WO1998021224A9

WO1998021224A9 - The cloning of duffy blood group antigen

Info

Publication number: WO1998021224A9
Application number: PCT/US1997/021067
Authority: WO
Filing date: 1997-11-14
Publication date: 1998-12-03

Abstract

The major subunit of the Duffy blood group antigenic system, gp-Fy protein, has been isolated, as have two variants, gp-Fyα and/or gp-Fyβ, which are produced from the Duffy FY gene by a differential splicing mechanism. gp-Fy protein contains the receptor by which P. vivax enters red cells and causes malaria. gp-Fy protein has therapeutic value in the prevention of malaria and in the regulation of erythrocyte, neural and renal functions and can be combined with physiologically acceptable diluents to yield a therapeutic agent suitable for these purposes. Peptides corresponding to a portion of gp-Fy protein that contains the receptor also have been synthesized. gp-Fy protein and derived peptides have utility in the production of therapeutics, e.g., antibodies, complementary peptides, etc., which are also useful to treat malaria and regulate essential erythrocyte, neural and renal functions. Also provided are nucleic acid primers and probes which specifically bind to or hybridize with portions of the FY gene or DNA or RNA derived therefrom for detecting the presence of gp-Fyα and/or gp-Fyβ nucleic acids in tissues samples. Methods for detecting Duffy protein expression, i.e., differential expression of gp-Fyα and/or gp-Fyβ proteins are provided.

Description

THE CLONING OF DUFFY BLOOD GROUP ANTIGEN

This application is a continuation-in-part of U.S. patent application Serial No. 08/140,797, filed on October 21, 1993, the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the gp-Fy protein, which is the major subunit of the Duffy blood group antigen, and use thereof. More specifically, the invention relates to the gp-Fy protein and related peptides, nucleic acids which encode the gp-Fy protein, and a method of their use.

The Duffy blood group system is a group of proteins expressed on the cell surfaces of erythrocytes in mammals. In humans, the Duffy blood group consists of two principal antigens Fy" and Fy^b produced by co-dominant alleles, FY*A and FY*B. Antisera specifically reactive with these two antigens, respectively anti-Fy^a and anti-Fy^b, have been used to define four phenotypes, Fy(a+b-), Fy(a-b+), Fy(a+b+) and Fy(a-b-) (Marsh 1975). The Fy(a-b-) phenotype, the predominant phenotype in Africans and African- Americans, is characterized in that neither of these antisera agglutinates Duffy Fy(a-b-) cells. Other antisera capable of defining the other Duffy phenotypes, Fy3, Fy4 and Fy5, are very rare. A murine monoclonal antibody, anti-Fy6, defines a Duffy antigenic determinant present in all red blood cells except Fy(a-b-) cells (Nichols 1987). Accordingly, erythrocytes reacting with Duffy antisera and anti-Fy6 are designated "Duffy-positive," while unreactive erythrocytes are designated "Duffy-negative."

The function of the Duffy protein on erythrocytes is not well understood. Evidence is accumulating suggesting that the Duffy blood group antigen and the erythrocyte chemokine receptor are the same protein (Horuk et al. 1993). The erythrocyte receptor apparently differs from the IL-8 receptors, IL-BRA and IL-ORB on neutrophils. The erythrocyte receptor binds a family of chemotactic and proinflammatory soluble peptides, including IL-8, melanoma growth stimulatory activity (MGSA), monocyte chemotactic protein 1 (MCP-1), and regulated on activation, normal T expressed and secreted (RANTES) protein. But to understand the relationships among the various receptors and ligands, and to understand the role Duffy protein plays in any of these interactions, a better understanding of the molecular structure of the protein and its expression is required.

The Duffy antigen is also involved in malaria, the most prevalent infectious disease of mankind. The widespread geographic distribution of malaria together with the severe pathologic consequences of the infection make the disease a major medical and financial burden for many of the developing nations.

There are several different kinds of malaria, one of which is caused by the parasite Plasmodium vivax, which attacks the red blood cells of susceptible individuals. A genetic trait of special interest with regard to P. vivax is the absence of antigens encoded by the blood group system called Duffy (Livingston 1984). It has been shown that individuals whose red blood cells lack the product of the Duffy genes are not susceptible to the penetration of P. vivax owing to the fact that Duffy molecules serve as the receptor for the parasite. (Miller et al. 1976).

Malarial parasites are transmitted from host to host by the feeding females of several species of the genus Anopheles. It is in the mosquito that the sexual phase of the life cycle of

P. vivax takes place leading to the production of sporozoites. After their introduction into a "new" host, these sporozoites reside in the parenchymal cells of the liver and multiply asexually causing the eventual rupture of hepatic cells and the release of asexual forms (merozoites) into the blood stream. There the merozoites actively penetrate into red blood cells in a nearly synchronous fashion and because the rate of growth and cell division of

P. vivax is essentially identical, the infected erythrocytes simultaneously reach the stage of parasite load at which they break. This produces the typical cycles of fever every 48 hours, a condition named "tertian" malaria.

P. vivax infection may persist without treatment for as long as five years. P. vivax parasitemias are relatively low-grade, primarily because the parasites favor the few young red blood cells or reticulocytes that exist in peripheral blood.

Immunity to P. vivax is commonly only partial in nature, which allows the occurrence of superinfections that evolve independently causing an overlap in the cycles of parasite release leading to the appearance of fever in shorter cycles. P. vivax exhibits considerable antigenic diversity and variation, as do other malarial Plasmodium species (Hommel 1985), although it has been recently shown that antigenic components of P. vivax sporozoites exist that are common to parasites from different isolates (Zavala et al. 1985).

In the context of the sources of antigenic differences between P. vivax isolates and their consequences with regard to vaccination, it is important that the merozoites of different strains of P. vivax share the same receptor for penetration into red blood cells, i.e., the Duffy molecule (Miller et al. 1976). In addition, regardless of its capacity to vary other antigenic molecules, the parasite recognition molecule, i.e., the molecule that binds to the Duffy molecule, must remain constant since it is the complementarity between it and the invariant receptor that allows the penetration of merozoites into erythrocytes and, thus, the continuity of the infection. Changes in the ligand specificity of this molecule would result in the loss of the parasite's capacity to infect, since P. vivax merozoites appear to be unable to utilize other human red blood cell receptors for their penetration in vivo, as shown by the resistance of Duffy-negative erythrocytes.

Persons, most commonly of African descent, with Fy(a-b-) erythrocytes cannot be infected by P. vivax. These cells are also resistant to the in vitro invasion by P. knowlesi, a simian parasite that invades Fy(a+b-) and Fy(a-b+) human erythrocytes (Miller et al. 1975). Receptors for red cell invasion by these parasites, therefore, are related to the Duffy blood group system.

In view of the above considerations, it is clear that existing knowledge is limited concerning the molecular structure and function of the Duffy protein, as well as its expression.

Moreover, the malarial binding site on erythrocytes has been insufficient to permit molecular approaches to the treatment and prophylaxis of malaria. Lacking an understanding of the molecular basis for the interaction between the merozoite and the Duffy antigen has been a serious impediment preventing the development of compositions and methods by which that interaction, and the concomitant infection, can be effectively inhibited or eliminated.

Accordingly, it is one of the purposes of this invention to overcome the above limitations in the functional manipulation and detection of the Duffy protein, as well as in the prevention and treatment of malaria, by providing a substantially improved understanding of the molecular structure and function of the Duffy protein. Another purpose of the invention is to provide molecular probes and methods capable of characterizing Duffy protein expression and function in biological systems. Furthermore, another purpose of the invention is to provide compositions and methods which promote the understanding of the inception and progression of malaria and to provide methods and compositions useful for combating this dread disease. Other purposes will present themselves to the skilled artisan to render the invention useful in particular contexts.

SUMMARY OF THE INVENTION

There is provided a nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof. The nucleic acid can be isolated natural or synthetic DNA or RNA encoding a gp-Fy protein. Preferably, when the nucleic acid is RNA, the nucleic acid is a non-spliced mRNA encoding gp-Fyα protein or a spliced mRNA encoding gp-Fyβ protein. One highly preferred nucleic acid of the invention is the Duffy cDNA, having a nucleotide sequence consisting of the sequence designated SEQ ID NO:l. Characteristic fragments of this sequence are also within the scope of the invention.

The invention also includes an isolated natural or synthetic undenatured gp-Fy protein or a characteristic fragment thereof. The gp-Fy protein can be encoded by a non-spliced gp-Fy mRNA or a spliced gp-Fy mRNA. Preferably, the gp-Fy protein has an amino acid sequence consisting of the sequence designated SEQ ID NO:2 or the sequence designated SEQ ID NO:3, or a characteristic fragment thereof.

Also provided by the invention is a peptide having an amino acid sequence comprising the sequence designated SEQ ID NO: 6. Preferably, the peptide has an amino acid sequence comprising the sequence designated SEQ ID NO: 5. More preferably, the peptide has an amino acid sequence comprising the sequence designated SEQ ID NO:4. Still more preferably, the peptide has an amino acid sequence comprising the sequence designated SEQ ID NO: 7.

The invention further includes a nucleic acid probe or primer, wherein the nucleic acid probe or primer hybridizes specifically with a nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof. Accordingly, the nucleic acid probe or primer can hybridize specifically with a non-spliced nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof. Alternatively, the nucleic acid probe or primer can hybridize specifically with a spliced nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof. Preferred nucleic acid probes or primers according to the invention have sequences selected from the sequences designated SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In one preferred embodiment, the nucleic acid probe or primer is attached to a detectable label moiety. The invention also includes a method of detecting gp-Fy-encoding nucleic acid, comprising hybridizing a nucleic acid probe or primer, capable of specifically hybridizing with a nucleic acid encoding a gp-Fy protein, with a biological sample comprising nucleic acid, and measuring an amount of hybridization of said nucleic acid probe or primer with said nucleic acid in the biological sample, wherein a measured amount of hybridization indicates that an amount of gp-Fy-encoding nucleic acid is present in said biological sample. The method can include detecting gp-Fy-encoding mRNA, preferably, employing a nucleic acid probe or primer which hybridizes specifically with a non-spliced nucleic acid encoding a gp-Fy protein or with a spliced nucleic acid encoding a gp-Fy protein. The method advantageously employs a nucleic acid probe or primer which is attached to a detectable label moiety. Furthermore, the invention includes a vector for transfecting a cell to express a heterologous protein, comprising a DNA segment encoding a gp-Fy protein or a characteristic fragment thereof conjugated to a promoter. Preferably, the vector comprises a DNA segment which encodes human gp-Fyα or gp-Fyβ protein or a characteristic fragment of either protein. A highly preferred DNA segment comprises the sequence designated SEQ ID NO:28. Moreover, the invention includes a transgenic animal modified to express a gp-Fy protein or a characteristic fragment thereof encoded by a heterologous DNA segment. Preferably, the animal is capable of expressing a heterologous DNA segment comprising the nucleic acid sequence designated SEQ ID NO:28. Also preferred is that the transgenic animal is a transgenic mouse. The invention also includes a method for making a transgenically modified animal, such as a transgenic mouse. The method comprises transfecting cells of an animal with a vector comprising a DNA segment encoding a gp-Fy protein or a characteristic fragment thereof conjugated to a promoter, to provide an animal capable of expressing a protein product encoded by said DNA segment. Again, a highly preferred DNA segment comprises a nucleic acid sequence designated SEQ ID NO:28. Relatedly, the invention includes a method for making a cell which expresses a heterologous protein, comprising transfecting a cell with a vector comprising a DNA segment encoding a gp-Fy protein or a characteristic fragment thereof conjugated to a promoter.

These and other advantages of the present invention will be appreciated from the detailed description and examples which are set forth herein. The detailed description and examples enhance the understanding of the invention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention have been chosen for purposes of illustration and description, but are not intended in any way to restrict the scope of the invention. The preferred embodiments of certain aspects of the invention are shown in the accompanying drawings, wherein:

Figure 1 is a schematic representation and partial restriction map of two long gp-Fy protein cDNA clones.

Figure 2 is a schematic representation of the nucleotide and amino acid sequences of the combined Fy^b71-81 cDNA clones encoding gp-Fy protein.

Figure 3 A is a hydropathy plot of the gp-Fy protein sequence; Figure 3B is a proposed model for the membrane orientation of the gp-Fy protein.

Figure 4 is a Northern blot analysis using either the Fy^b71 or Fy^b81 insert as a probe. Figure 5 is a Southern blot analysis using either the Fy^b71 or Fy^b81 insert as a probe. Figure 6 is a Northern blot analysis of poly(A)⁺ RNA obtained from human tissues probed with the insert of Fy^b81 clone.

Figure 7 is a schematic representation of the nucleotide sequence of the promoter region and the cDNA encoding gp-Fy protein.

Figure 8 A is a Northern blot analysis of the extended amplification product of non-spliced gp-FY mRNA; Figure 8B is a Northern blot analysis of the extended amplification product of spliced gp-Fy mRNA.

Figure 9 is a Northern blot analysis of the amplification products of spliced and non-spliced gp-Fy mRNAs synthesized in non-erythroid tissues.

Figure 10A is a Northern blot analysis of a quantitative amplification of spliced and non-spliced gp-Fy mRNAs; Figure 1 OB is a linear regression plot for the quantitation of non-spliced gp-Fy mRNA; Figure IOC is a linear regression plot for the quantitation of spliced gp-Fy mRNA.

Figure 1 1 is a bar graph presenting the relative amounts of the mRNAs produced in the tissues, with an inset showing a Northern blot analysis of a quantitative amplification of spliced gp-Fy mRNAs from erythroid and non-erythroid tissues.

Figure 12A is graph of a flow cytometric scan analysis of anti-Fy6 binding by expressed gp-Fy protein; Figure 12B is a Scatchard plot of the affinity of expressed gp-Fy protein for iodinated IL-8; Figure 12C is graph of a flow cytometric scan analysis of anti-Fy6 binding by expressed gp-Fy protein; Figure 12D is a Scatchard plot of the affinity of expressed gp-Fy protein for iodinated IL-8.

Figure 13 A and 13B together constitute a schematic representation of the nucleotide sequence of the genomic DNA fragment containing FY*B coding sequence used in the construction of transgenic mice.

Figure 14 shows an analysis of the PCR amplification products of genomic DNA from transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

Duffy antigens are multimeric red cell membrane proteins comprising several different subunits. The major subunit of the Duffy complex is a highly hydrophobic intramembrane glycoprotein designated "gp-Fy" (formerly "gpD"). The gp-Fy protein comprises more than 330 amino acid residues, and has a molecular mass (IvL) of about 35,000 daltons (~35kDa).

This protein is the product of the Duffy blood group gene designated FY, occurring as a single gene per haploid genome. The gp-Fy protein presents the antigenic determinants defined by anti-Fy^a, anti-Fy\ and anti-Fy6 antibodies. Hydropathy analysis predicts that the protein would have nine putative transmembrane α-helices, with an N-terminal exocellular domain of 64 amino acids and an intracellular C-terminal domain of 23 amino acids.

The characterization of this novel protein at the molecular level is crucial to determining its function on the red cell membrane, to understanding the parasite-erythrocyte recognition process, and to resolving the molecular mechanism of parasite invasion. The present invention relates to the isolation, sequence analysis and tissue expression of mRNA encoding gp-Fy protein. The present invention relates, in general, to a nucleic acid, e.g., DNA or RNA, encoding all, or a characteristic fragment, of a Duffy gp-Fy protein. When referring to a peptide encoded by a nucleic acid, the term "characteristic fragment" means a peptide molecule comprising at least five (or six) amino acid residues of a Duffy protein. A characteristic fragment of a nucleic acid according the invention, therefore, can comprise as few as 15 (or 18) nucleotides encoding such a characteristic portion of the gp-Fy protein. One of ordinary skill in the art, given the present disclosure, could easily identify and clone analogous genes in such other species without undue experimentation.

In one embodiment, the invention relates to a DNA segment comprising the entire Duffy FFgene, and encoding a gp-Fy protein. One highly preferred DNA is designated

SEQ ID NO: 1 (see Figure 2). Other preferred DNA segments encode the entire amino acid sequence of the gp-Fyα protein (SEQ ID NO: 2) or the entire amino acid sequence of the gp-Fyβ protein (SEQ ID NO:3). The DNA segment can be genomic DNA or cDNA. DNA segments to which this invention relates also include those encoding substantially the same gp-Fy proteins as those defined as SEQ ID NO:2 or SEQ ID NO:3, which include, for example, allelic forms of the given amino acid sequences and alternatively spliced products. Of course, given the known degeneracy of the genetic code, the DNA sequences of the invention include those which substitute other nucleotides in appropriate positions to encode the defined gp-Fy proteins. In another embodiment, the invention includes a messenger RNA (mRNA) molecule encoding a gp-Fy protein. In this embodiment, the invention takes advantages of the highly unusual transcriptional characteristics of the FY gene, in that both non-spliced and spliced mRNAs are naturally expressed. Thus, the invention includes a non-spliced mRNA encoding a gp-Fy protein having the sequence designated SEQ ID NO:2. Also, the invention includes a spliced mRNA encoding a gp-Fy protein having the sequence designated SEQ ID NO:3. This embodiment further includes naturally occurring variants of these mRNAs, including allelic forms, as well as mRNAs encoding gp-Fy proteins within the constraints of the degeneracy of the genetic code.

The invention further relates to a gp-Fy protein with the sequence designated SEQ ID NO: 2 or SEQ ID NO:3, an allelic variation thereof, or a chimeric protein thereof.

The gp-Fy protein can be obtained according to methods known in the art, including isolated natural protein, recombinantly produced protein, or synthetic protein. Preferably, the protein is undenatured gp-Fy protein. The present invention also relates to isolated natural, recombinantly-produced, and synthetic characteristic fragments of a gp-Fy protein.

Moreover, the invention includes a recombinant DNA molecule comprising a vector and a DNA segment encoding a gp-Fy protein having the sequence designated SEQ ID NO:2 or SEQ ID NO:3, or a characteristic fragment thereof. Using methodology well known in the art, recombinant DNA molecules of the present invention can be constructed, and numerous vectors, including eukaryotic and prokaryotic vectors are commercially (and otherwise) available to the artisan. The DNA segment encoding the gp-Fy protein or the characteristic fragment thereof can be present in the vector operably linked to regulatory elements, including, for example, a promoter.

The invention further includes host cells comprising the above-described recombinant DNA molecule. The recombinant DNA molecule may be stably transformed, stably transfected, or transiently transfected into the host cells or infected into the host cells by a live attenuated virus. The host cells can be, for example, prokaryotic cells such as Escherichia coli, Staphylococcus aureus, or eukaryotic cells such as a yeast, e.g., Saccharomyces cerevisiae, or cultured cells from multicellular organisms, e.g., Chinese hamster ovary cells (CHO) or Cos cells.

Applicants have isolated four cDNA clones that encode gp-Fy protein, the major subunit of the Duffy blood group antigenic system. From these four cDNA clones, the nucleotide sequence of a structural gene encoding gp-Fy protein has been determined. The nucleotide sequence is illustrated in Figure 2 and is designated SEQ ID NO: l. Due to the degeneracy of the genetic code and other phenomena, e.g., allelism, there may exist other natural DNA sequences that encode gp-Fy protein. The present invention, therefore, extends to such other naturally occurring DNA sequences, as well as to synthetic DNA sequences having the same sequence as SEQ ID NO: 1 and such other natural DNA sequences. The synthesis of DNA can be by any conventional means.

The clones that have been characterized provide the elements to establish: (i) the structural components of FY genes, (ii) the biosynthesis and expression of gp-Fy protein in human bone marrow and other tissues, (iii) the structure/function relationships exhibited by this red cell membrane protein that might also exist in other cell types and may imply function as a chemokine receptor, and (iv) the role of gp-Fy protein as the receptor for Plasmodium merozoite invasion.

Figure 2 also shows the amino acid sequence of one of two distinct gp-Fy proteins encoded by the human FY gene. This protein is produced through a non-spliced mRNA, and is designated gp-Fyα (SEQ ID NO:2), having the sequence given below:

Met Ala Ser Ser Gly Tyr Val Leu Gin Ala Glu Leu Ser Pro Ser

5 10 15

Thr Glu Asn Ser Ser Gin Leu Asp Phe Glu Asp Val Trp Asn Ser

20 25 30 Ser Tyr Gly Val Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp Ala

35 40 45

Asn Leu Glu Ala Ala Ala Pro Cys Asn Ser Cys Asn Leu Leu Asp

50 55 60

Asp Ser Ala Leu Pro Phe Phe lie Leu Thr Ser Val Leu Gly lie

65 70 75

Leu Ala Ser Ser Thr Val Leu Phe Met Leu Phe Arg Pro Leu Phe

80 85 90

Arg Trp Gin Leu Cys Pro Gly Trp Pro Val Leu Ala Gin Leu Ala

95 100 105 Val Gly Ser Ala Leu Phe Ser He Val Val Pro Val Leu Ala Pro

110 115 120

Gly Leu Gly Ser Thr Arg Ser Ser Ala Leu Cys Ser Leu Gly Tyr

125 130 135

Cys Val Trp Tyr Gly Ser Ala Phe Ala Gin Ala Leu Leu Leu Gly 140 145 150

Cys Asn Ala Ser Leu Gly Asn Arg Leu Gly Ala Gly Gin Val Pro

155 160 165

Gly Leu Thr Leu Gly Leu Thr Val Gly He Trp Gly Val Ala Ala

170 175 180 Leu Leu Thr Leu Pro Val Thr Leu Ala Ser Gly Ala Ser Gly Gly

185 190 195

Leu Cys Thr Leu He Tyr Ser Thr Lys Leu Lys Ala Leu Gin Ala

200 205 210 Thr Asn Thr Val Ala Cys Leu Ala He Phe Val Leu Leu Pro Leu

215 220 225

Gly Leu Phe Gly Ala Lys Gly Leu Lys Lys Ala Leu Gly Met Gly

230 235 240 Phe Gly Pro Trp Met Asn He Leu Trp Ala Trp Phe He Phe Trp

245 250 255

Trp Pro Asn Gly Val Val Leu Gly Leu Asp Phe Leu Val Arg Ser

260 265 270

Lys Leu Leu Leu Leu Ser Thr Cys Leu Ala Gin Gin Ala Leu Asp 275 280 285

Leu Leu Leu Met Leu Ala Glu Ala Leu Ala He Leu Asn Cys Val

290 295 300

Ala Thr Pro Leu Leu Leu Ala Leu Phe Cys Lys Gin Ala Thr Arg

305 310 315 Thr Leu Leu Pro Ser Leu Pro Leu Pro Glu Gly Trp Ser Ser Asn

320 325 330

Leu Asp Thr Leu Gly Ser Lys Ser

335

Another gp-Fy protein, not shown explicitly in Figure 2, is produced from the same structural gene through a mRNA splicing mechanism, and is designated gp-Fyβ (SEQ ID NO: 3), having the sequence given below:

Met Gly Asn Cys Leu His Arg Ala Glu Leu Ser Pro Ser Thr Glu

5 10 15

Asn Ser Ser Gin Leu Asp Phe Glu Asp Val Trp Asn Ser Ser Tyr 20 25 30

Gly Val Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp Ala Asn Leu

35 40 45

Glu Ala Ala Ala Pro Cys Asn Ser Cys Asn Leu Leu Asp Asp Ser

50 55 60

Ala Leu Pro Phe Phe He Leu Thr Ser Val Leu Gly He Leu Ala

65 70 75

Ser Ser Thr Val Leu Phe Met Leu Phe Arg Pro Leu Phe Arg Trp

80 85 90 Gin Leu Cys Pro Gly Trp Pro Val Leu Ala Gin Leu Ala Val Gly

95 100 105

Ser Ala Leu Phe Ser He Val Val Pro Val Leu Ala Pro Gly Leu

110 115 120 Gly Ser Thr Arg Ser Ser Ala Leu Cys Ser Leu Gly Tyr Cys Val

125 130 135

Trp Tyr Gly Ser Ala Phe Ala Gin Ala Leu Leu Leu Gly Cys Asn

140 145 150

Ala Ser Leu Gly Asn Arg Leu Gly Ala Gly Gin Val Pro Gly Leu 155 160 165

Thr Leu Gly Leu Thr Val Gly He Trp Gly Val Ala Ala Leu Leu

170 175 180

Thr Leu Pro Val Thr Leu Ala Ser Gly Ala Ser Gly Gly Leu Cys

185 190 195 Thr Leu He Tyr Ser Thr Lys Leu Lys Ala Leu Gin Ala Thr Asn

200 205 210

Thr Val Ala Cys Leu Ala He Phe Val Leu Leu Pro Leu Gly Leu

215 220 225

Phe Gly Ala Lys Gly Leu Lys Lys Ala Leu Gly Met Gly Phe Gly 230 235 240

Pro Trp Met Asn He Leu Trp Ala Trp Phe He Phe Trp Trp Pro

245 250 255

Asn Gly Val Val Leu Gly Leu Asp Phe Leu Val Arg Ser Lys Leu

260 265 270 Leu Leu Leu Ser Thr Cys Leu Ala Gin Gin Ala Leu Asp Leu Leu

275 280 285

Leu Met Leu Ala Glu Ala Leu Ala He Leu Asn Cys Val Ala Thr

290 295 300

Pro Leu Leu Leu Ala Leu Phe Cys Lys Gin Ala Thr Arg Thr Leu 305 310 315

Leu Pro Ser Leu Pro Leu Pro Glu Gly Trp Ser Ser Asn Leu Asp

320 325 330

Thr Leu Gly Ser Lys Ser

335 As was the case with respect to the DNA sequence of the structural gene, the present invention extends to gp-Fy proteins isolated from natural sources, and to gp-Fy proteins prepared by chemical synthesis. The chemical synthesis can be by any conventional means. In Figure 2, amino acid residues are numbered on the left; nucleotide positions are numbered on the right. The positions of specific peptide fragments of the gp-Fy protein that match predicted amino acids, are shown by solid single lines. The two potential carbohydrate binding sites to asparagine residues, are marked by up arrows (t). Glycosylation at the third glycosylation site (asparagine at position 37) is unlikely to occur since the asparagine is followed by aspartic acid. See Marshall (1972). The sequence at the 5' end marked by double underline is the sequence used to primer extend the 5' end, and the likewise marked sequence at the 3 ' end is the consensus poly(A) addition sequence.

The cognate gene is present in Duffy-positive and -negative individuals, but the bone marrow of Duffy-negative individuals does not synthesize gp-Fy-specific mRNA. In adult kidney, spleen and fetal liver, the mRNA has the same size as gp-Fy mRNA; however, in brain, the mRNA is much larger.

U.S. Patent No. 5,101,017 to Rubenstein et al. discloses an antibody designated anti-Fy6 which specifically blocks penetration of P. vivax merozoites into human erythrocytes in vitro. We have established that the anti-Fy6 antibody binds to a domain in the N-terminal region of the gp-Fy protein. More interesting — and entirely unexpected — is that this same domain is involved in the binding interaction with the malarial parasite itself. Therefore, it is likely that the anti-Fy6 antibody and the malarial Duffy-binding protein share similar stereochemistry. These molecular features of the gp-Fy protein could not have been identified absent cloning and sequencing the 7 gene and the gp-Fy proteins, as described herein. Applicants have found that the following peptides specifically bind the anti-Fy6 antibody in an ELISA assay:

(1) AELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYD (SEQ ID NO:4); (2) MASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYD (SEQ ID NO:5); and

(3) MASSGYVLQAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDAN LEAAAPCHSCNLLDDSALPF (SEQ ID NO:6); The amino acid sequence AELSPSTENSSQLDFEDVWNSS (SEQ ID NO:7), likely contains the epitope for the Rubinstein antibody. Therefore, the invention also extends to peptides comprising the amino acid sequence SEQ ID NO: 7.

The following peptides have been found not to bind to the anti-Fy6 antibody in an ELISA assay:

(4) DFEDVWNSSYGVNDSFPDGDYD (SEQ ID NO: 17);

(5) ANLEAAAPCHSCNLLDDSALPF (SEQ ID NO: 18); and

(6) AELSPSTENSSQL (SEQ ID NO: 19).

Note that peptides (6) (SEQ ID NO: 19) and (4) (SEQ ID NO: 17) together correspond to the whole length of peptide (1) (SEQ ID NO:4). That peptide (1) (SEQ ID NO:4) binds the Rubinstein antibody, whereas peptides (4) (SEQ ID NO: 17) and (6) (SEQ ID NO: 19) do not, suggests that the junction between the C-terminal of peptide (6) (SEQ ID NO: 19) and the N-terminal of peptide (4) (SEQ ID NO: 17) is important for binding.

Accordingly, the gp-Fy protein of the invention is also useful to prepare monoclonal antibodies having the same specificity as the Rubinstein antibody. The procedure for preparing such antibodies is essentially the same procedure that Rubinstein et al. employed except that instead of immunizing mice against human red cells, immunization is against gp-Fy protein er se or a related peptide exhibiting similar immunogenicity.

Additionally, the N-terminal (exocellular) region of the gp-Fy protein has now been identified as being involved in the interaction of the malaria parasite with the red cell. The region of the gp-Fy protein which serves as the malarial binding site has been identified and synthetic peptides comprising this important region are described in U.S. application Serial No. 08/749,526, filed on November 15, 1 96, the entire disclosure of which is incorporated herein by reference. The peptides designated SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and

SEQ ID NO: 7, as well as peptides containing SEQ ID NO: 7, bind the parasite in vivo and, therefore, can be used as therapeutic inhibitors of erythrocyte binding and/or inhibition by malarial Plasmodium species. The present invention is, consequently, also directed to such inhibitory peptides as well as to a method of protecting a warm-blooded animal, especially, a human, against infection by P. vivax by administering to such animal an effective amount of such peptides. Proteins that are complementary to gp-Fy protein or the synthetic peptides of the invention, e.g., antibodies specific to gp-Fy, will block the natural receptor(s) and, consequently, will also have the therapeutic utilities outlined above. In the preparation of such complementary proteins, the use of gp-Fy protein or the synthetic peptides of the invention are of value.

Peptides corresponding to a portion of gp-Fy protein that contains the receptor also have been synthesized. Such peptides have therapeutic usefulness comparable to that of gp-Fy protein itself and, as is the case with gp-Fy protein, the synthetic peptides can be combined with physiologically acceptable diluents to yield a composition effective against malarial infection, or a therapeutic agent useful to regulate essential erythrocyte, neural and renal functions. gp-Fy protein and synthetic peptides corresponding to a portion of gp-Fy protein also have utility in the production of therapeutics, e.g., antibodies, complementary peptides, and drugs modeled on the tertiary structure of the gp-Fy protein or synthetic peptides, which are also of therapeutic value in the treatment of malaria and in the regulation of essential erythrocyte, neural and renal functions.

The cloning and sequencing of the human and rhesus Duffy genes further enables other applications of the invention. For example, transgenic animals, preferably mammals such as mice, rats, goats, sheep, pigs, cats, dogs, rabbits, horses, etc., can be constructed using conventional transgenic techniques to express heterologous Duffy protein. Various such techniques are known, and certain of these techniques can yield heritability of the transgene. See, e.g., Pinkert et al. (1995) for an overview of these techniques, and the documents cited there for greater detail. For example, the invention includes a transgenic mammal, transformed by integration of an expressible transgene comprising a heterologous Duffy-related nucleic acid sequence into the genome of the mammal. Preferably, the transgene is heritable. Such a transgenic animal can then be used as an in vivo model for malarial infection in the species from which the Duffy gene is derived. Of particular importance, of course, is the development of animal models for human malarial infection. Such transgenic animal models would express a Duffy protein normally expressed in erythrocytes of humans, and would be capable of being bound by and infected by Plasmodium species capable of infecting humans, even if the normal host animal is not susceptible to those species. In one exemplary approach a transgenic test animal is administered a putative antimalarial substance and inoculated using a malarial organism which would normally measurably bind erythrocytes and/or produce measurable infection in the modified animal. Following a time sufficient to produce a measurable effect in an otherwise untreated animal, binding to and/or infection of erythrocytes is measured. A lower than normal rate of binding or infection indicates that the putative antimalarial inhibits the organism's capacity for binding and/or infection in vivo.

Thus, variants of the peptide of the invention (or other unrelated substances) can be tested as prospective therapeutic antimalarials in these model animals, avoiding deliberate exposure of humans to the malarial organism. Other uses for such modified organisms will be evident to the skilled artisan. gp-Fy protein shows significant homology to interleukin-8 (IL-8) receptors on rabbit and human erythrocytes. This is consistent with a recent report suggesting that the Duffy blood group antigen and the erythrocyte chemokine receptor are the same protein (Horuk et al. 1993). The erythrocyte receptor apparently differs from the IL-8 receptors, LL-BRA and

IL-ORB on neutrophils. The erythrocyte receptor binds a family of chemotactic and proinflammatory soluble peptides, including IL-8, melanoma growth stimulatory activity (MGSA), monocyte chemotactic protein 1 (MCP-1), and regulated on activation, normal T expressed and secreted (RANTES) protein. Administration of gp-Fy protein (or the synthetic peptides of the invention) interferes with the normal binding of these proteins to the erythrocyte receptor and, consequently, is useful to regulate the physiological effects of the secretion of these proteins. For example, it has been postulated that the erythrocyte receptor acts as a scavenger for certain inflammatory mediators, including IL-8. Administration of gp-Fy protein (or the synthetic peptides of the invention), therefore, would be expected to enhance scavenging of IL-8, thereby, lessening any IL-8 induced inflammation. For this purpose, the peptide of the invention, as described above, is suitable as a therapeutic agent. gp-Fy protein also shows significant homology to a human hippocampus cDNA clone HHCMF86 and, therefore, it is highly probable that gp-Fy protein or a homologous protein is present as a neuropeptide receptor in brain. Moreover, gp-Fy is present in all red cell progenitors and the possibility exists that it may function as a receptor for cell proliferation and/or differentiation. In human kidney, gp-Fy protein cDNA identifies a mRNA of the same size as that localized in bone marrow. Since the kidney is not, and has no potential to become, an erythropoietic organ, it is possible that this putative chemoattractant receptor has essential e.g., regulatory, renal functions. The therapeutic agent of the invention will, accordingly, also find use in the regulation of any or all of these neural, hematopoietic, or renal functions. The following examples are provided to assist in a further understanding of the invention. The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope thereof.

Example 1: Partial Amino Acid Sequence Analysis of gp-Fy protein

Red cells (Fy(a-b+)) were washed three times in cold phosphate-buffered saline (PBS) (pH 7.4), resuspended in the same solution, and mixed continuously overnight at 4°C with the

Rubinstein antibody at a concentration of 10 μg/mL of packed red cells. (This concentration, determined with radioiodinated antibody, exceeds the concentration required to saturate Duffy antigen sites.) Unbound antibody was removed by washing the red cells with cold PBS. Red cell ghosts were prepared by hypotonic lysis with 20 volumes of cold 5 mM sodium phosphate buffer (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride and 100 kallikrein inactivating units/mL TRASYLOL (aprotinin). Then the ghosts were washed exhaustively until they were light pink in color. Ghosts were centrifuged for 30 min at 43,000 x g; the supernatant was decanted, and the pellet was resuspended in 50 mM Hepes NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/mL TRASYLOL, and frozen at -20°C.

Frozen ghosts were later thawed and centrifuged for 30 min at 43,000 x g. The pellet was resuspended in 50 mM Hepes-NaOH, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/mL TRASYLOL to three times the initial volume of packed red cells. TRITON X-100 (peroxide free) detergent was added to a final concentration of 1%, and the solution was mixed gently for 1 h at room temperature. Shells were removed by centrifugation for 30 min at 43,000 x g. The supernatant was concentrated 4-fold in an Amicon concentrator using a PM Y10 filter (Amicon Corp.) under nitrogen pressure.

A 0.1 volume of PBS solution, 10 times the normal concentration, was added to the detergent extract. The detergent extract was then incubated with SEPHAROSE 4B beads coupled to anti-mouse IgG for 1 h at room temperature. The ratio of beads to detergent extract was 1 : 100 (v/v). The anti-mouse IgG-SEPHAROSE beads were removed by centrifugation, and washed in a solution containing PBS and 0.5% TRITON X-100 at a 1:20 (v/v) ratio of beads to washing solution. The washings were done at room temperature and repeated three times. Elution was done by incubating the beads in a solution containing 62.5 mM Tris-HCl (pH 6.8), 0.5% sodium dodecyl sulfate (SDS) at a 1 :2 (v/v) ratio of beads to eluant. The incubation was at 65 °C for 10 min and repeated three times. The eluted material was concentrated in an Amicon concentrator with PM Y10 filter (Amicon Corp.) under nitrogen pressure.

Polyacrylamide gel electrophoresis (PAGE) in the presence of 0.1% SDS was performed according to Laemmli (1970), with the following modifications: the acrylamide concentration was 10%, polymerization was done overnight to destroy oxidizing reagents, and 0.1 mM thioglycolate was added in the upper chamber. To the concentrated solution of affinity-purified material, the following chemicals were added: urea to 4 M, SDS to 2%, and β-mercapoethanol to 5%. After electrophoresis, the gels were fixed for 30 min in 10% isoamyl alcohol and 5% acetic acid and stained with 0.002%> Coomassie Blue R-250 until marker protein bands were seen. Regions that corresponded between the 36-46 kDa region and above the 96 kDa region were excised, destained with several changes of 5%> acetic acid, and washed with distilled water. Gel pieces were stored at -20° C or used immediately. Gel pieces, cut into 4 x 4 mm cubes, were delivered into the elution chamber of an ELUTRAP apparatus (Schleicher and Schuell) and eluted overnight in 50 mM ammonium bicarbonate, 0.1% SDS solution at 100 volts (constant). Fresh 50 mM ammonium bicarbonate, 0.1 %> SDS solution was added, and electroelution was continued for an additional 6-8 h. Eluted material was concentrated by CENTRICON microconcentrator (Amicon Corp.). The purified protein was alkylated and cleaved with cyanogen bromide (CNBr) as follows: The purified protein was precipitated with cold acetone in the presence of 1 mM HC1 at -20°C for 2 h. The precipitate was washed with 100% cold acetone, evaporated to dryness at room temperature, and was dissolved in 0.1 M Tris-HCl (pH 8.0) plus 0.5% SDS. Solid DTT was added to the solution to make a final DTT concentration of 10 mg/mL, and the solution was reduced for 2 h at 85 °C. One-tenth volume of 2.68 M iodoacetic acetamide was added to the solution and the tube was flushed with nitrogen, and incubated for 30 minutes at

- I I room temperature in the dark. After incubation, solid DTT was added to 10 mg/mL and dialyzed overnight against 0.1 M Tris-HCl (pH 8.0) plus 0.5%> SDS. Protein was precipitated with acetone as above and air dried. The precipitate was dissolved in 96 μL of 70%> formic acid and 4 μL of 1 M CNBr solution in 70%> formic acid, and incubated at room temperature for 48 h in the dark. Acid was evaporated to dryness and the pellet was washed with water and evaporated to dryness several times. The digested protein was subjected to either high performance liquid chromatography (HPLC) or polyacrylamide gel fractionation.

A peptide designated Pe 1, having the sequence PLFRWQLCPGWPVLAQ (SEQ ID NO:20), was obtained by sequencing the non-fractionated CNBr digest using the O-phthalaldehyde (OP A) blocking reagent (see Brauer et al. 1983). Another peptide, Pe 5 having the sequence MMILWAWFIFWWPNGVVLGLDFLV (SEQ ID NO:21), was the partial sequence of the only fragment (~4 kDa) that separated very well from the CNBr digest run of the three layer SDS-PAGE system (see Shagger et al. 1987). After the run, the peptide fragment was electroblotted onto PROBLOTT (Applied Biosystems) and sequenced (see LeGendre 1989). Another aliquot was digested with pepsin (50/1 ratio) at 37°C overnight and the fragments were separated by reverse-phase HPLC using a Vydac C-18 column. Peptides Pe 2 having the sequence FSIVV (SEQ ID NO:22), Pe 3 having the sequence FAQAL (SEQ ID NO: 23), Pe 4 having the sequence VGI (SEQ ID NO: 24), and Pe 6 having the sequence PSLPKGW (SEQ ID NO:25), eluted as single peaks from a reverse-phase HPLC column and were the few pepsin peptides yielded by reverse-phase HPLC. Sequencing was accomplished using an Applied Biosystems Protein/Peptide Sequencer, Model 470 or 477, according to the manufacturer's recommendations. Pepsin digestions at 100/1 ratio, at 4°C for 30 or 60 min, did not generate larger peptides.

Example 2: Primer Design and Polymerase Chain Reaction (PCR) Pe 5 (SEQ ID NO:21) was the most promising for generating a probe for the selection of gp-Fy protein clones. Peptides Pe 2 (SEQ ID NO:22), Pe 3 (SEQ ID NO:23), Pe 4 (SEQ ID NO:24) and Pe 6 (SEQ ID NO:25) were too short for PCR amplification, while the peptide Pe 1 (SEQ ID NO:20) was larger but it had three ill-defined residues.

The nucleotide sequence of the primers (23mer each) was deduced from the N-terminal and C-terminal amino acid sequences of Pe 5 (SEQ ID NO:21) (see Fig. 2). Since the Pe 5 (SEQ ID NO:21) peptide was produced by CNBr cleavage, a methionine was included at the N-terminus to increase the length of the peptide to 24 residues. Bases were chosen according to the codon preference described by Lathe (1985), and deoxyinosine (I) was incorporated at the position where degeneracy exceeded more than three-fold except towards the 3 ' end.

Two generated primers for amino acids at the N-terminal (primer A) and amino acids at the C-terminal (primer B) were chemically synthesized and used to amplify the coding sequence of Pe 5 (SEQ ID NO:21) peptide from pooled human bone marrow mRNA of Fy(a-b+) individuals. Primer A (sense) was specific for residues 245 to 252 (see Fig. 2) and consisted of 12-fold degeneracy 5 '-ATGAAYATHYTITGGGCITGGTT (where

I=deoxyinosine; Y=C or T; and H=C, T or A) (SEQ ID NO: 8). Primer B (antisense) was specific for residues 261-268 (see Fig. 2) and consisted of 32-fold degeneracy 5'-ACIAGRAARTCIAGICCIARNAC (where I=deoxyinosine; R=A or G; and N=G, A, T or C) (SEQ ID NO:9). First strand cDNA was synthesized from Fy(a-b+) phenotype mRNA using the preamplification kit from BRL (Bethesda, Maryland) and oligo-dT as primer. For enzymatic amplification, cDNA, Primer A, Primer B and Taq polymerase (Stratagene) were incubated in a Perkin-Elmer thermal DNA cycler. The amplification product of expected size (72 bp) was subcloned in pBluescript-SK vector (Stratagene). The deduced amino acid sequence of the insert, matched the sequence of Pe 5 (SEQ ID NO:21) peptide (see Fig. 2). From the sequence WFIFWWPH of peptide Pe 5 (i.e., residues 7-14 of SEQ ID NO:21), the oligonucleotide TGGTTTATTTTCTGGTGGCCTCAT (SEQ ID NO: 10) was chemically synthesized, ³ P-labeled at the 5 ' end with T4 polynucleotide kinase (New England Biolabs), and used as a probe to screen a human bone marrow cDNA library (see Example 4 below). This 24mer oligonucleotide probe having codon usage for amino acids 251 to 258, successfully identified true gp-Fy protein cDNA clones.

Example 3: Human mRNA and DNA isolation

Poly(A)⁺ RNA was isolated as follows: Human bone marrow aspirates were washed and cells were lysed in a solution of 5% β-mercaptoethanol plus 6 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 50 mM ethylenediaminetetraacetic acid (EDTA), so that the final guanidine concentration became 5 M. The solution was passed through a 25 G hypodermic needle to shear DNA, and Sarkosyl was added to 2%>. The solution was spun on a 5.7 M CsCl, 50 mM EDTA (pH 7.0) cushion in a SW 41 rotor at 32K rpm at 20°C for 18 h. The pellet was washed with 6 M guanidine hydrochloride and finally with absolute ethanol cooled in dry ice. The pellet was resuspended in diethylpyrocarbonate-treated water, adjusted to 0.3 M sodium acetate (pH 5.2) and ethanol-precipitated. The pellet was resuspended in proteinase K digestion buffer and digested for 2 h at 37° C, phenol-chloroform-extracted and ethanol-precipitated. The pellet was then dissolved in water, adjusted to 1 x DNase digestion buffer and treated with RNase-free DNase (BRL). Poly(A)⁺ RNA was isolated with mRNA isolation kit FAST TRACK from Invitrogen (San Diego, CA) according to the manufacturer's protocol. mRNAs from white adult liver, spleen, kidney, brain and fetal liver, as well as K562 erythroleukemia cells were obtained from Clontech Laboratories.

DNA was obtained from peripheral blood white cells of the four Duffy phenotypes by lysing the red blood cells in a whole blood unit with 0.83%> NH₄C1, pH 7.4, followed by the standard DNA extraction procedure described by Maniatis et al. (1982).

Example 4: Construction and Screening of a cDNA Human Bone Marrow Library

A mixture of mRNA pooled from several Fy(a-b+) individuals, the BRL SUPERSCRIPT CHOICE System and oligo-dT as a primer, were used to prepare cDNA. The cDNA was ligated into λZAP II vector and packaged with GIGAPACK GOLD (Stratagene) extract. About 1.9 x 10⁶ unamplified cDNA clones were screened with the

³²P-labeled probe described above in Example 2. cDNA inserts into pBluescript were isolated by the plasmid rescue method according to manufacturer's protocol. Both DNA strands were sequenced using vector primers; and primers were designed from the sequenced regions of the transcript.

Example 5: Nucleotide Sequence of the gp-Fy Protein cDNA Clones

The non-amplified human bone marrow cDNA library, constructed from pooled mRNA of Fy(a-b+) individuals as described in Example 4, was screened with the 24mer probe (SEQ ID NO: 10) synthesized in Example 2, above. Of the 1.9 x 10⁶ recombinant λZAP II phage, four positive clones were selected and sequenced. Each of these clones had sequence overlaps with at least one of the other clones, but none extended the full length of gp-Fy cDNA. Fy^b81 (1085 bp), which was the only clone that included the ultimate 5' end, and Fy^b71 (1083 bp), which extended from nucleotide position 185 to the poly(A)⁺ tail, were the two longest clones. (The "b" in the clone designations stands for Duffy phenotype Fy(a-b+).) Fy^b31 (989 bp) and Fy^b82 (726 bp) extended from nucleotide positions 275 and 527, respectively, to the poly(A) tail. Combination of Fy^b81 with any of the other three clones generated the full-length cDNA of gp-Fy protein. Figure 1 provides perspective on how the two longest clones (Fy^b71 and Fy^b81) overlap, as well as the combination (Fy^b71-81) of the clones. The joined Fy^b71-81 clone predicted an open reading frame (ORF) that started at position 176 and stopped at position 1 192, encoding a polypeptide of 339 amino acid residues (Fig. 2). A GENBANK sequence search (release 77) at the NCBI using the BLAST network service yielded a significant protein sequence homology to human and rabbit interleukin-8 receptors and quasi-total nucleotide sequence homology with a human hippocampus cDNA clone HHCMF86 (see below). The extended product of an antisense primer (from position 57 to 80, Fig. 2), yielded a sequence of 80 nucleotides which matched exactly with the predicted size at the 5' end of the Fy^b81 clone (see Example 9, and Figure 7). At positions 176-178, the initiation codon is not embedded within a sequence context most frequently associated with mammalian translation initiation. See Kozak (1987). We concluded that this is the true initiation codon since: (i) it is the only ATG codon at the 5 ' end; and (ii) from the first methionine residue, the polypeptide encoded by the combined clones has the same molecular mass as that of de-glycosylated gp-Fy protein. See Chaudhuri et al. (1993). At the 3' end, clone Fy^b71-81 included the consensus poly(A) addition signal AATTAAA (nt 1216-1222) (Fig. 2). Both the Fy^b71 and Fy^b81 clones had a perfect nucleotide sequence match except at the 5' ends, where several base substitutions yielded six different amino acid predictions. These discrepancies were not a sequencing error, since both DNA strands were sequenced several times. Rather, the differences reflected natural protein heterogeneity, since the cDNA library was constructed from the mRNA of several Fy(a-b+) individuals. We have identified the source of this heterogeneity as arising in a differential gene splicing mechanism, as described hereinbelow. To establish that Fy^b71-81 had a coding sequence specific to gp-Fy protein, the translated sequence was compared with the partial amino acid sequence data obtained from the six peptides Pe 1-Pe 6 identified in Example 1, above. Portions of the predicted amino acid sequence matched with Pe 1 peptide (SEQ ID NO: 20) sequenced by the OP A reagent; with four peptides isolated by reverse-phase HPLC, i.e., Pe 2 (SEQ ID NO:22), Pe 3

(SEQ ID NO:23), Pe 4 (SEQ ID NO:24) and Pe 6 (SEQ ID NO:25); and with the Pe 5 peptide (SEQ ID NO:21) isolated by SDS-PAGE. However, two of a total of 62 residues did not match. Thus, the residues at positions 92 and 327 were tryptophan by codon sequence analysis, but they were isoleucine and arginine, respectively, by amino acid sequence determination. Since tryptophan is a very unstable residue, the discrepancies may be a technical problem in amino acid sequence analysis. On the other hand, they may be due to the heterogeneity of the gp-Fy protein.

Additional evidence that Fy^b71-81 encoded gp-Fy protein was provided by Northern blot and ELISA analysis. Fy^b81 did not detect any mRNA in Duffy-negative individuals, but it detected a -1.27 kilobase (kb) transcript representing the full-length of gp-Fy mRNA in

Duffy-positive individuals (Fig. 4).

Moreover, anti-Fy6 antibody reacted with a 35mer synthetic peptide (residues 9 to 44, see Fig. 2), predicted by the Fy^b71-81 clone (not shown). The absence of gp-Fy-protein-specific mRNA in Fy(a-b-) phenotypes (see below) and the reaction of anti-Fy6 with a peptide derived from a gp-Fy cDNA, are strong indications that the clones that were isolated are true Duffy clones.

Example 6: Amino Acid Sequence and Membrane Topology of gp-Fy Protein

The predicted translation product of the Fy^b71-81 clone is an acidic protein of isoelectric point 5.65 and molecular mass M,. 35,733. The amino terminal region of the protein contains only two potential canonical sequences for N-glycosylation to asparagine residues. See Marshall (1972). This agrees with previous investigations indicating that N-glycosidase F digestion increases gp-Fy mobility on SDS-PAGE and with the chemical detection of N-acetylglucosamine. See Chaudhuri and Pogo (1995); Tanner et al. (1988); Wasniowaska et al. (1993). Predictions of transmembrane helix locations from sequence data using the hydropathy map of Engelman et al. (1986) and a scanning window of 20 residues show that the bulk of the protein is embedded in the membrane (Figs. 3 A and 3B). Nine transmembrane α-helices, a hydrophilic domain of 66 residues at the N-terminus, a hydrophilic domain of 25 residues at the C terminus, and short protruding hydrophilic connecting segments, were predicted. The pair of helices, D and E, is so closely spaced that they may be arranged as coupled antiparallel helices. A schematic illustration of gp-Fy protein topology, is shown in Figure 3B.

The charge-difference rule proposed by Hartmann et al. (1989), predicts that the N terminus is on the exocellular side and the C terminus of the protein is on the cytoplasmic side of the membrane. The N-terminal prediction is validated by the finding of the two potential

N-glycosylation sites on the N terminus. Moreover, the reaction of anti-Fy6 with a synthetic peptide deduced from this domain, establishes its exocellular location experimentally since the antibody binds to erythrocytes. The signal-anchor sequence for membrane insertion probably lies in the first transmembrane α-helix that follows the N-terminal domain. See Wessels et al. (1988); Blobel (1980). From there on, the protein is deeply buried in the membrane and exits at residue 314 on the cytoplasmic side of the membrane (Fig. 3B). The topological predictions of helices, hydrophilic connecting segments and the location of the C-terminal fragment, should be substantiated by direct biochemical and immunochemical analysis.

Duffy gp-Fy protein is deeply buried in the membrane like the membrane associated fragment of Band 3 (see Jay 1986), the human blood group Rh polypeptide (see Cherif-Zahar et al. 1990; Avent et al. 1990), bacteriorhodopsin (see Carlton et al. 1985), and lipophilin (see Stoffel et al. 1983). The significant homology of gp-Fy protein with interleukin-8 receptors is very intriguing. See Holmes et al. (1991) and Murphy et al. (1991). If gp-Fy protein bind chemokines and has the ability to activate a signal transduction cascade, this gives rise to gp-Fy protein as a new class of pro-inflammatory mediators. Thus, gp-Fy protein is not present in white blood cells, since a rabbit polyclonal antibody (anti-gp-Fy) against purified and denatured gp-Fy protein that reacts with erythrocytes and their precursors does not react with any white blood cells (unpublished results). Example 7: RNA Blot Analysis (Northern)

Poly(A)⁺ RNAs were run on formaldehyde/agarose gel and transferred onto HYBOND N+ nylon membranes (Amersham Corp.). They were hybridized in QUICKHYB (Stratagene) and washed according to the manufacturer's instructions. On Northern blot analysis, Fy^b71 or Fy^b81 clone detected a -1.27 kb mRNA species in the bone marrow of the three Duffy-positive phenotypes but not in individuals of Fy(a-b-) phenotype (Fig. 4). The absence of gp-Fy mRNA was consistent with the absence of gp-Fy protein in Duffy-negative individuals. Anti-gp-Fy antibody did not react with any red cell membrane protein of Fy(a-b-) erythrocytes (not shown). Duffy-negative individuals did not express gp-Fy protein, because they do not synthesize Duffy-specific mRNA.

In Figure 4, lane 1 contained 10 μg of Fy(a-b-) mRNA, lanes 2 and 3 contained 5 μg of Fy(a+b-) mRNA and Fy(a-b+) mRNA respectively, and lane 4 contained 2 μg of Fy(a+b+) mRNA. They were resolved on a 2% denaturing agarose gel, blotted, hybridized, and autoradiographed for 72 h at -80°C. RNA size markers shown: human 28S (5.1 kb) and 18S (2.0 kb) rRNA, and the 1.35 kb GIBCO BRL marker (Life Technologies), were used to calculate the size of gp-Fy mRNA. The actin probe at the bottom was used as a control of sample loading. RNA integrity was indicated by the presence of the two rRNA in the poly(A)⁺ fraction and the actin probe.

Example 8: DNA Blot Analysis (Southern) On Southern blot analysis Fy^b71 or Fy^b81 probe hybridized with DNA of

Duffy-positive and -negative individuals (Fig. 5). They identified a single band of 6.5 kb in BamHl, two bands of 12 kb and 2 kb in EcoRI, and two bands of 3.5 kb and 1.4 kb in Pstl digested DNA. These findings agree with the restriction map of the Fy^b71 and Fy^b81 clones and show a single copy gene. Determination of the structural differences among the genes of Duffy-positive and -negative individuals should clarify the mechanism of FY gene repression in

Duffy-negative individuals. A functional silencer element described in other systems may selectively repress transcription of FY gene in the erythrocytes of Fy(a-b-) individuals. See Li et al. (1993). The Duffy system is different from the ABO (Yamamoto et al. 1990) and Kell systems where mRNA has been found in individuals who do not express the blood group determinants. In Figure 5, each lane contained 10 μg of digested DNA; lanes 1-4 contained Fy(a-b-), lanes 5-8 contained Fy (a+b-), and lanes 9 to 12 contained Fy(a-b+) DNA. The enzyme digestions were as follows: lanes 1, 5 and 9 BamHl; lanes 2, 6 and 10 EcoRI; lanes 3, 7 and 11 Hinfi; and lanes 4, 8 and 12 Pstl. All restriction enzyme digestions were performed according to the conditions suggested by the supplier (New England Biolabs). Digested DNA was size-fractionated on 0.8% agarose gel and blotted as described for Northern analysis. Hybridization in QUICKHYB solution was carried out at 68 °C for 1 h according to the manufacturer's instructions. Gels were autoradiographed for seven days at -80°C. Sizes were calculated from the positions of the GIBCO BRL DNA markers.

Example 9: RNA Blot Analysis (Northern) in Non-Erythroid Tissues

In Figure 6, lanes 1, 3, 5 and 7 contained 2 μg of Fy(a-b+) bone marrow, fetal liver, adult spleen and erythroleukemia (K562) mRNAs, respectively. Lanes 2, 4 and 6 contained 7 μg of total brain, adult liver, and adult kidney mRNA, respectively. They were resolved on a 1.5%) denaturing agarose gel and autoradiographed for five days at -80°C. As indicated in Figure 6, a 1.27 kb mRNA species was found in adult spleen and kidney, as well as in fetal liver, but was not found in adult liver or K562 erythroleukemia cells. Hybridization with the β-globin probe showed a strong signal in bone marrow and fetal liver; it showed a weak signal in adult spleen, but no signal in adult liver, brain or kidney (not shown). The presence of gp-Fy mRNA in fetal liver was expected since the fetal liver is an erythropoietic organ. In human brain, a strong band of 8.5 kb and a faint band of 2.2 kb were detected.

This observation implies that a Duffy-related protein is expressed in brain tissue. This idea is supported by the quasi-total homology between Fy^b71-81 clone and a recently identified human hippocampus cDNA clone HHCMF86 (Adams MD et al. 1992). However, it is unlikely that the 8.5 kb brain mRNA codes for a Duffy protein with long 5 ' and 3 ' untranslated sequences. It is possible that the brain mRNA codes for a larger protein which has extensive homology with gp-Fy protein. The homologies of these mRNA species with gp-Fy-specific mRNA, remain to be demonstrated by sequence analysis; however, the findings strongly indicate that gp-Fy protein or a similar protein is produced in kidney, non-hemopoietic spleen cells, and probably in brain. Example 10: Spliced and non-spliced Duffy mRNAs in bone marrow

As described in Example 5, above, the mRNA of the major glycoprotein of the Duffy blood group system was cloned from a non-amplified, human bone marrow cDNA library. As noted, the two longest and overlapping clones were Fy^b71 of 1083 bp and Fy^b81 of 1085 bp. The combination of these clones generated the full-length cDNA of gp-Fy mRNA. The two clones (see Fig. 1) had a nucleotide sequence match, except at the 5' end, where base substitutions predicted six or eight different N-terminal amino acids in the products encoded by the two clones. This discrepancy was initially interpreted as protein heterogeneity because the cDNA library was constructed from pooled mRNA from several individuals. These two clones were subjected to further comparative analysis, to determine how the clones were related to one another. As described below, it has been found that the FY gene generates two distinct gene products by a differential splicing mechanism.

Human genomic DNA was isolated as explained in Example 3 above. The 5 ' upstream and 3 ' downstream regions, contiguous to the 5 ' and 3 ' untranslated flanking sequences of non-spliced gp-Fy mRNA, were cloned and sequenced. The 5 ' end was identified in a 1.4-kb

Pstl digested fragment hybridized with the 5' end of gp-Fy cDNA, and the 3 ' end was identified in a 1.9-kb EcoRI digested fragment hybridized with the 3' end of gp-Fy cDNA. The 5' upstream genomic DNA was cloned and sequenced using the method described in Chaudhuri et al. (1995). Figure 7 shows the structural organization of the coding and 5' upstream sequences of the Duffy gene. Figure 7 provides the nucleotide and amino acid sequences of the promoter region of FY. The portions of the sequence indicated by dashes and slashes, i.e., " — // — " are regions of the gene otherwise identified in Figure 2 above (SΕQ ID NO:l). Nucleotide positions are enumerated. The promoter region of FY was further analyzed. Consensus splicing sequences were identified at nt -277 (gt) and at nt +202 (ag) (each double underlined in Fig. 7). Moreover, a sequence of 21 bp was identified immediately upstream of the GT splicing sequence at nt -277. This sequence, having an open reading frame for 7 residues, encodes an amino acid sequence MGNCLHR (SΕQ ID NO:26) which is identical to the amino terminus of the peptide encoded by clone Fy^b71 (a single letter code is indicated below each codon in this

ORF in Fig. 7). By contrast, the amino terminal sequence of the peptide encoded by the Fy^b81 clone is MASSGYVLQ (SEQ ID NO:27). The Fy^b71 and Fy^b81 clones are otherwise identical, differing not at all in the region downstream from nt +203. These features indicate that the Fy^b71 clone was generated by the removal of an intron of 479 nts and splicing of an exon of 59 nts. Thus, Fy^b71 contains a short exon of 59 nts (exon 1) and a long exon of 1040 nts (exon 2), and as a result is 178 nts shorter than Fy^b81. Accordingly, it appears that the FY gene is capable of generating two different peptide products: one derived from spliced mRNA, carried in clone Fy^b71, and one derived from non-spliced mRNA, carried by clone Fy^b81. For convenience, the product encoded in the clone Fy^b81 is herein designated "gp-Fyα" (SEQ ID NO:2), and the product encoded in the clone Fy^b71 is designated herein "gp-Fyβ" (SEQ ID NO:3).

Other aspects of the promoter region of FY are also indicated in Figure 7. The 5' end (cap site) of gp-Fyα mRNA is indicated as +1, while negative numbers show upstream sequences. Bold uppercase letters within brackets show a 57 bp direct repeat. The open reading frames are indicated by a single-letter code below each codon. The NH₂-termini of gp-Fyα and gp-Fyβ are in bold lowercase and bold uppercase letters, respectively. Lowercase letters are the intron of the pre-gp-Fyβ mRNA. Regular uppercase letters denote the amino acid residues common to both proteins. The nucleotides at -3 nt and +4 nt relative to the ATG initiation codon are in bold and italicized letters. The potential cis-acting elements are underlined. Underlined arrows denote sense and antisense primers for PCR. The T → C mutation at nt -365 (GATA1 motif ) is shown in bold italics. The stop codon of the intron of what we have designated pre-gp-Fyβ mRNA is indicated (H<). The stop codon of gp-Fyα and gp-Fyβ mRNAs is also shown (#).

Verification of the 5' end of the transcripts was done by primer extension of bone marrow gp-Fyα and gp-Fyβ mRNAs. A ³²P-labeled 24mer antisense primer from nt -278 to nt -301 and nt +57 to nt +80 of the coding strand was extended on Fy(a-b+) mRNA using a preamplification kit (GIBCO BRL). The products were separated in a 6%> sequencing gel, shown in Figures 8 A and 8B. Figure 8 A shows the extended product of gp-Fyα mRNA. An M13 sequence ladder was used to determine product length. Figure 8B shows an extended product of gp-Fyβ mRNA. The same primer was used to run a sequence ladder on the FY clone to determine product length. The extended product of the transcript in clone Fy^b81 was 80 nts, matching with the predicted size at the 5' end of the clone (Example 5, above). The primer extension of Fy^b71, from the spliced junction site, yielded a major extended product of 59 nts and two minor products of 110 nts and 111 nts (Figs. 8A and 8B). Certain other features of the FY promoter region deserve comment. One feature of the upstream region of the ATG initiation codon of exon 1 was the absence of TATA and CAAT boxes (Fig. 7) (Tournamille et al. 1995). In this promoter region, we found a direct tandem repeat of 57 nts and we identified the potential binding sites of several transcription factors, such as apl, ap2, and spl, by comparison with the compilation of vertebrate-encoded transcription factors (Faisst et al. 1992). Also, the GATAl and CACCC motifs for globin gene transcription were found in this region (Fig. 7).

To determine the erythroid transcriptional initiation site of FY, Tournamille et al. (1995) have used 5'-rapid amplification of cDNA end (5'-RACE) procedure. Human bone marrow or erythroblast mRNA was the source of RNA, and they identified a cap site at 495 nts upstream from the first site identified by Chaudhuri et al. (1993). However, it now seems that what has been identified is the cap site and the intronic sequence of a non-spliced or "pre-gp-Fyβ" mRNA.

The detection of a pre-gp-Fyβ mRNA raises the question of whether or not this pre-mRNA can be translated. Thus, there is an ORF which starts at nt -298 (initiation codon of gp-Fyβ mRNA) and ends at nt +171 (stop codon) which will encode a novel Duffy peptide of 155 residues (Fig. 7). The first 7 residues of this peptide are the same as gp-Fyβ. The second ORF starts at nt +176 which is the initiation codon of gp-Fyα mRNA and will yield gp-Fyα protein. (Fig. 7). It is unlikely that this translation event will take place since most eukaryotic mRNAs have a single ORF and a single functional initiation site (Kozak 1989). There are few cases of polycistronic mRNA in eukaryotes (Kozak 1986).

A single T→C substitution of a GATAl motif abolishes erythroid gene expression in Duffy-negative individuals (Tournamille et al. 1995). Since neither variant of Duffy protein is expressed in these individuals, the GATAl element must control the erythroid cell-specific transcription of both mRNAs. This GATAl motif is located 364 bp and 28 bp upstream of the cap sites of gp-Fyα and gp-Fyβ mRNAs, respectively (Fig. 7). In gp-Fyα mRNA, the long distance from the cap site suggests that the mechanism of initiation of transcription requires DNA looping, in addition to the specific assembly of trans-acting factors (Rippe et al. 1995).

In addition, in both mRNAs, the initiation codon (AUG) was not embedded within the optimal context for translation initiation in eukaryotes (Kozak 1989). When the context around the first AUG codon is not optimal, positions -3 and +4 (defined with respect to A of the AUG codon as position +1) are especially critical (Kozak 1989). In gp-Fyβ mRNA, position -3 is a purine (G) which enables efficient translation. In gp-Fyα mRNA, position -3 is a pyrimidine (C) which restricts translation. In both mRNAs, position +4 is a purine (G), defining a strong initiation AUG codon (Fig. 7). Another difference between the Duffy spliced and non-spliced mRNAs which might influence their relative rates of translation are secondary structures (hairpins) located upstream of the initiator AUG codon (Kozak 1986). Computer analysis predicted ΔG = -46 and ΔG = -33 kcal/mol for potential hairpin structures of the gp-Fyα and gp-Fyβ mRNAs, respectively. In general, hairpin structures with predicted free energy of -50 kcal/mol or more will inhibit mRNA translation by interfering with the migration of the ribosomal initiation complex (Kozak 1986). Accordingly, the predicted structure of gp-Fyα mRNA would be less favorable for efficient translation than that of gp-Fyβ.

Example 11: Synthesis of gp-Fyα and gp-Fyβ mRNAs in non-erythroid organs

The Duffy gene is active in some cellular types of certain non-erythroid organs (Hadley et al. 1994; Peiper et al. 1995). Applicants have found that, in the kidney, the endothelium of glomeruli, peritubular capillaries, vasa recta and principal cells (epithelium) of the collecting duct, all express the Duffy protein (Chaudhuri et al. (1997)). However, in the thyroid, only the endothelial cells of the small vasculature express the protein. In the lung, endothelial cells of large venules and epithelial cells (type 1) of pulmonary alveoli express the Duffy antigen. Not all endothelial cells and epithelial cells synthesize this protein.

We studied the production of non-spliced gp-Fyα mRNA and spliced gp-Fyβ mRNA in several non-erythroid organs and how such expression relates to that observed in bone marrow. A search of the mRNAs was done by RT-PCR and amplification was performed using two 5' sense primers, one specific for each of the gp-Fyα and gp-Fyβ mRNAs, and a 3 ' antisense primer, common to both mRNAs. The primers were designed so that the amplification products were 543 nts and 387 nts long for gp-Fyα and gp-Fyβ mRNAs, respectively. The amplification was performed in a single reaction to minimize artefactual discrepancy in the product yields.

The poly(A)⁺ RNA samples (1 μg) were treated with DNAase ("Amplification Grade," GIBCO BRL, Gaithersburg, MD) to remove any DNA contamination. The first strand synthesis was produced with the SUPERSCRIPT Preamplification System (GIBCO BRL) following the manufacturer's protocol. The 5' sense primers plsα having the sequence GCTTCCCCAGGACTGTTCCTGCTC (SEQ ID NO: l 1), i.e., nucleotides +2 to +25 (Fig. 7), and plsβ having the sequence CTGCGGGCCTGAACCAAACGG (SEQ ID NO: 12), i.e., nucleotides -323 to -303 (Fig. 7), were constructed from the untranslated 5' regions of gp-Fyα and gp-Fyβ mRNAs, respectively. In addition, a 3 ' antisense primer pi as having the sequence ACCTAGCCCTGGGGCCAAGACGGG (SEQ ID NO: 13), i.e., nucleotides +521 to +544 (Fig. 7), was designed from the common 3 ' region of both transcripts.

The enzymatic (PCR) amplification employed Taq DNA polymerase (Promega, Madison, WI), a cDNA sample equivalent to 0.1 μg of mRNA, and the plsα

(SEQ ID NO: 11), plsβ (SEQ ID NO: 12), and pi as (SEQ ID NO: 13) primers. Amplification cycles were as follows: one cycle of 5 min at 94°C; 30 cycles of 30 s at 94°C, 30 s at 60°C, and 2 min at 72°C; followed by 10 min of elongation at 72°C. The amplification products were separated in a 1.5% agarose gel, shown in Figure 9. In Figure 9: lane 1 is bone marrow; 2 is fetal kidney; 3 is fetal liver; 4 is thyroid; 5 is adult kidney; 6 is adult lung; 7 is adult brain;

8 is fetal brain; 9 is fetal lung; 10 is skeletal muscle; 1 1 is HEL cells; and 12 is HEL cells without reverse transcriptase.

As shown in Figure 9, both species of gp-Fy mRNA were produced in non-erythroid organs. Like bone marrow, the non-erythroid organs produced much less gp-Fyα mRNA than gp-Fyβ mRNA. Fetal kidney and HEL did not produce gp-Fyα mRNA. Fetal liver, as an erythroid organ, produced both species of mRNA. FY is inactive in adult liver (Chaudhuri et al. 1993; Neote et al. 1994). The adult brain expressed much less gp-Fyα mRNA than did the fetal brain. The predominance of gp-Fyβ mRNA over gp-Fyα mRNA in erythroid and non-erythroid organs is a particular feature of Duffy gene expression. Whether or not distinct cell types differentially express the proteins has yet to be determined. Example 12: Quantitation of gp-Fyα and gp-Fyβ mRNAs by competitive RT-PCR

The study described in Example 11 made it evident that gp-Fyα mRNA was less abundant than gp-Fyβ mRNA at saturation levels of amplification. Although all the samples had the same amplification conditions, the level of amplification may not reflect the true quantity of each mRNA since there are many variables that influence the kinetics of amplification.

We employed a technique that obviates these problems and involves co-amplification of a standard template, using the same primers as those of the target cDNA. The standard templates (competitors ) were designed by deleting 264 nts from the respective target cDNAs. RT-PCR was performed using a constant amount of cDNA equivalent to a known quantity of poly(A)⁺ RNA, increasing amounts of competitor and sense and antisense primers (Fig. 10A).

A competitive PCR procedure was used for the quantitation of mRNA according to the method of Gilliland et al. (1990). Standard templates (competitors) were made by using restriction enzymes to delete an internal sequence of 264 nucleotides from the cDNA clones of each mRNA. The plasmids were religated and grown. The inserts were excised enzymatically, and gel-purified. For amplification, the sense primers were p2sα having the sequence ATGGCCTCCTCTGGGTATGTCCTC (SEQ ID NO: 14), i.e., nucleotides +176 to +199 (Fig. 7), and p2sβ having the sequence ATGGGGAACTGTCTGCACAGG (SEQ ID NO: 15), i.e., nucleotides -298 to -278 (Fig. 7), which were specific for gp-Fyα and gp-Fyβ, respectively. A common antisense primer, p2as having the sequence

TCAGGTTGACAGGTGGGAAGA (SEQ ID NO: 16), i.e., nucleotides +1196 to +1216 (Fig. 7), was also used. An increasing amount of a standard template was added to a fixed amount of cDNA equivalent to 100 ng of poly(A)⁺ RNA. Target and standard templates were amplified with the specific sense and antisense primers and labeled with α-³²P-dCTP. Aliquots of amplified products were run into a 1% agarose gel, bands were excised, and the amount of radioactivity was determined. The amplified target and standard templates were 1040 and 776 nts, respectively for gp-Fyα mRNA. The amplified target and standard templates were 1035 and 771 nts, respectively for gp-Fyβ mRNA. The ratios of radioactivity of target/standard were plotted against the amounts of standard added. The size ratio of target versus standard was used to adjust the radioactivity in the standard. Figures 10A-10C illustrate the quantitation of gp-Fyα and gp-Fyβ mRNA by competitive RT-PCR. In Figure 10 A, lanes 1 and 7 do not contain competitor; lanes 2-5 contain 12, 25, 50, and 100 fg of competitor, respectively; and lanes 8-11 contain 0.6, 1.2, 2.5, 5, and 10 pg of competitor, respectively. Lanes 1-5 are gp-Fyα mRNA; lanes 6-11 are gp-Fyβ mRNA. Figure 10B shows a linear regression curve for the quantitation of gp-Fyα mRNA. The ratio of amplified competitor or standard (A to target (A,) is plotted against amounts of competitor added. A_s/A_t=l determined the amount of target mRNA. Figure 10C shows the linear regression for the quantitation of gp-Fyβ mRNA. Values are plotted as in Figure 10B. The amount of target template was estimated from the sample in which the amplified product of target and standard templates was the same. As is shown Figure 10B and 10C, in 100 ng of bone marrow mRNA there were 28 fg and 8 pg of gp-Fyα mRNA and gp-Fyβ mRNA, respectively. Since both mRNAs were of approximately the same size, bone marrow gp-Fyβ mRNA was 285 times more abundant than gp-Fyα mRNA. In the kidney and thyroid, FY produced 250 times more gp-Fyβ mRNA than gp-Fyα mRNA (not shown). Clearly, it is the spliced transcript, gp-Fyβ mRNA, which is the most abundant Duffy mRNA species in bone marrow and other organs.

Example 13: Quantitation of gp-Fyβ expression in erythroid and non-erythroid tissues

An experimental approach similar to that described in Example 11 was used to determine the relative amounts of gp-Fyβ mRNA in erythroid and non-erythroid organs. cDNA equivalent to 100 ng of poly(A)⁺ RNA from bone marrow (1), adult kidney (2), and thyroid (3) were mixed with a constant amount (1 pg) of standard template. Target and standard templates were amplified with same primers as described in Example 11. The ratios of radioactivity for target/standard were determined. As shown in Figure 11, bone marrow produced five times more gp-Fyβ mRNA than adult kidney and twenty times more than thyroid. We believe that this result can be attributed to fewer cells manufacturing gp-Fy in the kidney and thyroid than in bone marrow.

The different amounts of the two Duffy mRNAs may be due to different rates of synthesis or different stabilities. Since the Duffy protein does not appear to perform a regulatory function, it is assumed that the protein has a low rate of turnover. Therefore, the regulatory control in both variants is achieved by the rate of transcription.

Example 14: Binding of interleukin-8 and anti-Fy6 to K562 cells expressing gp-Fyβ

The non-spliced Duffy mRNA has been transfected and expressed in K562 cells (Chaudhuri et al. 1994) and a human embryonic kidney cell line, 293 (Neote et al. 1994). The gp-Fyα expressed in these cell lines was shown to react with anti-Fy6 and several chemokines such as interleukin-8 (IL-8), MGSA, RANTES, and MCP-1 (Neote et al. 1994; Chaudhuri et al. 1994). To better understand the relationships between the two types of Duffy protein, we compared the anti-Fyό reactivity of gp-Fyβ with that of gp-Fyα. Two clones, designated Fy^b71-81 and Fy^b71 as described above, were used for the construction of expression vectors containing the gp-Fyα and gp-Fyβ transcripts, respectively, using the method described by Chaudhuri et al. (1993). The transcripts were amplified by PCR, using 5'- and 3 '-specific primers, containing Hindϊll and Ba tll restriction sites, respectively. The amplified products were digested with Hindlϊl and BamYΑ enzymes and ligated to pREP4 expression vector (Invitrogen, San Diego, CA). K562 cells were transfected with 2 μg of plasmid cDNA. The transfection was performed by adding 20 μg of Lipofectamine transfection reagent (GIBCO BRL) according to the manufacturer's protocol. Stable transfected cells were selected with an antibiotic, hygromycin, and monitored for gp-Fy expression as described in Chaudhuri et al. (1994). K562 cells stably expressing the gp-Fyα and gp-Fyβ variants were used to assay each of the products for anti-Fyό reactivity and IL-8 binding. Anti-Fy6 binding was tested as described by Chaudhuri et al. (1994). K562 cells (2 x 10⁶ cells/mL) expressing each of the two variant proteins were incubated with 1 nM ¹²⁵I-[ser-IL-8]₇₂ (DuPont NEN, Wilmington, DE) and increasing concentrations of unlabeled IL-8 at room temperature for 1 h. Non-specific binding was measured in the presence of excess unlabeled ligand and data were analyzed as explained (Chaudhuri et al. 1994).

The results of these assays are illustrated in Figures 12A-12D. Figure 12A shows the FACS scan analysis of anti-Fy6 binding, and Figure 12B shows the Scatchard plot for IL-8 affinity for gp-Fyα antigen. In comparison, Figure 12C shows the corresponding FACS scan analysis and Figure 12D shows the corresponding Scatchard plot for gp-Fyβ antigen. As can be seen in Figures 12A and 12C, expressed gp-Fyβ, like gp-Fyα, efficiently reacts with anti-Fy6, indicating that the change of six or eight residues at the NH₂ terminus does not affect antibody binding. In order to determine whether the affinity of IL-8 for gp-Fyβ is different, we studied the binding of IL-8 to K562 transfectants expressing gp-Fyα and gp-Fyβ proteins. Both forms of gp-Fy bound the proinflammatory peptide with approximately the same K_D values; 7.69 nM for gp-Fyα and 7.51 nM for gp-Fyβ (Figures 12B and 12D). Accordingly, both variants of Duffy protein exhibited the same immunological and physiological (scavenger) properties.

In summary, the Duffy gene, originates functional mRNAs by non-spliced and spliced mechanisms, In this sense, the Duffy system is similar to other such systems including, for example, chicken link protein (Deak et al. 1 91), cod and trout immunoglobin light locus (Daggfeldt et al. 1993), HIV-1 Rev protein (Stutz et al. 1994), bacu ovivus Autographa californica (Kovacs et al. 1991), and Marek's disease virus (Peng et al. 1992).

The spliced mRNA (gp-Fyβ mRNA) described here is the major transcript present in bone marrow and non-erythroid organs. The non-spliced mRNA (gp-Fyα mRNA) is produced in bone marrow and some organs. The transcripts generate two proteins with identical immunological (binding to anti-Fyό) and physiological (scavenger of proinflammatory peptides) properties.

The structural difference between the two variants occurs at the sequences of six and eight residues at the N-terminal domains of gp-Fyβ and gp-Fyα, respectively. We have determined that the bone marrow is the major source of gp-Fyβ protein in Duffy-positive individuals. Similar methods can be used to identify which organ(s) is(are) the major producer(s) of this protein in Duffy-negative individuals.

Example 15: Preparation of Transgenic Mice Expressing gp-Fy^b Protein Transgenic mice have been constructed to express the human Duffy gp-Fy^b antigen. A

3523 bp genomic DNA fragment containing FY*B coding sequence and -1.5 kb upstream and -1 kb downstream flanking sequences (SEQ ID NO:28; shown in Figures 13A and 13B) was amplified by the polymerase chain reaction using PY-specific primers (sense: 5'-CTGCAGGGGTAGATGCCCTTTCTC-3 ' (SEQ ID NO:29); antisense: 5'-GAATTCCAAGCAGAAGATGAATC-3' (SEQ ID NO:30)). The amplified fragment was cloned in the pBluescript vector (Strategene). Plasmid DNA was purified by two-round centrifugation in CsCl gradients. The fragment containing the inserted genomic FY*B gene was excised by appropriate restriction enzymes and separated on a gel followed by DNA purification. The pure DNA fragment was reconstituted to a concentration of approximately 5 μg mL and was used to construct transgenic mice.

The purified DNA fragment was micro-injected into the male pronucleus of fertilized eggs of the B6/CBA FI mouse (Jackson Laboratory, Bar Harbor, ME), which had been removed from the oviducts of a female mouse that had mated the night before. The zygotes with the insertion were transferred to the oviducts of 0.5-day pseudo-pregnant females and allowed to develop into embryos. Ten females became pregnant, producing 60 pups.

Four weeks after birth, DNA was prepared from tail clips of each baby animal using proteinase K digestion and ethanol precipitation. The DNA was tested for FY sequence integration by dot blot hybridization with a probe derived from the Duffy genomic DNA or by PCR amplification using PT-specific primers. In the PCR, 200 ng of the genomic DNA was amplified with the Duffy-specific primers with Taq polymerase. The PCR reaction was carried out for 30 cycles as follows: 30 s at 94°C, 30 s at 65 °C, and 3 min at 72°C. Ten microliters (10 μL) of the reaction mixture was run on a 1%> agarose gel, using a 1 kb DNA marker, with a non-transgenic mouse sample as a control. Figure 14 shows representative PCR results, with a DNA marker (lane 1), DNA from a non-transgenic mouse control (lane 2), and DNA samples from 12 transgenic mice (lanes 3-14). The dot blot hybridization and PCR amplification showed that 11 out of the 60 mice (18%> transduction rate) had successful integration of the human Duffy genomic DNA into their chromosome, and more than one copy ofPTwas observed (data not shown).

Expression of the exogenous gene was examined by hemagglutination. Serological studies were performed by collecting blood from each animal showing successful integration of human Duffy DNA by puncture of the orbital plexus under an anesthetized condition. The isolated RBCs were tested for the presence of human Fy^b antigen by hemagglutination using urine MoAbs anti-Fy3, anti-Fyό, and human anti-Fy^b. Of the 1 1 transgenic mice having the Duffy gene incorporated, RBCs from four mice showed the expression of the expected cognate antigens. These results are shown in Table 1, below, which summarizes immunological data concerning red cells from the 11 mice as compared to human red cells. TABLE 1

Erythrocytes anti-Fy3 Anti-Fy6 Anti-Fy^b PCR

Human ++ ++ Positive

Mouse #1 ^■++ ++ Positive Mouse #2 ++++ ++ Positive Mouse #3 Positive Mouse #4 Positive Mouse #5 ++ ++ ++ Positive Mouse #6 Positive Mouse #1 +++ + ++ Positive Mouse #8 Positive Mouse #9 Positive Mouse #10 Positive Mouse #11 Positive

These data indicate that not all of the integration o£FY*B gene occurred at the chromosomal site which is being actively trnascribed. However, approximately 7% of the transfected animals actively transcribed FY and synthesized (expressed) the Duffy Fy^b protein.

Furthermore, it is demonstrated that the integrated DNA sequence contains all of the information necessary for Duffy promoter activity and its expression in erythroid specific manner. The red cells of the transgenic mice are serologically identical to a Duffy-positive human having Fy(a-b+) erythrocytes. These agglutination data imply that the expressed human Duffy protein was folded onto the mouse RBC membrane preserving its native (i.e., human) conformational structure and antigenic sites. Transgenic mice and other animals expressing other forms of the gp-Fy protein can also be constructed given the information herein concerning the molecular basis for the Duffy polymorphism.

These transgenic mice are, therefore, suitable for use as an animal model of malaria and for the development of therapeutic materials and methods to prevent merozoite binding and/or invasion of erythrocytes. For example, the invention includes testing methods such as the following method for testing the capacity of an analyte for inhibiting erythrocyte binding or infection by a malarial organism in a mammal in vivo. The method can comprise: administering an analyte suspected of having specific binding affinity for a malarial Duffy-binding ligand to a test mammal (i.e., a transgenic animal) modified to express on erythrocytes a heterologous Duffy protein having a malarial binding domain, the erythrocytes being susceptible to binding or infection by a malarial organism; inoculating said test mammal with a malarial organism in an amount and under conditions sufficient to measurably infect said mammal absent any added substance capable of specifically inhibiting binding or infection of erythrocytes of said mammal by said malarial organism; and determining whether said malarial organism has bound to or infected erythrocytes of said test mammal, a lack of infection indicating that the analyte inhibits malarial binding or infection of erythrocytes in said mammal in vivo. In the method, the administering step can be performed prior to or after the inoculating step.

Thus, while there have been described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further embodiments can be made without departing from the spirit of the invention, and it is intended to include all such further modifications and changes as come within the true scope of the claims set forth herein.

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TJ, Lu ZH, Hesselgesser J and Horuk R, "The Duffy antigen/receptor for chemokines

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Riwom S, Janvier D, Navenot JM, Benbunan M, Muller JY and Blanchard D, "Production of a new murine monoclonal antibody with FY6 specificity and characterization of the immunopurified N-glycosylated Duffy-active molecule," Vox Sang. 66:61 (1994). Shagger H et al., Anal. Biochem. 168:368 (1987). Stoffel W et al., Hoppe-Seyler's Z. Physiol. Chem. 364: 1455 (1983).

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Pogo, A Oscar

Chaudhuri, Asok

(ii) TITLE OF INVENTION: THE CLONING OF DUFFY BLOOD GROUP

ANTIGEN

(iii) NUMBER OF SEQUENCES: 30

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Hoffmann & Baron, LLP

(B) STREET: 350 Jericho Turnpike

(C) CITY: Jericho

(D) STATE: New York

(E) COUNTRY: USA

(F) ZIP: 11753

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 1.44 Mb storage

(B) COMPUTER: IBM compatible

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/749,543

(B) FILING DATE: 15-NOV-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: O'Dea, Sean W.

(B) REGISTRATION NUMBER: 37690

(C) REFERENCE/DOCKET NUMBER: 454-9 PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (516) 822-3550

(B) TELEFAX: (516) 822-3582

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1267 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA

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

GGCTTCCCCA GGACTGTTCC TGCTCCGGCT CTTCAGGCTC 40

CCTGCTTTGT CCTTTTCCAC TGTCCGCACT GCATCTGACT 80

CCTGCAGAGA CCTTGTTCTC CCACCCGACC TTCCTCTCTG 120

TCCTCCCCTC CCACCTGCCC CTCAGTTCCC AGGAGACTCT 160

TCCGGTGTAA CTCTG ATG GCC TCC TCT GGG TAT GTC CTC 199

Met Ala Ser Ser Gly Tyr Val Leu

CAG GCG GAG CTC TCC CCC TCA ACT GAG AAC TCA AGT CAG 238 Gin Ala Glu Leu Ser Pro Ser Thr Glu Asn Ser Ser Gin

CTG GAC TTC GAA GAT GTA TGG AAT TCT TCC TAT GGT GTG 277 Leu Asp Phe Glu Asp Val Trp Asn Ser Ser Tyr Gly Val

AAT GAT TCC TTC CCA GAT GGA GAC TAT GAT GCC AAC CTG 316 Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp Ala Asn Leu

GAA GCA GCT GCC CCC TGC CAC TCC TGT AAC CTG CTG GAT 355 Glu Ala Ala Ala Pro Cys Asn Ser Cys Asn Leu Leu Asp

GAC TCT GCA CTG CCC TTC TTC ATC CTC ACC AGT GTC CTG 394 Asp Ser Ala Leu Pro Phe Phe lie Leu Thr Ser Val Leu

GGT ATC CTA GCT AGC AGC ACT GTC CTC TTC ATG CTT TTC 433 Gly lie Leu Ala Ser Ser Thr Val Leu Phe Met Leu Phe

AGA CCT CTC TTC CGC TGG CAG CTC TGC CCT GGC TGG CCT 472 Arg Pro Leu Phe Arg Trp Gin Leu Cys Pro Gly Trp Pro

GTC CTG GCA CAG CTG GCT GTG GGC AGT GCC CTC TTC AGC 511 Val Leu Ala Gin Leu Ala Val Gly Ser Ala Leu Phe Ser

ATT GTG GTG CCC GTC TTG GCC CCA GGG CTA GGT AGC ACT 550 lie Val Val Pro Val Leu Ala Pro Gly Leu Gly Ser Thr

CGC AGC TCT GCC CTG TGT AGC CTG GGC TAC TGT GTC TGG 589 Arg Ser Ser Ala Leu Cys Ser Leu Gly Tyr Cys Val Trp

TAT GGC TCA GCC TTT GCC CAG GCT TTG CTG CTA GGG TGC 628 Tyr Gly Ser Ala Phe Ala Gin Ala Leu Leu Leu Gly Cys

CAT GCC TCC CTG GGC CAC AGA CTG GGT GCA GGC CAG GTC 667 Asn Ala Ser Leu Gly Asn Arg Leu Gly Ala Gly Gin Val

CCA GGC CTC ACC CTG GGG CTC ACT GTG GGA ATT TGG GGA 706 Pro Gly Leu Thr Leu Gly Leu Thr Val Gly He Trp Gly

GTG GCT GCC CTA CTG ACA CTG CCT GTC ACC CTG GCC AGT 745 Val Ala Ala Leu Leu Thr Leu Pro Val Thr Leu Ala Ser

GGT GCT TCT GGT GGA CTC TGC ACC CTG ATA TAC AGC ACG 784 Gly Ala Ser Gly Gly Leu Cys Thr Leu He Tyr Ser Thr

GAG CTG AAG GCT TTG CAG GCC ACA CAC ACT GTA GCC TGT 823 Lys Leu Lys Ala Leu Gin Ala Thr Asn Thr Val Ala Cys

CTT GCC ATC TTT GTC TTG TTG CCA TTG GGT TTG TTT GGA 862 Leu Ala He Phe Val Leu Leu Pro Leu Gly Leu Phe Gly

GCC AAG GGG CTG AAG AAG GCA TTG GGT ATG GGG CCA GGC 901 Ala Lys Gly Leu Lys Lys Ala Leu Gly Met Gly Phe Gly

CCC TGG ATG AAT ATC CTG TGG GCC TGG TTT ATT TTC TGG 940 Pro Trp Met Asn He Leu Trp Ala Trp Phe He Phe Trp

TGG CCT CAT GGG GTG GTT CTA GGA CTG GAT TTC CTG GTG 979 Trp Pro Asn Gly Val Val Leu Gly Leu Asp Phe Leu Val

AGG TCC AAG CTG TTG CTG TTG TCA ACA TGT CTG GCC CAG 1018 Arg Ser Lys Leu Leu Leu Leu Ser Thr Cys Leu Ala Gin

CAG GCT CTG GAC CTG CTG CTG AAC CTG GCA GAA GCC CTG 1057 Gin Ala Leu Asp Leu Leu Leu Met Leu Ala Glu Ala Leu

GCA ATT TTG CAC TGT GTG GCT ACG CCC CTG CTC CTC GCC 1096 Ala He Leu Asn Cys Val Ala Thr Pro Leu Leu Leu Ala

CTA TTC TGC CAC CAG GCC ACC CGC ACC CTC TTG CCC TCT 1135 Leu Phe Cys Lys Gin Ala Thr Arg Thr Leu Leu Pro Ser

CTG CCC CTC CCT GAA GGA TGG TCT TCT CAT CTG GAC ACC 1174 Leu Pro Leu Pro Glu Gly Trp Ser Ser Asn Leu Asp Thr

CTT GGA AGC AAA TCC TAGTTCTCTT CCCACCTGTC AACCTGAATT 1219 Leu Gly Ser Lys Ser

AAAGTCTACA CTGCCTTTGT GAAAAAAAAA AAAAAAAAAA AAAAAAAA 1267

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 338 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Ser Ser Gly Tyr Val Leu Gin Ala Glu Leu Ser Pro Ser 1 5 10 15

Thr Glu Asn Ser Ser Gin Leu Asp Phe Glu Asp Val Trp Asn Ser

20 25 30

Ser Tyr Gly Val Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp Ala

35 40 45

Asn Leu Glu Ala Ala Ala Pro Cys Asn Ser Cys Asn Leu Leu Asp

50 55 60

Asp Ser Ala Leu Pro Phe Phe He Leu Thr Ser Val Leu Gly He

65 70 75

Leu Ala Ser Ser Thr Val Leu Phe Met Leu Phe Arg Pro Leu Phe

80 85 90

Arg Trp Gin Leu Cys Pro Gly Trp Pro Val Leu Ala Gin Leu Ala

95 100 105

Val Gly Ser Ala Leu Phe Ser He Val Val Pro Val Leu Ala Pro

110 115 120

Gly Leu Gly Ser Thr Arg Ser Ser Ala Leu Cys Ser Leu Gly Tyr

125 130 135

Cys Val Trp Tyr Gly Ser Ala Phe Ala Gin Ala Leu Leu Leu Gly

140 145 150

Cys Asn Ala Ser Leu Gly Asn Arg Leu Gly Ala Gly Gin Val Pro

155 160 165

Gly Leu Thr Leu Gly Leu Thr Val Gly He Trp Gly Val Ala Ala

170 175 180

Leu Leu Thr Leu Pro Val Thr Leu Ala Ser Gly Ala Ser Gly Gly

185 190 195

Leu Cys Thr Leu He Tyr Ser Thr Lys Leu Lys Ala Leu Gin Ala

200 205 210

Thr Asn Thr Val Ala Cys Leu Ala He Phe Val Leu Leu Pro Leu

215 220 225

Gly Leu Phe Gly Ala Lys Gly Leu Lys Lys Ala Leu Gly Met Gly

230 235 240

Phe Gly Pro Trp Met Asn He Leu Trp Ala Trp Phe He Phe Trp

245 250 255 Trp Pro Asn Gly Val Val Leu Gly Leu Asp Phe Leu Val Arg Ser

260 265 270

Lys Leu Leu Leu Leu Ser Thr Cys Leu Ala Gin Gin Ala Leu Asp

275 280 285

Leu Leu Leu Met Leu Ala Glu Ala Leu Ala He Leu Asn Cys Val

290 295 300

Ala Thr Pro Leu Leu Leu Ala Leu Phe Cys Lys Gin Ala Thr Arg

305 310 315

Thr Leu Leu Pro Ser Leu Pro Leu Pro Glu Gly Trp Ser Ser Asn

320 325 330

Leu Asp Thr Leu Gly Ser Lys Ser

335

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 338 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Gly Asn Cys Leu His Arg Ala Glu Leu Ser Pro Ser Thr Glu 1 5 10 15

Asn Ser Ser Gin Leu Asp Phe Glu Asp Val Trp Asn Ser Ser Tyr

20 25 30

Gly Val Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp Ala Asn Leu

35 40 45

Glu Ala Ala Ala Pro Cys Asn Ser Cys Asn Leu Leu Asp Asp Ser

50 55 60

Ala Leu Pro Phe Phe He Leu Thr Ser Val Leu Gly He Leu Ala

65 70 75

Ser Ser Thr Val Leu Phe Met Leu Phe Arg Pro Leu Phe Arg Trp

80 85 90

Gin Leu Cys Pro Gly Trp Pro Val Leu Ala Gin Leu Ala Val Gly

95 100 105

Ser Ala Leu Phe Ser He Val Val Pro Val Leu Ala Pro Gly Leu 110 115 120

Gly Ser Thr Arg Ser Ser Ala Leu Cys Ser Leu Gly Tyr Cys Val 125 130 135

Trp Tyr Gly Ser Ala Phe Ala Gin Ala Leu Leu Leu Gly Cys Asn 140 145 150

Ala Ser Leu Gly Asn Arg Leu Gly Ala Gly Gin Val Pro Gly Leu 155 160 165

Thr Leu Gly Leu Thr Val Gly He Trp Gly Val Ala Ala Leu Leu 170 175 180

Thr Leu Pro Val Thr Leu Ala Ser Gly Ala Ser Gly Gly Leu Cys 185 190 195

Thr Leu He Tyr Ser Thr Lys Leu Lys Ala Leu Gin Ala Thr Asn 200 205 210

Thr Val Ala Cys Leu Ala He Phe Val Leu Leu Pro Leu Gly Leu 215 220 225

Phe Gly Ala Lys Gly Leu Lys Lys Ala Leu Gly Met Gly Phe Gly 230 235 240

Pro Trp Met Asn He Leu Trp Ala Trp Phe He Phe Trp Trp Pro 245 250 255

Asn Gly Val Val Leu Gly Leu Asp Phe Leu Val Arg Ser Lys Leu 260 265 270

Leu Leu Leu Ser Thr Cys Leu Ala Gin Gin Ala Leu Asp Leu Leu 275 280 285

Leu Met Leu Ala Glu Ala Leu Ala He Leu Asn Cys Val Ala Thr 290 295 300

Pro Leu Leu Leu Ala Leu Phe Cys Lys Gin Ala Thr Arg Thr Leu 305 310 315

Leu Pro Ser Leu Pro Leu Pro Glu Gly Trp Ser Ser Asn Leu Asp 320 325 330

Thr Leu Gly Ser Lys Ser 335

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 amino acids (B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

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

Ala Glu Leu Ser Pro Ser Thr Glu Asn Ser Ser Gin Leu Asp Phe 1 5 10 15

Glu Asp Val Trp Asn Ser Ser Tyr Gly Val Asn Asp Ser Phe Pro

20 25 30

Asp Gly Asp Tyr Asp

35

(2) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Ala Ser Ser Gly Tyr Val Leu Gin Ala Glu Leu Ser Pro Ser 1 5 10 15

Thr Glu Asn Ser Ser Gin Leu Asp Phe Glu Asp Val Trp Asn Ser

20 25 30

Ser Tyr Gly Val Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp

35 40

(2) INFORMATION FOR SEQ ID NO: 6

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 66 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Ala Ser Ser Gly Tyr Val Leu Gin Ala Glu Leu Ser Pro Ser 1 5 10 15 Thr Glu Asn Ser Ser Gin Leu Asp Phe Glu Asp Val Trp Asn Ser

20 25 30

Ser Tyr Gly Val Asn Asp Ser Phe Pro Asp Gly Asp Tyr Asp Ala

35 40 45

Asn Leu Glu Ala Ala Ala Pro Cys His Ser Cys Asn Leu Leu Asp

50 55 60

Asp Ser Ala Leu Pro Phe

65

(2) INFORMATION FOR SEQ ID NO: 7

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Ala Glu Leu Ser Pro Ser Thr Glu Asn Ser Ser Gin Leu Asp Phe 1 5 10 15

Glu Asp Val Trp Asn Ser Ser

20

(2) INFORMATION FOR SEQ ID NO: 8

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATGAAYATHY TITGGGCITG GTT 23

(2) INFORMATION FOR SEQ ID NO: 9

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACIAGRAART CIAGICCIAR NAC 23

(2) INFORMATION FOR SEQ ID NO: 10

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TGGTTTATTT TCTGGTGGCC TCAT 24

(2) INFORMATION FOR SEQ ID NO: 11

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCTTCCCCAG GACTGTTCCT GCTC 24

(2) INFORMATION FOR SEQ ID NO: 12

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTGCGGGCCT GAACCAAACG G 21 (2) INFORMATION FOR SEQ ID NO: 13

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ACCTAGCCCT GGGGCCAAGA CGGG 24

(2) INFORMATION FOR SEQ ID NO: 14

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ATGGCCTCCT CTGGGTATGT CCTC 24

(2) INFORMATION FOR SEQ ID NO: 15

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ATGGGGAACT GTCTGCACAG G 21

(2) INFORMATION FOR SEQ ID NO: 16

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TCAGGTTGAC AGGTGGGAAG A 21

(2) INFORMATION FOR SEQ ID NO: 17

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Asp Phe Glu Asp Val Trp Asn Ser Ser Tyr Gly Val Asn Asp Ser 1 5 10 15

Phe Pro Asp Gly Asp Tyr Asp

20

(2) INFORMATION FOR SEQ ID NO: 18

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Ala Asn Leu Glu Ala Ala Ala Pro Cys His Ser Cys Asn Leu Leu 1 5 10 15

Asp Asp Ser Ala Leu Pro Phe

20

(2) INFORMATION FOR SEQ ID NO: 19

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Ala Glu Leu Ser Pro Ser Thr Glu Asn Ser Ser Gin Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO: 20

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Pro Leu Phe Arg Trp Gin Leu Cys Pro Gly Trp Pro Val Leu Ala 1 5 10 15

Gin

(2) INFORMATION FOR SEQ ID NO: 21

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

Met Met He Leu Trp Ala Trp Phe He Phe Trp Trp Pro Asn Gly 1 5 10 15

Val Val Leu Gly Leu Asp Phe Leu Val

20

(2) INFORMATION FOR SEQ ID NO: 22

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Phe Ser He Val Val

1 5

(2) INFORMATION FOR SEQ ID NO: 23

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23;

Phe Ala Gin Ala Leu 1 5

(2) INFORMATION FOR SEQ ID NO: 24

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Val Gly He 1

(2) INFORMATION FOR SEQ ID NO: 25

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

Pro Ser Leu Pro Lys Gly Trp 1 5

(2) INFORMATION FOR SEQ ID NO: 26 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Met Gly Asn Cys Leu His Arg 1 5

(2) INFORMATION FOR SEQ ID NO: 27

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: protein (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

Met Ala Ser Ser Gly Tyr Val Leu Gin 1 5

(2) INFORMATION FOR SEQ ID NO: 28

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3523 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

CTGCAGGGGT AGATGCCCTT TCTCTCTGCT GGCCAGCTCT GCCCCTCAGT 50

GAGAAACTTT ACATATTGCT AAGATGCCTG GCCAATGAAA CAGTTCCAGA 100

GACTTTATGT CCCCAGTAGA AATATGAATA GAAATCACCC TGTGCTCAAT 150

GGTCCCATTT TAAAATATGC TGTCCCATTG TCCCCTAGAG CCTATCTTAA 200

CTTGTCAGAC CATGTATTCC ACTTCATATG CAAGAGGCAT GCACTGAGCC 250

CATAGGTGGC TAGGCAAACA CCCAATAGCT CCCTGAAATG GCTTCATTAT 300

GGAGGCTCGA CAGCCACCCC AACCCTCCCA CTCTCACACT GAAACACCCA 350 GACCTAGAGA TAGCTAGACA CACCCAGACA CCCGCCAAGC CCCTCACATA 400

CAGATATGTG CACAATGATA CACAGCAAAT GTACACAGAG TTCAGTACAC 450

ACAAAGAGCT CACGCCCACG TGCACACACC CCTCAGTTGG GACAGAGTTG 500

ACCACCACCA CCTTTCTCCC AAACACATGG CTTTGGAACT GCCTTTCCTT 550

GGATCCAGTT CAAGGGGATG GAGGAGCAGT GAGAGTCAGC CGCCCTTCCA 600

CTCCAATTTC CCAGCACCTC CCTTATCTCT GCCTCACAAG TCACCCAGCC 650

CCCCTCTCTT CCTTCCTTGT GCTTGAAGAA TCTCTCCTTG CTGGAAAGCC 700

CCCTGTTTTC TCAATCTCCC TTTCCACTTC GGTAAAATCT CTCCTTGCTG 750

GAAAGCCCCC TGTTTTCTCA ATCTCCCTTT CCACTTCGGT AAAATGCCCA 800

CTTTCTGGTC CCCACCTTTT TCCTGAGTGT AGTCCCAACC AGCCAAATCC 850

AACCTCAAAA CAGGAAGACC CAAGGCCAGT GACCCCCATA GGCCTGAGGC 900

TTGTTGCAGG CAGTGGGCGT GGGGTAAGGC TTCCTGATGC CCCCTGTCCC 950

TGCCCAGAAC CTGATGGCCC TCATTAGTCC TTGGCTCTTA TCTTGGAAGC 1000

ACAGGCGCTG ACAGCCGTAC CAGCCCTTCT GTCTGCGGGC CTGAACCAAA 1050

CGGTGCCATG GGGAACTGTC TGCACAGGGT GAGTATGGGG CCAGGCCCCA 1100

GAGTCCCTTA TCCCTATGCC CCTCATTTCC CCTGCTGTTT GCCCCTCAGT 1150

CTTTATATCT CTTCCTTTTC CTCCTCATCT TTTCTCCCTT CCTGCTTTTT 1200

TCCTCTTCCT TCAAAGTCTT TTTCCTTTTC TCCTTCCTAT GCTAGCCTCC 1250

TAGCTCCCTC TTGTGTCCCT CCCTTTGCCT TTGAGTCAGT TCCATCCTGG 1300

TCTCTTGGTG CCTTTCCTTC TGACCTTGCA CTGCTCCTCC AGCCCCAGCT 1350

GCCCTGGCTT CCCCAGGACT GTTCCTGCTC CGGCTCTTCA GGCTCCCTGC 1400

TTTGTCCTTT TCCACTGTCC GCACTGCATC TGACTCCTGC AGAGACCTTG 1450

TTCTCCCACC GCACCTTCCT CTCTGTCCTC CCCTCCCACC TGCCCCTCAG 1500

TTCCCAGGAG ACTCTTCCGG TGTAACTCTG ATGGCCTCCT CTGGGTATGT 1550

CCTCCAGGCG GAGCTCTCCC CCTCAACTGA GAACTCAAGT CAGCTGCAGT 1600

TCGAAGATGT ATGGAATTCT TCCTATGGTG TGAATGATTC CTTCCCAGAT 1650 GGAGACTATG ATGCCAACCT GGAAGCAGCT GCCCCCTGCC ACTCCTGTAA 1700

CCTGCTGGAT GACTCTGCAC TGCCCTTCTT CATCCTCACC AGTGTCCTGG 1750

GTATCCTAGC TAGCAGCACT GTCCTCTTCA TGCTTTTCAG ACCTCTCTTC 1800

CGCTGGCAGC TCTGCCCTGG CTGGCCTGTC CTGGCACAGC TGGCTGTGGG 1850

CAGTGCCCTC TTCAGCATTG TGGTGCCCGT CTTGGCCCCA GGGCTAGGTA 1900

GCACTCGCAG CTCTGCCCTG TGTAGCCTGG GCTACTGTGT CTGGTATGGC 1950

TCAGCCTTTG CCCAGGCTTT GCTGCTAGGG TGCCATGCCT CCCTGGGCCA 2000

CAGACTGGGT GCAGGCCAGG TCCCAGGCCT CACCCTGGGG CTCACTGTGG 2050

GAATTTGGGG AGTCCGTGCC CTACTGACAC TGCCTGCTAC CCTGGCCAGT 2100

GGTGCTTCTG GTGGACTCTG CACCCTGATA TACAGCACGG AGCTGAAGGC 2150

TTTGCAGGCC ACACATACTG TAGCCTGTCT TGCCATCTTT GTCTTGTTGC 2200

CATTGGGTTT GTTTGGAGCC AAGGGGCTGA AGAAGGCATT GGGTATGGGG 2250

CCAGGCCCCT GGATGAATAT CCTGTGGGCC TGGTTTATTT TCTGGTGGCC 2300

TCATGGGGTG GTTCTAGGAC TGGATTTCCT GGTGAGGTCC AAGCTGTTGC 2350

TGTTGTCAAC ATGTCTGGCC CAGCAGGCTC TGGACCTGCT GCTGAACCTG 2400

GCAGAAGCCC TGGCAATTTT GCACTGTGTG GCTACGCCCC TGCTCCTCGC 2450

CCTATTCTGC CACCAGGCCA CCCGCACCCT CTTGCCCTCT CTGCCCCTCC 2500

CTGAAGGATG GTCTTCTCAT CTGGACACCC TTGGAAGCAA ATCCTAGTTC 2550

TCTTCCCACC TGTCAACCTG AATTAAAGTC TACACTGCCT TTGTGAAGCG 2600

GGTGGTTTCT TATTTTGTCT GGGGAGAAGA AGGAGAATGG AGAGAGAGAC 2650

ATTTTTATGT CAGACTTTCT TGCCAGTGTC TGCTTCTATA GCTGGCTTGG 2700

GAAGAAGGTG AATGATGAAT AAATACCCTC AGGGTACACA GATGTTCTCT 2750

TGAGGTGTGG GGTCAGGCCA TCTCAAGGGA GAAGAGAAGA GGAACTAGAG 2800

CATGAGGGGA GTCATTAAAC CAAAAAAAAC AGAAGGGATG GCTTAGCTGG 2850

AAAAAAAGCT GTTCTGGGAA GCAAATGGAA TAGGAACTCA AACTGAGAGA 2900

TAAACAGTGA AGAGTGATGA CAAAGCCCAG AGCAATACCA CCTCCCCCTG 2950 TCCAACCTGC CCAGCCTCTG TCTTCTGTCT CCTCTCTGGC TTTGTTTAGT 3000

GATTAGGACA GTGGTGGGGA AGGTGAAAGA AGCATCCCAG GGGATGTTAC 3050

TCAGTTCAGG GAACATATCA AGGTAATTTA AAAAGCCACT TCCTGGGAGT 3100

CATCTCTCCC AGGTTCCTCA GCATGACCTG AATGTGTGTG TGTGCGTGTG 3150

TGTGTGTGTG TGTACACATC TGTTTCTCGA TCTGTTAGAA TCTACCTTTA 3200

TGTTAGATGT ATGCATGTAA AAACATATGT CCACCCATGA GCTTGCATCT 3250

CTGCTAGCAC CTGAACTGCG ACACCTGTGC GTGTGCACTG ACTTTTCTCA 3300

GGACCCAAAC CCCCACTCAA TTCTGCACTC ATCCCTGTTC ACAGGATATA 3350

GAATCGGGAT TTATGACTCA CTCCTTACCC AAATGAGTTT TCTTTACCCT 3400

GGTTTTTAAG CCTAGTCTTT TCTGTGTAGG ATGTGTGGAG GGAAGAAAAG 3450

ATCAAGAAGT TGTGAGGGGT GGAGAAACTT GAAGGGGGAG GCCCTGATTT 3500

GATTCATCTT CTGCTTGGAA TTC 3523

(2) INFORMATION FOR SEQ ID NO: 29

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: CTGCAGGGGT AGATGCCCTT TCTC 24

(2) INFORMATION FOR SEQ ID NO: 30

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 nucleotides

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GAATTCCAAG CAGAAGATGA ATC 23

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid, encoding a gp-Fy protein or a characteristic fragment thereof.

2. A nucleic acid according to Claim 1, wherein said nucleic acid is isolated natural or synthetic DNA encoding a gp-Fy protein.

3. A nucleic acid according to Claim 1, wherein said nucleic acid is isolated natural or synthetic RNA encoding a gp-Fy protein.

4. A nucleic acid according to Claim 3, wherein RNA is a non-spliced mRNA encoding gp-Fy╬▒ protein or a spliced mRNA encoding gp-Fy╬▓ protein.

5. A nucleic acid according to Claim 1, having a nucleotide sequence comprising the sequence designated SEQ ID NO: 1, or a characteristic fragment thereof.

6. An isolated natural or synthetic undenatured gp-Fy protein or a characteristic fragment thereof.

7. An isolated natural or synthetic protein according to Claim 6, wherein the gp-Fy protein is encoded by a non-spliced gp-Fy mRNA or a spliced gp-Fy mRNA.

8. An isolated natural or synthetic protein according to Claim 7, wherein the gp-Fy protein has an amino acid sequence comprising the sequence designated SEQ ID NO: 2 or a characteristic fragment thereof.

9. An isolated natural or synthetic protein according to Claim 7, wherein the gp-Fy protein has an amino acid sequence consisting of the sequence designated SEQ ID NO:3 or a characteristic fragment thereof.

10. A peptide, having an amino acid sequence comprising the sequence designated SEQ ID NO:6.

11. A peptide according to Claim 10, having an amino acid sequence comprising the sequence designated SEQ ID NO: 5.

12. A peptide according to Claim 10, having an amino acid sequence comprising the sequence designated SEQ ID NO:4.

13. A peptide according to Claim 10, having an amino acid sequence comprising the sequence designated SEQ ID NO: 7.

14. A nucleic acid probe or primer, wherein said nucleic acid probe or primer hybridizes specifically with a nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof.

15. A nucleic acid probe or primer according to Claim 14, wherein said nucleic acid probe or primer hybridizes specifically with a non-spliced nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof.

16. A nucleic acid probe or primer according to Claim 14, wherein said nucleic acid probe or primer hybridizes specifically with a spliced nucleic acid encoding a gp-Fy protein or a characteristic fragment thereof.

17. A nucleic acid probe or primer according to Claim 14, wherein said nucleic acid probe or primer has a sequence comprising SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO:12, SEQ JD NO: 13, SEQ JX> NO:14, SEQ JD NO:15, or SEQ ID NO: 16.

18. A nucleic acid probe or primer according to Claim 14, wherein said nucleic acid probe or primer is attached to a detectable label moiety.

19. A method of detecting gp-Fy-encoding nucleic acid, comprising hybridizing a nucleic acid probe or primer, capable of specifically hybridizing with a nucleic acid encoding a gp-Fy protein, with a biological sample comprising nucleic acid, and measuring an amount of hybridization of said nucleic acid probe or primer with said nucleic acid in the biological sample, wherein a measured amount of hybridization indicates that an amount of gp-Fy-encoding nucleic acid is present in said biological sample.

20. The method according to Claim 19, wherein said method includes detecting gp-Fy-encoding mRNA.

21. The method according to Claim 19, wherein said nucleic acid probe or primer hybridizes specifically with a non-spliced nucleic acid encoding a gp-Fy protein.

22. The method according to Claim 19, wherein said nucleic acid probe or primer hybridizes specifically with a spliced nucleic acid encoding a gp-Fy protein.

23. The method according to Claim 19, wherein said nucleic acid probe or primer is attached to a detectable label moiety.

24. A vector for transfecting a cell to express a heterologous protein, comprising a DNA segment encoding a gp-Fy protein or a characteristic fragment thereof conjugated to a promoter.

25. A vector according to Claim 24, wherein said DNA segment encodes human gp-Fy╬▒ or gp-Fy╬▓ protein or a characteristic fragment of either protein.

26. A transgenic animal genetically modified to express a gp-Fy protein or a characteristic fragment thereof encoded by a heterologous DNA segment.

27. A transgenic animal according to Claim 26, wherein the heterologous DNA segment comprises SEQ ID NO:28.

28. A transgenic animal according to Claim 26, wherein the transgenic animal is a transgenic mouse.

29. A method for making a transgenically modified animal, comprising transfecting cells of an animal with a vector comprising a DNA segment encoding a gp-Fy protein or a characteristic fragment thereof conjugated to a promoter, to provide an animal capable of expressing a protein product encoded by said DNA segment.

30. A method according to Claim 29, wherein said DNA segment comprises SEQ ID NO:28.

31. A method according to Claim 29, wherein said animal is a mouse.

32. A method for making a cell which expresses a heterologous protein, comprising transfecting a cell with a vector comprising a DNA segment encoding a gp-Fy protein or a characteristic fragment thereof conjugated to a promoter.