown as dark boxes, while those for which only a cosmid assignment is known are shown as open boxes. The approximate location of STS 506 (D17S126) and the PCR marker HP5 (in the 3' non coding region of OR17-OR17-210) are also shown. None of the cosmids tested positively with the flanking chromosome 17 STS's PFN1 and 50813. The approximate DNA breakpoints of Miller-Dieker Syndrome patient BR8 is shown, within cosmid 7 by Southern blot analysis of DNA from a cell line containing chromosome 17 of this patient on mouse background, with radiolabelled cosmid 7 DNA;
Fig. 3 shows the deduced amino acid sequences of olfactory receptor clones from human chromosome 17 and their alignment. The deduced protein sequences of the PCR-generated clones encoding putative human ORs were aligned using the GCG package pileup program (Genetic Computer Group, Madison, Wisconsin, U.S.A.). Rat OR I141 is also included for comparison and positioning of the sequences in the full length open reading frame. The numbering system used is based on the 114 sequence. Transmembrane domains were determined by an averaged hydropathy plot procedure combined with a hydrophobic moment analysis and are marked by lines with roman numerals. Dark boxes represent amino acids identity, while different shades of gray stand for two different degrees of conservative substitutions. Marked by a • are positions of full identity among all the sequences. ORs 17-23 and 17-90 have a deletion of one nucleotide compared to all other sequences, which leads to an immediate stop codon to be in frame. Therefore, amino acid 166 in these sequences was marked as *, and the remainder of the protein is the translation of the phase deduced by homology to the other OR sequences;
Fig. 4 shows the DNA sequences of the olfactory receptors from the human chromosome 17 cluster shown in Fig. 3; and
Fig. 5 shows the expression of OR17-93 in human olfactory epitheli¬ um. PCR analysis was performed using OR17-93 specific primers on the following templates; OR17-93 and OR17-2 clones as positive controls (OR), cDNA of olfactory epithelium (OR), cDNA of respiratory epithelium (RE), cDNA from nasal inverted papilloma (PA). Arrows are pointing at the PCR products of OR17-93 and OR17-2 clones (260 and 230 bp, respectively). M=100 bp ladder markers (BRL, Gaithersburg, MD).
DESCRIPTION OF A SPECIFIC EMBODIMENT
The invention will now be described by outlining the experiments which led to the discoveries which form the basis of the present invention. This section is intended for illustration of the invention and should not be construed to limit the invention in any way.
Materials and Methods OR Probe preparation
Genomic DNA was extracted from male Wistar rats and from blood of a human individual. The rat probe (ORI) was obtained by PCR using oligonucleotides OR5B (TM2; CCC ATG TA(T/C) TT(G/CVT) TT(C/T) CTC (A/G/T)(G/C) (C/T) AA(OT)(T/C)T(G/A) TC C) and OR3B (TM7; OR3B AG(A/G) C(A/T)(A/G) TAI ATG AAI GG(A/G) TTC AIC AT). The human probe, ORIII, was amplified by nested PCR, using first OR5B/OR3B and then reamplified by OR5A (GGC (QT)TA TGA CCG IT(A/T) (T/C)(G/C)T (G/A)GC (OT)AT ITG) and OR3B. PCR amplifica¬ tion was carried out in a buffer containing 50mM KC1, lOmM Tris pH 8.3, 0.01% gelatine, 0.2 mM each deoxyribonucleotide, l M each primer and 2.5 U of Taq DNA polymerase (Cetus, Branchburg, NJ) lOOμl as follows: 30 cycles of denaturation at 94°C, annealing at 55°C and extension at 72°C, each step for 1 min. The first step of denaturation and the last step of extension were 4 min. long. Both PCR-generated probes were purified by Geneclean II (BIO 101, La Jolla, CA).
Library Screening
A 5 -fold coverage Chromosome 17 cosmid library was used, prepared previously from flow-sorted chromosome 17 DNA, isolated from human cell line LCL12712. DNA probes were radiolabelled to a specificity activity of 10s-109 cpm/μg by random hexamer priming using [ -32P] dCTP (Amersham, Buckinghamshire, UK). Hybridization to filters was carried out overnight in 50% formamide, 50 mM sodium phosphate buffer, pH 7.2, 1 mM EDTA, 10X Denhardt's, 4X SSC, 1% SDS, 10% dextran sulphate, 50 μg/ml sonicated salmon sperm DNA, and 100 μg/ml sonicated human placental DNA at 42°C. Cosmid and genomic filters were washed in IX SSC, 0.1% SDS at room temperature for 60 minutes and exposed to Kodak XAR 5 X-ray film at -80°C. The positive cosmids isolated were numbered sequentially from 1 to 76. The isolation of 70 OR-positive cosmids (average insert length 35 kb) in a region of 350 kb suggests a 7- fold coverage of the library, over this region, in agreement with the previously estimated coverage.
'Partial digest' mapping of the cosmids
Cosmid DNA was purified using a Qiagen column (Diagen, Chatsworth, CA) according to the manufacturer's recommendations. For the cosmid blots, 1 μg of DNA was digested by the appropriate restriction enzyme and 400 ng run on an 0.65% agarose gel. The DNA was visualized by staining with ethidium bromide and transferred onto nylon membranes (Hybond N+, Amersham, Buckinghamshire, UK), according to the manufacturer's recommendations. For partial digest mapping, 10 ug of cosmid DNA was linearised by cleavage with lambda terminase (lU/mg DNA) for 2 hours at room temperature and 2 μg partially digested with EcoRl or HindϊJl in a total volume of 30 μl at 37°C (0.2 U/ g DNA, removing 5 μl aliquots every 5 minutes). Partially digested cosmid DNA were hybridized in solution with radiolabelled oligonucleotides complemen¬ tary to the protruding 12bp single-stranded sequences at the cleaved cos site and the samples fractionated on a 0.5% agarose gel. The annealing of labelled oligonucleotides specific for the 'left' or 'right' termini of the cos sites allowed the autoradiographic visualization of restriction fragments of increasing length extending from each side of the cosmid. A restriction map was constructed by calculating and compiling the digitized migration distances of partial digestion restriction fragments. Minimal Complexity Index (MCI) Analysis
In order to find how many OR coding regions lie in each cosmid, PCR amplification using OR5B and OR3B as primers was performed on individual cosmids. The PCR products were digested with a frequent cutter restriction enzymes (e.g. Hinfl, Haelϊl, Hhal). After agarose gel separation and ethidium bromide staining, the lengths of the fragments were summed and divided by the length of the undigested product, as described 1>2- The value obtained is the Minimal Complexity Index (MCI), indicating the minimal number of putative receptors encoded by the cosmid.
Sequencing of cloned PCR products
Cosmid DNA was extracted by the miniprep alkaline lysis procedure. PCR was performed separately on each, using OR5B/OR3B primers. An equimolar pool of PCR products from all cosmids, as well as products amplified from individual cosmids were ligated to pCRlOOO or pCRII plasmids (Invitrogene, San Diego, CA). Recombinant clones were identified by Southern blot hybridization and PCR. Sequencing was performed using Taq DNA polymerase and dye-terminators on an ABI DNA sequencer model 373 A, with universal vector primers and HOR1, an internal primer (GA/(C/T) G(A/G)(A/C) T(A/T)(CVT) (G/QTG GCC ATG TGC).
Fluorescence in situ hybridization (FISH)
Hybridization and detection were carried out according to a modification of the technique described by Pinkel et al.u. For metaphase in situ hybridization, 1 μg of cosmid DNA was biotinylated using a Bionick kit (BRL, Gaithersburg, MD). 80 ng of labelled DNA was mixed with 4 μg Cot-1 DNA (BRL, Gaithersburg, MD), dried and resuspended in hybridiza¬ tion buffer (50% formamide, 2xSSC, 10% dextran sulphate, 1% Tween 20). The sample was denatured by heating to 80°C for 3.5 minutes and incubated at 37°C for 60 minutes. Chromosome slides were baked for two hours at 65°C and then denatured in 70% formamide, 2xSSC for 3 minutes at 75°C. Slides were dehydrated by washing with 70%, 95% and absolute ethanol. Hybridization took place overnight at 37°C under sealed coverslip. Post hybridization washes were with 2xSSC. 50% formamide (3 x 5 min.) and 2xSSC (3 x 5 min.) both at 42°C. The slides were then washed in 4xSSC, 0.05% Tween 20, pH 7 (SSCT) for 3 minutes at 37°C and preincubated in SSCT containing 5% low fat dried milk (Marvel) (SSCTM) prior to the addition of FITC-conjugated avidin DCS (Vector labs) at 5 g/ml in SSCT. The signal was amplified by addition of biotinylated anti-avidin (5 μg/ml) and a second application of FITC-avidin (DCS (5 μg/ml). Finally the slides were washed with phosphate buffered saline and dehydrated through an ethanol series. Chromatin was released from routinely fixed nuclei according to a recently developed method15. For two color FISH one probe was labelled with biotin and one with digoxygenin, as above. Detection was by incubation with mouse anti-digoxygenin monoclonal antibody (Boehringer Manheim, Manheim, Germany) at 0.8 μg/ml plus avidin Texas red at 2 μg/ml, both in SSCTM. This was followed by anti-mouse IgG conjugated to FITC at 30 μg/ml. The biotinated anti-avidin was used alone and finally another round of avidin Texas red was performed. Post hybridization washes were as described. Slides were mounted in Citiflour (Citiflour Ltd., London) with propidium iodide 0.5 μg/ml for one color visualization or DAPI (diamidino-2-phenyl-indole dihydrochloride; Sigma, St. Louis, MO) 0.1 μg/ml for two colors, and photographed using a Zeiss Axiophot microscope. Somatic cell hybrid mapping
Somatic cell hybrids BR8 and AY1 were constructed by fusion of lymphoblastoid cell lines of Miller-Dieker syndrome patients MDS-9 and MDS-12, respectively, with TK' rodent cells followed by selection in HAT media. Hybrid clones containing a single, detected chromosome 17 were identified by RLFP analysis of polymorphic loci in distal 17p and confirmed by cytogenetic analysis.
OR tissue expression Human tissues were surgically removed from a female patient having inverted papilloma and were immediately frozen in liquid nitrogen. Total RNA was extracted using TRI-REAGENT (NRC Inc., Cincinnati, OH) following the manufacturer's procedure. Yields were 3.3, 0.9 and 1.6 mg RNA/g tissue for papilloma, olfactory epithelium and respiratory epithelium, respectively. Total RNA (2 μg) was treated with 0.2 units reverse transcriptase (BRL, Gaithersburg, MD) for 20 mins. at 37°C and was amplified using OR17-93 specific primers (OR17-93A: ATT GCT GCA GAA CAT GCA GAA; OR17-93B: ACA TGG CAT GGA AGG TGG TC). A control experiment was conducted to verify that the PCR signals are RNA-dependent, and not due to a genomic DNA contamination. Since Taq DNA polymerase is known to have a significant reverse transcriptase activity, the omission of reverse transcription was considered less desirable. Instead, control PCR reactions were performed using primers for 3-actin spanning intron 2.
Results
OR-positive cosmids from human chromosome 17
Conserved regions in the 2nd and 7th transmembrane domains of the published rat OR sequences1'2 were used to design oligonucleotide primers (OR5B and OR3B, respectively). These were utilized to amplify a complex set of OR-related sequences by polymerase chain reaction (PCR), using rat and human genomic DNA as templates (probes ORI and ORIII, respectively). The resulting PCR products were radiolabelled and used to screen cosmid libraries prepared from flow-sorted human chromosomes 17, 21, 22 and X12. A large number of positively hybridizing clones was obtained only for chromosome 17: 75 cosmids out of approximately 20,000 tested gave a clear hybridization signal with the human genomic probe (ORIII), of which about half (36) contained OR genes sufficiently conserved to be detected by the ORI rat PCR probe as well. The clones were individually tested for the presence of OR coding sequences by Southern hybridization and a PCR amplification with the primers described above. The latter is feasible since OR genes have no introns in their coding regions1'2,6. Seventy hybridization positive cosmids gave the expected ~ 700 bp long product generated by the primers OR5B and OR3B, and were further characterized. Each of the PCR products was then digested with frequent-cutting restriction enzymes, and subjected to a Minimal Complexity Index (MCI) analysis (see Materials and Methods), giving an estimate for the minimal number of OR coding regions per cosmid. The results showed an average of 2.3 ORs per ~ 35 kb cosmid genomic insert, or approximately an average of one OR coding region per 15 kb of genomic DNA. There was a correlation between the intensity of hybridization signal in the library screen and the inferred number of ORs present on a given cosmid (not shown). Six cosmids showing disparate OR restriction patterns (numbers
1,2,4,7,8,10) were used for fluorescence in situ hybridization to metaphase spreads of human chromosomes (Fig. 1). All four mapped to a similar position, 17pl3, close to the p-telomere of chromosome 17. This result, together with the frequent presence of more than one OR coding region per cosmid provided initial indications that at least some of the identified OR genes were clustered.
Chromosomal mapping of the OR gene cluster The approximate size of the genomic OR locus was determined by hybridizing the 5B/3A PCR products from cosmids, 1,2,4 and 7 to a Southern blot containing human genomic DNA digested with a variety of infrequently cutting restriction enzymes (BssHII, SacII, Mlul, Nrul and Notϊ). All four PCR products identified a ~ 400 kb .BssHII restriction fragment, giving an estimate for the maximal size of the region. This indicated that the clustered OR genes do not have CpG islands at their 5' flanking ends. Hindϊil and EcoRl restriction maps were constructed for 40 cosmids using a partial digest mapping protocol17, and 35 of them arranged into three contigs with a total length of 347 kb (Fig. 2). All the other cosmids were judged by full restriction digest patterns with Hindlll to be overlapping with the mapped subset. Two small gaps were seen, which probably arise due to the large distance between neighboring OR genes in these regions. The gap sizes between cosmids 53-45, 53-68 and 28-65 were estimated at 10-20 kb, and the contigs oriented relative to each other, by two-color FISH15 to free chromatin using pairwise combination of cosmids from the ends of the three established contigs (data not shown). This produced clear linear signals from cosmids hybridized to chromatin fibers, enabling the relationship between adjacent or overlapping probes to be determined. A more accurate localization and an absolute orientation of the
OR cluster within the chromosomal band 17pl3.3 was obtained using DNA from somatic cell hybrids containing the deleted human chromosome 17 homolog of two Miller-Dieker Syndrome (MDS) patients16. DNA from hybrid BR8 was positive with the ORI 7-93 PCR primers but negative with PCR marker HP5 (near OR17-210) as well as with STS marker 50616 which is found only on cosmid 73 (Fig. 2). Southern hybridization with cosmids 7, 44 and 59 further confirmed the location of the BR8 breakpoint at the centromeric end of the OR cluster, within cosmid 7. DNA from hybrid AYl was positive with all three PCR markers, consistent with its breakpoint being telomeric to STS marker 506. Thus, the OR cluster is localized between the BR8 breakpoint and STS 506, immediately centromeric to the MDS critical region.
Mapping and sequencing of OR coding regions
In order to localize the OR genes in the cluster, and to obtain DNA sequence information for them, an equimolar mixture of coding regions amplified by PCR (with primers OR5B-OR3B) from each of the 70 cosmids, was used to generate a library of OR clones. Of 17 clones sequenced, 11 were different. Restriction patterns of OR5B-OR3B PCR products with up to six frequent-cutting restriction enzymes allowed a comparison of these clones to those seen in PCR products from the individual cosmids. This suggested the existence of 6 additional OR genes in the cluster, and provided cosmid assignments for each of the 16 OR coding regions identified. By relating this information to the cosmid physical map, it was possible to localize most of the receptors with an accuracy of ±4 kb within the cluster. Further positional information was obtained from Southern blot hybridization of the ORIII probe to HindlU, and Hindlll/EcoRi digests of the cosmids (Fig. 2). The 17 OR protein sequences (between transmembrane domains
2 and 7) are shown in Fig. 3 and the corresponding DNA sequences are shown in Fig. 4. The ORs have 36-39% mutual amino acid sequence identity. Two of the sequences (ORI 7-2 and ORI 7-4) are identical to recently published human OR clones HUMHGM0715 and HGM07E11, respectively. The latter gene was independently mapped to 17pl3 — >pl2n. All the OR gene sequences were co-linear, except OR17-93 which had a 9 amino acid insert between positions 129 and 130, within intracellular loop 2. In addition, there is a single amino acid deletion at position 268 in four of the OR sequences. In two cases, pairs of OR genes shared >99% identity, with members of each pair mapping to the same position within the accuracy of our determination. For the first pair (OR17-2 and OR17-31), the difference was one silent mutation (at amino acid 218). For the second pair (ORI 7-23 and OR17-90, both pseudogenes with the same single base deletion at amino acid 166) the difference was a trinucleotide deletion in OR17-90, which affected two coded amino acids (positions 243-4). These two OR gene pairs may be alleles, occurring at respectively identical loci on cosmids derived from the parental and maternal chromosomes. Alternative¬ ly, they could be the result of a recent tandem duplication.
Tissue expression
The OR genes described here were isolated from cloned genomic DNA fragments. To examine whether any of them are actually expressed in human olfactory epithelium, samples of olfactory and non-sensory nasal tissues from a human subject were obtained. Total RNA was extracted, and examined for OR expression by a reverse transcriptase - polymerase chain reaction (RT-PCR) assay (Fig. 5). A control PCR with -actin primers spanning an intron was used to demonstrate that signals are not significantly generated from a genomic DNA contamination. Since OR genes are rather similar to each other, it was necessary to verify that a specific OR of the presently described cluster is the one actually amplified. The verification made use of the unusual 27 nucleotide insertion in OR17-93 and this, together with the design of a specific primer pair allowed to ascertain that only one gene product was amplified.

Claims

CLAIMS:
1. A DNA molecule selected from the group consisting of: (a) an isolated cluster of olfactory receptor genes; (b) an isolated non-coding fragment of the DNA sequence of (a);
(c) a DNA sequence included in (a) or (b) and included within a sequence coding for an olfactory receptor with the proviso that the sequences designated 17-2 and 17-4 in Fig. 4 are excluded;
(d) a DNA sequence different than the DNA sequence of (c) and encoding the same protein or polypeptide;
(e) a DNA sequence of (c) or (d) in which one or more coding triplets from the open reading frame have been added, deleted or replaced, the protein or polypeptide encoded by said sequence having similar properties to that encoded by the DNA sequence of (c) or (d); and
(f) a DNA sequence capable of hybridizing under conditions of stringency to the DNA sequence of (a), (b), (c), (d) or (e).
2. A DNA molecule according to Claim 1, wherein the isolated gene cluster is derived from human chromosome 11 or 17.
3. A DNA molecule having a coding sequence being a member selected from the group consisting of:
(a) a DNA sequence depicted in Fig. 4 with the proviso that the sequences designated 17-2 and 17-4 be excluded;
(b) a DNA sequence encoding a functional polypeptide being a fragment of the polypeptide encoded by the DNA sequence of
(a);
(c) a DNA sequence comprising the DNA sequence of (a) or (b);
(d) a DNA sequence of (a), (b) or (c) wherein one or more triplets in the open reading frame have been added, replaced or deleted, the protein or polypeptide encoded by the sequence having essentially the same properties as the protein encoded by the respective sequence of (a), (b) or (c); (e) a DNA sequence of (a), (b), (c) and (d) comprising also non- coding stretches of nucleic acid residues; and (f) a DNA sequence capable of hybridization under conditions of stringency with the DNA sequence of (a), (b), (c) or (d).
4. A method of production of a protein or a polypeptide in a host cell comprising transforming a host cell by a DNA molecule of Claim 3, growing the host cells under conditions allowing for expression of the protein or polypeptide encoded by said DNA molecule and harvesting the expressed product.
5. A protein or polypeptide being a member selected from the group consisting of:
(a) a protein or polypeptide having an amino acid sequence depicted in Fig. 3;
(b) a protein or polypeptide wherein one or more amino acid residues has been added, deleted or chemically modified, the protein or polypeptide having essentially the same biological properties as the respective protein or polypeptide under (a); and (c) a protein comprising a polypeptide having a sequence of (a) or
(b).
6. A host cell transformed by a DNA molecule according to any one of Claims 1 or 3.
7. A probe for use in assaying fragrances, aromas or flavors in a medium, comprising a protein or polypeptide according to Claim 5.
8. A probe for use in testing of the presence of a gene cluster among individuals in a population, comprising a DNA molecule as defined in Claim 1(b).
9. An antibody directed against a protein or polypeptide according to Claim 5.
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