WO1998001580A1

WO1998001580A1 - RECOMBINANT HTm4 GENE, PROTEIN AND USE THEREOF IN METHODS FOR DETECTING HEREDITARY ATOPY

Info

Publication number: WO1998001580A1
Application number: PCT/US1996/011479
Authority: WO
Inventors: Bing Lim; Chaker N. Adra; Jean-Michel Lelias
Original assignee: Beth Israel Deaconess Medical Center
Priority date: 1996-07-10
Filing date: 1996-07-10
Publication date: 1998-01-15
Also published as: AU6487296A

Abstract

The invention relates to a recombinant DNA molecule which encodes a HTm4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HTm4 protein and a recombinant HTm4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

Description

RECOMBINANT HT_m4 GENE, PROTEIN AND USE THEREOF IN METHODS FOR DETECTING HEREDITARY ATOPY

Background of the Invention

Atopic diseases, which include allergy, asthma, atopic der atititis (or eczema) and allergic rhinitis, together constitute one of the largest group of clinical disorders requiring medical intervention. In the United Kingdom alone, atopy gives rise to 3-5 million cases and as many as 2,000 deaths each year.

Atopy is generally defined as a disorder of Immunoglobulin E (IgE) responses to common antigens, such as pollen or house dust mites. It is frequently detected by either elevated total serum IgE levels, antigen specific IgE response or positive skin tests to common allergens. In principle atopy can result from dysregulation of any part of the pathway which begins with antigen exposure and IgE response to the interaction of IgE with its receptor on mast cells, the high affinity Fc receptor Fc_eRI, and the subsequent cellular activation mediated by that ligand- receptor engagement (Ravetch, Nature Genetics, 7:117-118 (1994). Cookson et al . , Lancet, 333:1292-1295 (1989) have reported a genetic link between generalized atopic IgE responses and a locus on human chromosome llq.

Fc_eRI, is part of a tetrameric receptor complex consisting of an or chain, a β chain and two γ chains (Kinet et al . , Proc. Natl . Acad. Sci . USA, 15:6483-6487 (1988)). Together, they mediate interaction with IgE-bound antigens leading to dramatic cellular responses, such as the massive degranulations of mast cells. Thought until recently to be expressed only in mast cells and basophils, the high- affinity receptor Fc_€RI has been shown to be present also in Langerhans cells (Kinet, J.-P. et al . , Proc. Natl . Acad. Sci . USA 85:6483-6487 (1988)), eosinophils (Sutton, B.J. and Gould, H.J., Na ture (London) 366: 421 -428 (1993)) and peripheral monocytes (Gounni, A.S. et al., Ref 4 ). The β subunit, Fc_eRI/3, is a 4-transmembrane protein with both the amino and carboxyl termini residing in the cytoplasm. The human CD20 antigen (Tedder, T.F., et al . , Proc. Natl . Acad. Sci . USA 55:208-212 (1988)), as well as its murine equivalent Ly-44 (Tedder, T.F. et al . , J. Immunol . 141:4388-4394 (1988)), are expressed only in B-cells. Functional studies with different CD20 antibodies indicate that CD20 is involved in the regulation of B-cell activation (Clark, E.A. and Lane, J.L. Annu . Rev. Immunol . 9 : 97 - 127 (1991)). The CD20 protein also contain four transmembrane domains with the amino and carboxyl ends on the same cytoplasmic side of the cell membrane. There is an overall amino acid similarity of 16% between CD20 and Fc.Rlβ . Furthermore, the murine Fc_eRI-? gene maps to the same region in chromosome 19 as the Ly-44 gene (Huppi, K. e al., J. Immunol . 143 : 3787-3791 (1989)).

The identification of genes that may play a role in IgE responses or atopic diseases would be desirable. It would also be desirable to develop an assay which can detect hereditary atopy.

Summary of the Invention

The invention relates to a recombinant DΝA molecule which encodes a HT_m4 protein, a transfected host cell which has been stably transfected with a DΝA molecule which encodes a HT_rn4 protein and a recombinant HT_m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

Brief Description of the Drawings

Figures 1A and IB depict the cDΝA sequence of the HT_m4 gene and the amino acid sequence of the encoded protein. Νucleotide sequence is numbered on the left. The amino acid sequence of the longest open reading frame is numbered on the right beginning with the first presumed initiating methionine. An upstream in-frame stop codon, TAA, is indicated in bold letters at position 85. A TAA stop codon (END) is followed by a 3' untranslated region containing an AATAAA poly adenylation signal. The four putative transmembrane domains are underlined. Two phosphorylation sites are underlined with dotted lines.

Detailed Description of the Invention The invention relates to the discovery and cloning of the HT_m4 cDNA. The 1672-nucleotide long cDNA contains a long open reading frame, beginning at nucleotide position 97. The expression product of the cDNA is a 4 transmembrane spanning protein with a calculated molecular mass of about 25 kDa and 214 amino acids. The sequences are set forth in Figures 1A and IB.

HT_m4 contains four hydrophobic domains of 20 to 21 amino acids . The amino terminal region before the beginning of the first hydrophobic domain contains four prolines. Each of the hydrophilic regions between the transmembrane segments contains a single proline. Several substrates for Casein kinase 2 phosphorylation (Pina, L.A. Biochim . Biophys . Acta . 1054 : 267 -284 (1990)) of serine/threonine are found at residues 24 (TGPE) , 155 (SSSE) , 181 (TLLE) , and 203 (SREE) and for Protein Kinase C phosphorylation at residue 149 (SLR) . The sequence is consistent with a polypeptide chain that crosses the membrane four times, projecting two small loops extracellularly, and retaining the amino- and carboxyl- terminal portions in the cytoplasm.

The HT_m4 protein, as defined herein, encompasses an expression product which possesses one or more of the functions of the native protein. Included are functional protein or polypeptide fragments of the native protein and/or proteins or polypeptides where one or more amino acids have been deleted, added or substituted. Preferably, the protein or polypeptide shares at least about 50 % homology and more preferably at least about 75 % homology with the corresponding sequences of the native protein of Figures 1A and IB.

Recombinant DNA molecules of the invention, in one embodiment, encode an HT_m4 protein, as defined herein. In one embodiment, the molecule shares at least about 50 % homology, and preferably at least about 75 % homology (such as at least about 90 % homology) with the corresponding sequences of the native gene, particularly in highly conserved regions of the 4-transmembrane protein family of HT_ra4, CD20 and Fc_eRI_/S. Preferably, the recombinant DNA molecule comprises the corresponding encoding nucleotide sequences of Figures 1A and IB .

In another embodiment recombinant DNA molecules, such as probes, can be employed, for example, to isolate genes encoding transmembrane proteins or receptors, such as the Fc.RI. Such molecules comprise recombinant DNA molecules which hybridize to all of or a fragment of the sequences of Figures 1A and IB. Preferably, the molecules hybridize under stringent conditions, such as those set forth in Sambrook et al . Molecular Clonine: A Laboratory Manual , 2nd Edition (Cold Spring Harbor Lab., Cold spring Harbor, NY (1989) .

The recombinant DNA molecules can contain coding and non-coding sequences. Preferably, the molecules comprise at least about 25 nucleotides and more preferably at least about 60 nucleotides with 95-100 % to pull out gene.

Preferably, DNA probes comprise sequences the same as or homologous corresponding to the region encoding the N- or C-termini of the protein.

The predicted structure of HT_m4 as discussed herein demonstrates the relationship of this protein to Fc,RI/3 and the CD20 antigen and provides evidence for a family of 4- transmembrane spanning proteins. The conservation of amino acids between all three proteins is highest in the four transmembrane domains. While much greater divergence exist in the hydrophilic amino and carboxyl termini, several amino acids within these regions are conserved such as the presence of 4 to 5 prolines in the amino terminus of all three proteins. Two conserved cysteine residues in the second extracellular domain between Tm-3 and Tm-4 suggest that intra- or inter-molecular di-sulphide bonds in this domain are present in all three proteins. HT_m4 also contains two phosphorylation sites (threonine²⁴ and serine²⁰³) in the cytoplasmic region of the protein. Finally, there is a well conserved SP(P) motif near the carboxyl end of all three proteins. The difference between CD20 and the other two proteins is contributed significantly by several long stretches of non-homologous amino acids. The carboxyl terminus of Fc_eRI/3 contains the Reth or antigen receptor activation motif (ARAM) (Reth, M. Na ture (London) 338 : 383 - 384 (1989)), which is not present in CD20 or HT_m4. The ARAM sequence is found in the cytoplasmic tail of several receptor subunits including CD3 γ, δ, e and ζ , Igo; and IgS, in MB-1 and B29 antigen, and in the β and γ chain of Fc_eRI (Weiss, A. and Littman, D.R. Cell 76:263-274 (1994)). Tyrosine residues in ARAM sequences are believed to be critical inducers of and substrates for phosphorylation by cytoplasmic tyrosine kinases, allowing for the recruitment of additional effector molecules (Weiss, A. and Littman, D.R. Cell 76:263-274 (1994); Paolini, R. et al . , Na ture (London) 353:855-858 (1991); Eiseman, E. and Bolen, J.B. Nature (London) 355 : 78 - 80 (1992)). The common exon-intron organization of the genes containing the ARAM sequence has led to the suggestion that they might have evolved from the same gene family (Weiss, A. and Littman, D.R. Cell 76:263- 274 (1994)). However, the structural similarity of Fc_eRIβ to CD20 and HT_m4 suggests that the ARAM sequence was acquired by the Fc.RI_/S gene during evolution.

Chromosome mapping localized the HT_m4 gene to chromosome llql2-13.1, the location of the CD20 gene. However, the murine Fc_eRIβ and the murine equivalent for CD20, Ly-44, are both located in the same position in mouse chromosome 19 (Tedder, T.F. et al . , J. Immunol . 141 : 4388 - 4394 (1988); Clark, E.A. and Lane, J.L. Annu . Rev. Immunol . 9:97-127 (1991); Huppi, K. et al . , J^". Immunol . 143 : 3787- 3791 (1989)). Therefore, the three genes are believed to have been originated and evolved from the same locus, further supporting the proposition that they are members of the same family of related proteins. They also form a family of proteins that is quite distinct from another large family of 4-transmembrane proteins related to TAPA- KFearon, D.T. Curr. Op. Immunol . 5:341-348 (1993); Barclay, A.N. et al . , The Leucocyte Antigen Facts Book, (Academic Press Inc., San Diego, CA) (1993)) which include CD9, CD37, CD532, CD63 and R2.

The identification of a gene product like HT_m4 related to Fc_eRI3 is significant. While simultaneous cotransfection of the α, β and γ genes are necessary to induce surface expression of the murine Fc_cRI receptor, cotransfection of the human α and γ genes without the β gene is sufficient to induce expression of high-affinity Fc receptors (Miller, L. et al . , Science 244 : 334 -337 (1989)). Furthermore, recent evidence indicated that functional high-affinity IgE Fc receptors can be found on monocytes in the absence of the β chain (Maurer, D. et al., J. Exp. Med. 179:745-750 (1994) ) .

As such, the HT_m4 gene and protein can be useful in the research and study of the induction of expression of Fc.RI and the particular function of Fc_€RI_/3. As such, the HT_m4 gene and protein can be useful in, for example, the design of drugs which can block or inhibit induction of Fc_eRI, thereby treating atopic diseases.

Further, the diverse association of subunits in Fc receptors of different hematopoietic cells has been established. For example, Fc_£RI/3 was found to be associated with the low-affinity Fc receptor for IgG, FcγRIII (CD16) , in mast cells (Kurosaki, T. et al . , J. Exp. Med . 175:447-451 (1992)). Fc_eRIγ has also been found as a homodimer in association with FcγRIII in macrophages (Ra, C. et al . , Na ture 341:752-754 (1989)) or as a heterodimer with f and η chains in T cell receptor complex (Orloff, D.G. et al . , Nature 347 : 189 (1990)). In NK cells, Fc_£RIγ may be found as homodimers and as a heterodimer with the ζ- chain of T cell receptor (Letourneur, O. et al . , J. Tmmunol. 147:2652-2656 (1991)). More recently others have shown that the γ chain can also form an association with the high-affinity receptor for IgG, FcγRI (CD64) , in monocytic cell lines and neutrophils (Scholl, P.R. and Geha, R.S. Proc . Natl . Acad . Sci . USA 90:8847-8850 (1993); Ernst, L.K., et al . , Proc . Na tl . Acad. Sci . USA 90 : 6023 - 6027 (1993)). These findings suggest that a variety of signal transduction complex composed of different subunits might mediate similar effector functions but with different functional consequences. Association of these subunits with alternative ligand recognition subunits in a multimeric receptor complex would allow coupling of distinct ligands to common signaling pathways .

The expression of HT_m4 in all hematopoietic lineages and not in any of the non-hematopoietic cells tested indicates that HT_m4 participates in biochemical pathways unique to hematopoietic lineages.

Nucleic acids, such as DNA probes, comprising sequences of the HT_m4 genes can be used in an assay to detect patients suffering from hereditary atopic disorders. Either DNA or RNA can be used in the present assay method. The DNA which can be used in the method can be cDNA or genomic DNA. The source of DNA can be from any cell or cells removed from the individual and can include cultured progeny thereof, such as somatic cells, blood cells, sperm, fibroblasts or other somatic or germline cells. Also because the nucleic acid which is preferably analyzed is germline DNA, the method can be carried out prior or subsequent to onset of disease or disease symptoms. Where cDNA or RNA is to be used, the nucleic acid source should be from hematopoietic cells.

The presence of mutation can be determined using methods generally known in the art, such as by PCR (described herein below) . Alternatively, the nucleic acid comprising the site of mutation or its complement can be sequenced, thereby identifying the presence of a mutation. In yet another embodiment, the protein encoded by the DNA can be sequenced or identified, thereby establishing the presence of the mutation. In yet another embodiment, an antibody which selectively binds to one of the mutated sequence or wild-type sequence can be made and used to screen a protein fraction for the respective proteins.

One such method is PCR methods using a pair of primers specific for sequence flanking the mutation region. The resulting products can be sequenced, analyzed on gels, such as polyacrylamide or agarose gels, or evaluated by physical characteristics such as melting temperature or secondary structure. Other methods for determining nucleic acid mutations or modifications can be employed, as well. Co-amplification of two alleles in a heterozygote can generate PCR products which differ in the gene and therefore their melting and/or secondary structure characteristics are likely to differ. Under conditions as described in, e.g., Mutter and Boynton (Nucleic Acids Res. 23:1411 (1995)), amplification efficiency of the two alleles is near-equivalent, generating PCR products in a ratio proportional to that of the genomic template. Variability and biasing can be diminished by substitution of 7-deaza-2' -dGTP for dGTP during amplification, an intervention which reduces stability of intramolecular and intermolecular GC basepairing.

Allelic PCR fragments are easily separated, for example, by gel electrophoresis and detected by intercalating dye staining (e.g., ethidium bromide). As an alternative procedure, capillary electrophoresis can be employed. One example of capillary electrophoresis is in a polymer network consisting of 8% polyacryloylamino- ethoxyethanol in the absence of cross-linker, and offers a simple procedure for separation and on-line detection via UV absorbance at 254 nm, thus avoiding additional staining steps. The capillary column can be used repeatedly and the electropherogram can be stored on magnetic support. Comparisons among different runs can be obtained aligning all tracings to an internal standard of a known base pair size added as a marker (Nesi et al . , Electrophoresis, 15:644-6 (1994) ) .

In yet another embodiment, the presence of a mutation can be determined according to the method of Yamamoto et al. (Biochem. Biophys. Res. Comm. , 182:507 (1992)). The DNA or RNA obtained from the subject to be tested is amplified by standard PCR, a primer extension is carried out following addition of dideoxy ATP to the reaction mixture. The extension of the end-labeled reverse primer adjacent to 3' end of the site of mutation stops at a selected nucleotide after the sequence to be tested and the resultant primer products can be analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography .

Additional PCR based methods which can be used include random rapid amplification of cDNA ends (RACE) , described by Carney et al . (Gene, 155:289, 1995); single strand confor ation polymorphism analysis (Ris-Stalpers et al . , Pediatric Res., 36:227 (1994)) and reverse transcriptase PCR (Nakamura et al . , J. Neurological Sci. 122:74 (1994)). Additional hybridization techniques include the use of probes labeled with the same or different radioactive or fluorescent dyes, for example. This method allows for the direct detection of a mutation or wild type sequences (see, e.g., Sanpei et al., Biochem. Biophys. Res. Comm. 212:341-6 (1995); Taneja, J. Cell Biology, 128:995-1002 (1995) and Saito, Japanese Journal of Human Genetics, 39:421-5 (1994) ) .

In yet another embodiment the protein which is encoded by the gene or fragment thereof, or in the alternative, the nucleic acid, can be separated by size using art-recognized separation media and methods. Standard polyacrylamide gels or a modified SDS-PAGE protocol using low concentration of methylenebisacrylamide and long runs (Ide et al . , Biochem. Biophys. Res. Comm. 209:1119 (1995)).

The mutation associated with hereditary atopy can be identified by isolating and sequencing the HT_m4 gene of a population of individuals suspected of having hereditary atopy and of a population of individuals believed to not possess hereditary atopy. The sequences thus provided can be correlated and compared (e.g., obtaining consensus sequences or aligning sequences). "Mutation", as defined herein, is a consistent deviation in sequence shared by a population (or subpopulation thereof) of individuals suspected of having hereditary atopy, in comparison to the sequences found in the population (or subpopulation) of individuals believed to not possess hereditary atopy.

Also, the nucleic acids of the invention can be useful as probes to map genes on the human chromosome, such as employing the methods of fluorescence in si tu hybridization (Kobayashi e ai., Blood, 81 : 3027-3033 (1993)). The HT_m4 protein can be employed in the preparation of antibodies, such as monoclonal antibodies, according to methods known in the art. The antibodies can be used to block or mimic ligand binding to the receptor comprising HT_m4 or other receptors, such as Fc_£RI, isolate the antibodies can be used to the HT_m4 protein or hematopoietic cells which contain the HT_m4 protein.

The antibodies can also be useful in the detection of hematopoietic cells in a sample. For example, the method comprises contacting the sample with the antibody under conditions sufficient for the antibody to bind to the HT_ιn4 protein and detecting the presence of bound antibody.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

Exemplification Materials and Methods

Cell Lines and Primary Cells: Hematopoietic cell lines used in this study included lymphomyeloid (DU528) , erythroleukemic (K562,OCIR), promyelocytic (HL60) , myeloblastic (KG-1) , monoblastic (U937) , T-cell leukemia/lymphoma (MOLT-4, Lyl7, Lyl3) and myeloma (OCI- My5) lines. Non-hematopoietic cell lines used included bone marrow stromal (BS-1) , hepatoma (HepG2) , melanoma (HS294) , skeletal muscle (HuSk) , neuroblastoma (SKNSH) , cervical cancer (HeLa) and lung cancer (Calu-1) cells. All cell lines were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 1 mM L- glutamine, except for DU528, in which horse serum replaced bovine serum. Total RNAs from a human mast cell line, HMC-1 (Butterfield, J.H. et al., Leuk. Res . 12:345-355 (1988)), and a human factor dependent megakaryocytic line, M07e, were kindly provided by Dr. Karl Nocka, Cytomed Inc., Cambridge, MA. Normal bone-marrow cells were harvested from transfusion-filters after bone marrow transplantations. Primary leukemic cells with over 90% blasts were harvested from the peripheral blood of a patient with M4 acute myeloid leukemia. Total RNAs of neutrophils and eosinophils from normal individuals and eosinophils from a patient with hypereosinophilic syndrome were kindly provided by Dr. Peter Weller and Dr. Kaiser Lim, Harvard Medical School.

Preparation of Probes from Subtractive cDNA Libraries for Differential Screening: The construction of four subtractive cDNA libraries (DU528/BS-1, K562/BS-1, KG-l/BS- 1, and BS-l/BS-1) , from three human hematopoietic cell lines (DU528, K562 and KG-1) and one non-hematopoietic human cell line (BS-1) , using the PT3T719U multiphagemid vector (Pharmacia) was described previously (Lelias, J.M. et al . , Proc . Natl . Acad. Sci . USA 90:1479-1483 (1993)). cDNA inserts released from two of the hematopoietic (DU528/BS-1 and KG-l/BS-1) and the non-hematopoietic (BS- 1/BS-l) subtractive libraries were purified, labeled with ³²P, and used as probes to screen the K562/BS-1 library

(Sambrook, J. et al . , Molecular Cloning: A Laboratory Manual , 2nd edition (Cold Spring Harbor Lab., Cold Spring Harbor, NY (1989) ) .

Induction of Cell Line U937: The U937 cell line was grown to a concentration of 5 x 10^s cells per ml and differentiation was induced with 50 nM phorbol 12-myristate 13-acetate (PMA; Sigma) .

Chromosomal Localization of the HT^ Gene: The chromosomal location of the HT_m4 gene was determined by fluorescent in situ hybridization (FISH) as previously described

(Kobayashi, H. et al., Blood ,91:3027-3033 (1993)). Human metaphases were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes. The HT_m4 cDNA was labeled by nick-translation with biotin-11-UTP (Enzo Diagnostics, Syosset, NY) . The biotin-labeled probe was hybridized to metaphase cells and detected with fluorescein-conjugated avidin (Vector Lab, Burlingame, CA) . Slides were examined by two independent observers without knowledge of the probe used.

Reverse-transcriptase Polymerase Chain Reaction (RT-PCR) : Reverse transcriptase reaction was carried out as described (Sanger, F. et al., Proc. Natl . Acad. Sci . USA 74:5463-5467 (1977)) with RΝAs from cell lines or cells enriched for various cell types to obtain first strand cDΝAs. The cDΝAs were subjected to PCR amplification reactions as described (Wulf, G.M. et al . , EMBO J . 12:5065-5074 (1993)), using primers spanning nucleotide 721 to 1087 of HT_m4 to give a predicted PCR product of 388 nucleotides. The sense primer used was 5' -TCACCTCCCAATTCTGTGTAATCAAGA-3 ' (SEQ ID NO: 1), and the anti-sense primer was 5' -GATTATACCGCCTTCGTTCCTTA AACC-3' (SEQ ID NO: 2) . PCR reactions were carried out with 100 nM primers for 30 cycles of denaturation (1 minute at 94 °C) , annealing (1 minute at 54°C) and extension (2 minutes at 72 °C) .

General Methods : RNA was isolated using the RNAzol method (Biotecx Laboratories, Houston, TX) . DNA sequencing was done by the dideoxynucleo-tide chain-termination technique (Sanger, F. et al . , Proc . Natl . Acad. Sci . USA 74:5463-5467 (1977) ) after subcloning appropriate DNA fragments into

M13.

Results Isolation of Clone HT_m;1 : By differential screening of the K562 hematopoietic library with cDNA probes from two hematopoietic libraries (enriched for hematopoietic cDNAs) and cDNA probes from the non-hematopoietic BS-1 library, clones that hybridized positively only to the hematopoietic probes were isolated. One of these, denoted clone HT_m4 was used as a probe to screen a Northern blot panel consisting of total RNAs from various hematopoietic and non- hematopoietic cell lines.

Expression pattern of HT_m : A combination of Northern blot and RT-PCR analysis was used to determine the spectrum of tissue and lineage expression of the gene. The cDNA insert of clone HT_m4 hybridized to a transcript of about 1.7 kb in five hematopoietic lines which included myeloid and erythroid lineages and to normal human bone marrow cells. The HT_m4 and mRNA was not detectable in a T cell lymphoma line (Lyl7) and in a lymphomyeloid leukemic line with T and granulocytic differentiation potential (DU528) . In all of the seven non-hematopoietic cell lines which included lung, cervical, brain, skeletal muscle, melanoma, hepatoma, and bone marrow stromal cells, no hybridizing mRNA could be detected. These non-hematopoietic lines included cells of ectodermal, endodermal and mesodermal origin. The mRNA was also absent in the primary blast cells of a patient diagnosed to have M4 acute myeloid leukemia (AMD .

To facilitate screening of RNA samples, particularly those derived from cells in quantities too limited for Northern blot analysis, we examined expression by RT-PCR. The quality of the first strand cDNAs obtained after reverse transcription was satisfactory as evaluated by using primers for the housekeeping gene HPRT. Based on nucleotide sequence of HT_m4 cDNA, oligonucleotides were synthesized and used as specific primers for PCR amplification. The predicted PCR product of a 388 nucleotide-long DNA was obtained in normal bone marrow cells and the HL60 cell line but not in the HeLa and Lyl7 cell line, confirming the Northern blot analysis. RNAs from a human mast cell line (HMC-1) and a megakaryocytic line (M07e) were also positive for HT_m4 mRNA. RNAs from normal eosinophils or neutrophils and eosinophils from a patient with hype -eosinophilie syndrome (HES) also yielded the predicted PCR product. Two leukemic T-cell lines (Lyl3 and MOLT4) and a myeloma cell line (OCI-My5) were also found to be positive for HT_m4. All PCR-derived DNAs hybridized positively to radio-labeled HT_m4 in subsequent Southern analysis.

Molecular analysis of HT_ml human cDNA: The nucleotide sequence of the HT_m4 cDNA and the predicted amino acid sequence are shown in Figures 1A and IB. The 1672- nucleotide long cDNA contains a long open reading frame, beginning at nucleotide position 97, encoding a protein of 214 amino acids with a calculated molecular mass of 25 kDa. A hydrophilicity analysis with the Kyte-Doolittle algorithm (Kyte, J. and Doolittle, R.F. J. Mol . Biol . 157:105-132 (1982)), reveals that HT_m4 contains four hydrophobic domains of 20 to 21 amino acids. The amino terminal region before the beginning of the first hydrophobic domain contains four prolines. Each of the hydrophilic regions between the transmembrane segments contains a single proline. Several substrates for Casein kinase 2 phosphorylation (Pina, L.a. Biochim. Biophys . Acta 1054 : 267- 284 (1990)) of serine/threonine are found at residues 24 (TGPE) , 155 (SSSE) , 181 (TLLE) , and 203 (SREE) and for Protein Kinase C phosphorylation at residue 149 (SLR) . The sequence is consistent with a polypeptide chain that crosses the membrane four times, projecting two small loops extracellularly, and retaining the amino- and carboxyl-terminal portions in the cytoplasm.

Expression of HT_ml during differentiation of cell line U937 : To ascertain if expression of the HT_m4 mRNA may vary depending on the stage of cellular differentiation, we examined the consequences of induced differentiation in the monoblastic cell line U937. Exposure of the cells to PMA rapidly induced differentiation to macrophages, as confirmed morphologically and molecularly by monitoring the marker for terminally differentiated macrophages, CDllb (Arnout, M.A. Immunol . Rev. 114:145-180 (1990)). The expression of HT_m4 mRNA over a period of 48 hours showed an initial increase followed by a down regulation so that by day three, HT_m4 transcripts were detectable at a very low level .

HT_rnl is located on chromosome llql2-13: Forty-one chromosomes from 30 metaphases were scored for the positive chromosomal band. Band llql2 was labeled on eighteen of chromosome 11 homologues, band llql3.1 on twenty-one of chromosome 11 homologues and band llql3.2 on two of chromosome 11 homologues. No signal was detected on other chromosomes in these cells. Similar results were obtained in an additional experiment using this probe. Thus, HT_m4 is localized to chromosome Ilql2-ql3.1.

Equivalents

Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Beth Israel Hospital, Boston

(B) STREET: 330 Brookline Avenue

(C) CITY: Boston

(D) STATE/PROVINCE: Massachusetts

(E) COUNTRY: USA

(F) POSTAL CODE/ZIP: 02215

(G) TELEPHONE: (617) 667-2045 (I) TELEFAX: (617) 667-4774

(ii) TITLE OF INVENTION: RECOMBINANT HTM4 GENE, PROTEIN AND ASSAYS

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Hamilton, Brook, Smith & Reynolds, P.C.

(B) STREET: Two Militia Drive

(C) CITY: Lexington

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02173

(vj COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Brook, David E.

(B) REGISTRATION NUMBER: 22,592 (C) REFERENCE/DOCKET NUMBER: BIH94-03A PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 861-6240

(B) TELEFAX: (617) 861-9540

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TCACCTCCCA ATTCTGTGTA ATCAAGA 27

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

GATTATACCG CCTTCGTTCC TTAAACC 27

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1661 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 97..741

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GTGATCTTTT CTGAGTGTCT CCTACTTGCG ACAAGGTGGA CTTGGGAGGA AAGCCGTCTG 60

CCAAAGCCTG AAGCCTCCAA GCCATAAACA ACCCCA ATG GCC TCC CAC GAA GTT 114

Met Ala Ser His Glu Val 1 5

GAT AAT GCA GAG CTG GGG TCA GCC TCT GCC CAT GGT ACC CCA GGC AGT 162 Asp Asn Ala Glu Leu Gly Ser Ala Ser Ala His Gly Thr Pro Gly Ser 10 15 20

GAG ACG GGA CCA GAA GAG CTG AAT ACT TCT GTC TAC CAC CCC ATA AAT 210 Glu Thr Gly Pro Glu Glu Leu Asn Thr Ser Val Tyr His Pro lie Asn 25 30 35

GGA TCA CCA GAT TAT CAG AAA GCA AAA TTA CAA GTT CTT GGG GCC ATC 258 Gly Ser Pro Asp Tyr Gin Lys Ala Lys Leu Gin Val Leu Gly Ala lie 40 45 50

CAG ATC CTG AAT GCA GCA ATG ATT CTG GCT TTG GGT GTC TTT CTG GGT 306 Gin lie Leu Asn Ala Ala Met lie Leu Ala Leu Gly Val Phe Leu Gly 55 60 65 70

TCC TTG CAA TAC CCA TAC CAC TTC CAA AAG CAC TTC TTT TTC TTC ACC 354 Ser Leu Gin Tyr Pro Tyr His Phe Gin Lys His Phe Phe Phe Phe Thr 75 80 85

TTC TAC ACA GGC TAC CCG ATT TGG GGT GCT GTG TTT TTC TGT AGT TCA 402 Phe Tyr Thr Gly Tyr Pro lie Trp Gly Ala Val Phe Phe Cys Ser Ser 90 95 100

GGA ACC TTG TCT GTT GTA GCA GGG ATA AAA CCC ACA AGA ACA TGG ATA 450 Gly Thr Leu Ser Val Val Ala Gly lie Lys Pro Thr Arg Thr Trp lie 105 110 115 CAG AAC AGT TTT GGA ATG AAC ATT GCC AGT GCT ACA ATT GCA CTA GTG 498 Gin Asn Ser Phe Gly Met Asn He Ala Ser Ala Thr He Ala Leu Val 120 125 130

GGG ACT GCT TTT CTC TCA CTA AAT ATA GCA GTT AAT ATC CAG TCA TTA 546 Gly Thr Ala Phe Leu Ser Leu Asn He Ala Val Asn He Gin Ser Leu 135 140 145 150

AGG AGT TGT CAC TCT TCA TCA GAG TCA CCG GAC CTA TGC AAT TAC ATG 594 Arg Ser Cys His Ser Ser Ser Glu Ser Pro Asp Leu Cys Asn Tyr Met 155 160 165

GGC TCC ATA TCA AAT GGC ATG GTG TCT CTA CTG CTG ATT CTC ACC TTG 6 2 Gly Ser He Ser Asn Gly Met Val Ser Leu Leu Leu He Leu Thr Leu 170 175 180

CTG GAA TTA TGC GTA ACT ATC TCT ACC ATA GCC ATG TGG TGC AAT GCA 690 Leu Glu Leu Cys Val Thr He Ser Thr He Ala Met Trp Cys Asn Ala 185 190 195

AAC TGC TGT AAT TCA AGA GAG GAA ATT TCC TCA CCT CCC AAT TCT GTG 738 Asn Cys Cys Asn Ser Arg Glu Glu He Ser Ser Pro Pro Asn Ser Val 200 205 210

TAATCAAGAA TACCTCCTTA TGAAAATAAT TCTGAGAGCA TGAATATTTG ACCTTAAATC 798

TCCAGTGACT CAGAGCTTCA CCCACAAACT CAGGAGAACA TAAGCCTGCT CGTAAAGCTC 858

AATCCTTCTA TCATGGCACC AATCACAAGA ACCTTGGACG TTTGACTGAC TCTATCCTTT 918

CTCTCCTAAC TATAAATCCT ATTTGTGTGT CGTGGGTATG GAAGGACAGA TATATTTCTT 978

TAGGCATTCT TGGATATCTG TAACTTCTAT GATCATTACT CCAAAGTTGT TTCCAGAAAT 1038

TGGTTCTATT TCTTCTTATC CACCTACTCC ATTGCTTTAT GAGGTTTAAG GAAGGAAGGC 1098

GGTATAATCC CTATTCAATA TATTTTTTCT AAAATCCAAC TTCTGACCGC CCAGTAGGAA 1158

GAAAAATGAG ACATTTTTTC CATTACAGAG AAATGCTTCT TGACTTTAAC ATCAGCATTA 1218 TAAAAAGTGT CAAATAAAAA ATTACCATCA TTATCATTAA AATAAATTTT CACTGTATTT 1278

GAGATGGGAG GGTTAAGGCT CAGGGATTTT ATTTCAGTGA ACTGCTGGAA CTCACACATG 1338

CCCTGATATG TAAATGATGA TTTATGTTGG CGAGTCTGAG AGCAAGCCCA AATGTGTTCT 1398

TCAAAGGACA ATGGGAAACT GTAAAGTAGA GAACTAAAGA ATAAGGCCTT TAGAATCTGA 1458

CACATCTGGG TTCAAATTCT GAAACTGTCA CTTATTACCT GTATGAACAT GGGCAAATTA 1518

TCTAATCTCT CTGATCTATT TTTCCTCATC TGTAAAATAG GTGTAATAAT AACAACTACT 1578

TTGTCGGTTG CTCTGAGGGT TAAATGAAAA TAAAAAGAAA ATGTGAAACA GCACCACAGG 1638

TACTTGAAAA AAAAAAAAAA AAA 1661

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 214 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ala Ser His Glu Val Asp Asn Ala Glu Leu Gly Ser Ala Ser Ala 1 5 10 15

His Gly Thr Pro Gly Ser Glu Thr Gly Pro Glu Glu Leu Asn Thr Ser 20 25 30

Val Tyr His Pro He Asn Gly Ser Pro Asp Tyr Gin Lys Ala Lys Leu 35 40 45

Gin Val Leu Gly Ala He Gin He Leu Asn Ala Ala Met He Leu Ala 50 55 60

Leu Gly Val Phe Leu Gly Ser Leu Gin Tyr Pro Tyr His Phe Gin Lys 65 70 75 80 His Phe Phe Phe Phe Thr Phe Tyr Thr Gly Tyr Pro He Trp Gly .Ala 85 90 95

Val Phe Phe Cys Ser Ser Gly Thr Leu Ser Val Val Ala Gly He Lys 100 105 110

Pro Thr Arg Thr Trp He Gin Asn Ser Phe Gly Met Asn He Ala Ser 115 120 125

Ala Thr He Ala Leu Val Gly Thr Ala Phe Leu Ser Leu Asn He Ala 130 135 140

Val Asn He Gin Ser Leu Arg Ser Cys His Ser Ser Ser Glu Ser Pro 145 150 155 160

Asp Leu Cys Asn Tyr Met Gly Ser He Ser Asn Gly Met Val Ser Leu 165 170 175

Leu Leu He Leu Thr Leu Leu Glu Leu Cys Val Thr He Ser Thr He 180 185 190

Ala Met Trp Cys Asn Ala Asn Cys Cys Asn Ser Arg Glu Glu He Ser 195 200 205

Ser Pro Pro Asn Ser Val 210

Claims

CLAIMS What is claimed is:

1. A method for detecting hereditary atopy in a subject comprising: (a) determining the presence of a mutation in a nucleic acid obtained from the subject which encodes a HT_m4 protein; and (b) correlating the presence of the mutation with hereditary atopy.

2. The method of Claim 1 wherein the nucleic acid is genomic DNA.

3. The method of Claim 2 wherein the DNA is obtained from somatic cells.

4. The method of Claim 3 wherein the DNA is obtained from a tissue or blood sample.

5. The method of Claim 2 wherein the presence of the mutation is determined by PCR.

6. The method of Claim 2 wherein the nucleic acid is compared with one or more nucleic acids encoding HT_m4 obtained from subjects suspected of or diagnosed with hereditary atopy.

7. The method of Claim 2 wherein the nucleic acid is compared with one or more nucleic acids encoding HT_m4 obtained from subjects neither suspected of nor diagnosed with hereditary atopy.

8. A method for determining the presence of a mutation in a nucleic acid encoding an HT_m4 protein or its complement in a subject comprising:

(a) obtaining DNA or RNA from the subject wherein the DNA or RNA comprises the HT_m4 coding sequence or its complement ; and

(b) determining sequence of the DNA or RNA; and

(c) comparing the sequence with the sequence the HT_m4 in a population of individuals.

9. The method of Claim 8 wherein the nucleic acid is genomic DNA.

10. The method of Claim 9 wherein the DNA is obtained from somatic cells.

11. The method of Claim 10 wherein the DNA is obtained from a tissue or blood sample.

12. The method of Claim 9 wherein the presence of the mutation is determined by PCR.

13. The method of Claim 9 wherein the nucleic acid is compared with one or more nucleic acids encoding

HT.,,₄ obtained from subjects suspected of or diagnosed with hereditary atopy.

14. The method of Claim 9 wherein the nucleic acid is compared with one or more nucleic acids encoding ^HTm₄ obtained from subjects neither suspected of nor diagnosed with hereditary atopy.

15. The method of Claim 9 wherein the DNA or RNA hybridizes to all or a portion of the sequence of SEQ ID NO: 1 or the complement thereof.

16. The method of Claim 15 wherein the DNA or RNA hybridizes to all or a portion of the sequence of SEQ ID NO: 1 or the complement thereof under stringent conditions.

17. The method of Claim 16 wherein the DNA molecule comprises the sequence of SEQ ID NO: 1.