WO2003016341A2

WO2003016341A2 - Regulation of iron uptake

Info

Publication number: WO2003016341A2
Application number: PCT/IB2002/003647
Authority: WO
Inventors: Nadia Hubert; Matthias Hentze
Original assignee: European Molecular Biology Laboratory
Priority date: 2001-08-17
Filing date: 2002-08-19
Publication date: 2003-02-27
Also published as: WO2003016341A3; AU2002328117A1; GB0120148D0

Abstract

The invention relates to the isolation and characterization of a novel exon of the DMT1 (Divalent Metal Transporter 1) mRNA encoding a novel N-terminal domain of the DMT1 protein.

Description

REGULATION OF IRON UPTAKE

The present invention relates generally to iron metabolism and transport. More specifically, the invention relates to the isolation and characterization of a novel exon of the DMTl (Divalent Metal Transporter 1) mRNA encoding a novel N-terminal domain of the DMTl protein.

All publications, patents and patent applications cited herein are incorporated in full by reference.

BACKGROUND

In mammals, iron is essential for life. Because of its toxicity at high concentrations, iron homeostasis is tightly regulated. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases.

A critical step in iron regulation is intestinal iron absorption. Iron may be absorbed from the diet by duodenal enterocytes. Non-haem iron is reduced by a ferric reductase in the brush border and is transported into the cell through the transmembrane iron transporter DMTl (also known as Nramp2 or DCT1 ). In conditions of iron deficiency the expression of DMTl is upregulated.

Two groups have reported the characterisation of the human DMTl gene from exon 1 to exon 16 (Kishi and Tabushi, 1998) and to exon 17 (Lee et al., 1998). The function of the DMTl protein has been described simultaneously by two different groups (Fleming et ah, 1997; Gunshin et al, 1997). In their characterisation of DMTl, they have shown that DMTl mRNA is upregulated in the duodenum and in the kidney in animals with an iron deficient diet. DMTl has an unusually broad substrate range that includes Fe²⁺, Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺, Cu²⁺, Ni²⁺ and Pb²⁺. Some common human diseases are due to pathological increases and decreases in intestinal iron uptake. The most prevalent iron overload disorder is Hereditary Haemochomatosis (HH) which is genetically inherited and is characterised by increased iron absorption due to impaired regulation of absorption in the duodenum. It is thought that DMTl probably accounts for the increased entry of iron into enterocytes in hemochromatosis. DMTl is believed to function abnormally in the presence of mutant HFE protein (the hemochromatosis protein) or other factors that play a role in hemochromatosis. In HH patients, the duodenal enterocytes are believed to have an iron deficient phenotype, which leads to inappropriately high intestinal iron absorption. HH is caused by mutations in HFE, a gene linked to the human major histocompatibility complex on chromosome 6p. Although the HFE protein resembles an atypical MHC class 1 protein and forms a heterodimer with D2-microglobulin, it does not contain a functional peptide-binding groove. It has been shown to interact with the transferrin receptor to form a high-affinity complex but the role of this complex in the regulation of intestinal iron absorption is not yet fully understood. However, animal models have provided some insights into the pathogenesis of HFE-associated haemochromatosis. Several important conclusions can be drawn from the study of Hfe mutant mice. Three independent knockout experiments have shown the Hfe null mice absorb more iron than normal mice and deposit it in hepatocytes. Comparison of Hfe knockout mice with Hfe knock- in animals, show that the missense mutation results in less iron loading than the null mutation. Also, although mice heterozygous for Hfe mutations accumulate far less iron than Hfe null mutants, they load more iron than wild-type mice, indicating that human HFE mutation carriers may also be predisposed to some iron overloading.

Finally, studies of compound-mutant mice, in which mutations in HFE have been combined with mutations in the genes that encode DMTl or hephaestin, show that these mice have decreased iron loading, indicating that iron loading in mouse haemochromatosis results from increased iron flux through a pathway involving both DMTl and hephaestin. This suggests that subtle mutations in proteins of the iron-transport apparatus may act as genetic modifiers of the severity of diseases such as haemochromatosis.

In view of the involvement of DMTl in iron regulation, and the existence of various diseases associated with aberrant intestinal iron uptake, there is a great need to modulate DMTl activity in the intestine and to identify patients suffering from iron absorption disorders.

THE INVENTION

The invention concerns the identification of an alternative exon of DMTl, herein referred to as exon 1 A, which appears to be expressed in a tissue specific manner and which is conserved in different mammalian species (including human, mouse and rat) and is regulated by iron concentration.

The presence of a novel exon in human Caco2 cells (derived from colon carcinoma) has been determined using 5'RACE PCR. In these cells, two isoforms of the DMTl mRNA have been found, which differ with respect to the 5' sequence. One of these isoforms corresponds to the published DMTl cDNA and the second has a different 5' sequence. This alternative 5' sequence corresponds to the usage of an alternative exon 1 , namely exon 1A, instead of the previously described first exon (IB). By comparison with the human genomic sequence, this novel exon has been mapped at 1.9 kb upstream of the previously determined exon 1 (figure 1 ). This observation leads to a revised genomic organization of the human DMTl gene (figure 2).

Interestingly, the alternative human isoform of the DMTl mRNA, containing the exon 1A, possesses an open reading frame coding for 29 a ino acids in frame with the DMTl protein sequence, leading to an N-terminal extended DMTl protein.

RT-PCR experiments performed with primers specified for each isoform (figure 4) suggest a different expression pattern in the cell lines tested. In the Caco2 cells, the isoform 1A is strongly expressed in comparison to HeLa or 293 cells. The iron treatment experiment of the Caco2 cells shows that this isoform is strongly upregulated in iron deficiency (reflected by the desferal treatment).

Additionally, an alternative 5' variant f DMTl mRNA has been identified in mouse intestine using the same method (figure 3B). The sequence of the exon 1A and IB do not share any similarity. However the mouse and human exon 1A sequences share 40% similarity and the amino acid sequence deduced from the 5' sequence of the new mouse isoform is 66% similar to the human extended N-terminal sequence.

RT-PCR experiments with specific primers show that the distribution of the isoform 1A and IB is different. Whereas the exon IB containing mRNA is expressed ubiquitously, the expression of the exon 1A containing mRNA appears to have a more restrictive distribution pattern (figure 5). Previous authors have stated that expression of DMTl (DMTl isoform IB) is iron-regulated (see e.g. Gunshin et al. (1997) and Cannone-Hergaux (1999). However, their methods could not distinguish between exon 1 A or exon IB containing mRNA. Our studies have identified a new isoform of DMTl , DMTl isoform 1A, that is strongly iron-regulated, while our studies have indicated that expression of the DMTl isoform IB is only weakly iron-regulated. As mentioned above, various iron disorders result from aberrant iron absorption from the intestine. For instance, in HH the duodenal enterocytes are believed to have an iron deficient phenotype, which leads to inappropriately increased iron intestinal absorption. Since the DMTl isoform 1A is strongly iron-regulated and is expressed in the intestine then DMTl isoform 1A (or exon 1A) provides a useful target for modulating intestinal iron absorption. DMTl isoform IB, in contrast, is only weakly iron regulated, is more ubiquitous in its expression and appears to be expressed in the duodenum at relatively modest levels. Both of these properties make DMTl isoform IB a less favourable target for the modulation of intestinal iron absorption than DMTl isoform 1A.

Similarly, because HH is thought to be caused by duodenal enterocytes exhibiting an iron deficient phenotype and because the expression of the DMTl isoform 1A is strongly iron- regulated, then it can be expected that the level of expression of DMTl isoform 1 A (or exon 1A) will be a more sensitive indicator of HH disease. Accordingly, expression of the DMTl isoform 1 A / exon 1 A provides a useful indicator of HH disease.

The use of the DMTl isoform 1 A and exon 1A may have similar advantages in treating and/or diagnosing various other iron disorders. For example, blocking expression of the DMTl isoform 1A may be useful in case of patients suffering from secondary iron overload such as, but not limited to, patients treated by blood transfusion (for example in the treatment of thalassaemia). More broadly, the 1A isoform may play a role in non-intestinal tissues, such as, but not limited to, the brain, the endothelium, the kidney and the joints and may therefore be a target in other tissues where iron metabolism plays a relevant role and/or in disease states such as, for example, Parkinson's, Alzheimer's, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis). In a first aspect, the invention provides an isolated polypeptide, which polypeptide is:

(i) a polypeptide comprising the amino acid sequence encoded by exon 1 A of a naturally occurring DMTl gene and a portion of exon 2 of the naturally occurring DMTl gene, the portion of exon 2 of the DMTl gene being that which is located upstream of, and not including, the ATG start codon used in the IB isoform; or (ii) a functional equivalent of (i).

The polypeptide defined in (i) above is herein referred to as the DMTl exon 1 A polypeptide.

The amino acid sequence that is encoded by exon 1 A and a portion of exon 2 of a naturally occurring DMTl gene, the portion of exon 2 of the DMTl gene being that which is located upstream of, and not including, the ATG start codon used in the IB isoform is herein referred to as the polypeptide 1 A sequence.

The term "exon 1A" includes a naturally occurring DNA sequence which, with regard to the sense strand of a DNA molecule, is in nature located upstream of exon IB (i.e. 5' of exon IB) and which is an alternative exon to exon IB such that, in nature, exon 1A or exon IB are expressed in a mutually exclusive manner, following transcription and splicing to exon2.

The term "exon 1A" also includes a nucleic acid sequence which comprises different bases from a naturally exon 1A DNA sequence but which nevertheless encodes the same amino acid sequence by virtue of the degeneracy of the genetic code or by virtue of the fact that the base alterations are located in a non-coding portion of the exon 1A sequence.

Examples of exon 1A include sequences listed herein as SEQ ID Nos. 3, 4 and 6 which correspond to a human, mouse and rat exon 1 A respectively.

SEQ ID NO.3: ATATATAGAGGCAGGAGCTGGCATTGGGAAAGTCAAACT AGTTCTGCACC ATG AGG AAG AAG CAG CTG AAG ACG GAG GCA GCT CCA CAC TGT GAA CTA A

SEQ ID NO.4: GAAAGTACTCCTCTGCATATATAGAGGCTGCGCTGCTCTG AAAAGCCAAACCAGTCCTGCACC ATG GGG AAG AAG CAG CCA AGG GCG GCA GCA GCT GCT CCC AAC TGT GAG CTA A Further examples of exon 1A include exons that are equivalent to the human, mouse or rat exon 1 A (i.e. homologs of one or more of SEQ ID Nos. 3, 4 or 6) and which may be identified in other animals by those skilled in the art using, for example, database searches, RT-PCR cDNA or genomic library screening.

By an "amino acid sequence encoded by" a given nucleic acid molecule / nucleic acid sequence we refer to the amino acid sequence which corresponds to the codons of the nucleic acid molecule / nucleic acid sequence. The amino acid sequence may be generated in any manner including, for example, by chemical synthesis, DNA replication or reverse transcription.

In contrast, an "amino acid obtained by" a given nucleic acid molecule / nucleic acid sequence refers to an amino acid sequence which is obtained by transcription and / or translation from the said nucleic acid molecule / nucleic acid sequence.

In the case of the amino acid encoded by the human exon 1A and a portion of the human exon 2, the portion of the exon 2 being that which is located upstream of the ATG used in the IB isoform, this amino acid sequence is: M R K K Q L K T E A A P H C E L K S Y S K N S A T Q V S T [SEQ ID 1].

In the case of the amino acid encoded by the mouse exon 1 A and a portion of the mouse exon 2, the portion of the exon 2 being that which is located upstream of the ATG used in the IB isoform, this amino acid sequence is: M G K K Q P R A A A A A P N C E L K S Y S K S T D P Q V S T [SEQ ID 2].

In the case of the amino acid encoded by the rat exon 1 A and a portion of the rat exon 2, the portion of the exon 2 being that which is located upstream of the ATG used in the IB isoform, this amino acid sequence is: MGKKQPRAAASAAPNCELKSYSKSTDPQVST [SEQ ID 5].

The word "comprising", and grammatical variants thereof, is used herein to mean including or consisting.

By an "isolated polypeptide " is meant a polypeptide which is devoid of, in whole or part, tissue or cellular components with which the polypeptide is normally associated in nature. Thus, a polypeptide contained in a tissue extract would constitute an "isolated" polypeptide, as would a polypeptide synthetically or recombinantly produced.

The term "isolated" does not denote the method by which the polypeptide is obtained or the level of purity of the preparation. Thus, such isolated species may be produced recombinantly, isolated directly from the cell or tissue of interest or produced synthetically based on the determined sequences.

Preferably, the DMTl gene is a human, mouse or rat DMTl gene.

Preferably, the DMTl exon 1 A polypeptide further includes an amino acid sequence additional to the polypeptide 1A sequence. Amino acid sequences which are additional to the exon 1A amino acid sequence are herein referred to as "additional amino acid sequences". Preferably, the additional amino acid sequence is an amino acid sequence found in nature at the C-terminal end of a polypeptide 1A sequence. Thus, the additional amino acid sequence may comprise the amino acid sequence encoded by the portion of exon 2 which is located downstream of, and including, the ATG used in the IB isoform. Optionally the additional amino acid sequence may further comprise an amino acid sequence encoded by a nucleic acid sequence located, in nature, downstream of, and preferably contiguous with, exon 2.

Thus, a polypeptide of the first aspect of the invention may comprise the following amino acids:

(i) a polypeptide 1 A sequence;

(ii) the amino acid sequence of a DMTl isoform 1A polypeptide truncated at its C- terminus, wherein the amino acid sequence comprises a polypeptide 1 A sequence.

(iii) the amino acid sequence of a DMTl isoform 1 A polypeptide. By a "DMTl isoform 1A polypeptide" we refer to an isoform 1A polypeptide encoded by a naturally occurring DMTl gene, preferably a human, mouse or rat DMTl gene. A "DMTl isoform 1 A polypeptide" is encoded by a "full complement" of exons, i.e. it is encoded by the set of the exons which encode a naturally occurring, full-length DMTl isoform 1A polypeptide.

By a "human DMTl isoform 1 A polypeptide" we include the amino acid sequence encoded by a nucleic acid sequence consisting of exon 1 A, exons 2 to 15 (using the Lee et al. (1998) or the Kishi and Tabuchi, (1998) exon numbering system) and exons 16 and 16 A (using the Lee et al. (1998) exon numbering system) or exon 16 (using the Kishi and Tabuchi, (1998) exon numbering system).

By a "human DMTl isoform 1A polypeptide" we also include the amino acid sequence encoded by a nucleic acid sequence consisting of: exon 1 A, exons 2 to 15 (using the Lee et al. (1998) or the Kishi and Tabuchi, (1998) exon numbering system), and exons 16 and 17 {using the Lee et al. (1998) exon numbering system). A reference hereinafter to exons 2 to 15 of the human DMTl gene is a reference to exons 2 to 15 using the exon numbering system used in Lee et al (1998) or the exon numbering system used in Kishi and Tabuchi, (1998).

A reference hereinafter to exon 16 of the human DMTl gene is a reference to exon 16 using the exon numbering system used in Lee et al (1998) or a reference to the first 54 nucleotides of exon 16 using the exon numbering system used Kishi and Tabuchi (1998).

A reference hereinafter to exon 17 of the human DMTl gene is a reference to exon 17 using the exon numbering system used in Lee et al (1998).

A reference hereinafter to exons 2 to 17, is a reference to exons 2 to 16, exon 16A and exon 17.

The human DMTl gene sequence can be obtained from GenBank accession #'s AF064475- AF064483. The DMTl gene sequence also appears in DNA Database of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/), EMBL (htttp://www.ebi. ac.uk/embl/) and GenBank Nucleotide Sequence Databases under the accession numbers AB015355 and AF064484.

The sequences of the mouse and rat DMTl genes are not yet publicly available, although the mouse and rat cDNA sequences are available. A reference herein to a mouse or rat DMTl gene includes reference to the mouse and rat DMTl cDNA respectively.

The database accession numbers for the mouse DMTl cDNAs are AF029758 (GenBank) and L33415 (GenBank). The database accession numbers for the rat DMTl cDNAs are AF008439 (GenBank) and AF029758 (GenBank). The first 66 nucleotides of AF008439 (Gunshin et al 1997) may comprise the rat exon 1A: CCACGCGTCCGATGGGGAAGAAGCAGCCGAGGG CAGCAGCAAGTGCTGCTCCAAACTGTGAGCTAA (SEQ ID No. 6). The rat polypeptide 1 A sequence is MGKKQPRAAAS AAPNCELKSYSKSTDPQVST (SEQ ID No. 5).

The reference AF029758 contains a different 5'end. The rat exon IB sequence corresponds to the first 28 nucleotides of the reference AF029758:

CGGGCGGCGTGTGTGGAGGTGGAGGACG.

The terms "polypeptide" and "protein" are used herein interchangeably and refer to any polymer of amino acids linked through peptide bonds or modified peptide bonds, i.e. peptide isosteres. Thus, the terms "polypeptide" and "protein" include oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.

The polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide. In such polypeptides, the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.

Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally- occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.

The modifications that occur in a polypeptide often will be a function of how the polypeptide is made. For polypeptides that are made recombinantly, the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.

The polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically- produced polypeptides or polypeptides that are produced by a combination of these methods.

The functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that, in place of a polypeptide 1A sequence, comprise an amino acid sequence that is homologous to the human, mouse or rat polypeptide 1 A sequence.

The functionally-equivalent polypeptides of the first aspect of the invention also include polypeptides comprising a polypeptide 1 A sequence or an amino acid sequence homologous to the human, mouse or rat exon 1A polypeptide 1A sequence, in addition to an amino acid sequence homologous to an "additional amino acid" sequence as described above. Two amino acid sequences are said to be "homologous", as the term is used herein, if the sequence of one of the amino acid sequences has a high enough degree of identity or similarity to the sequence of the other amino acid sequence. "Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity" indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

Homologous amino acid sequences therefore include mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the polypeptide 1A sequence and the "additional" amino acid sequences. Such mutants may include amino sequences in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of a polypeptide comprising the homologous amino acid sequence in question when compared with a polypeptide comprising a non-mutated version (ie fully functional version) of that amino acid sequence. Also especially preferred in this regard are conservative substitutions. Such homologous amino acid sequences also include amino acid sequences in which one or more of the amino acid residues includes a substituent group. In the case of an amino acid sequence homologous to the amino acid sequence encoded by the human, mouse or rat polypeptide 1A sequence it is preferred that the amino acid sequences have degrees of identity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 98% or 99%.

In the case of an amino acid sequence homologous to an "additional amino acid" sequence, it is preferred that the amino acids have a degree of sequence identity of at least 30%, preferably at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 98% or 99% with the human, mouse or rat sequences.

Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=l 1 and gap extension penalty=l].

The functionally-equivalent polypeptides of the first aspect of the invention also include polypeptides comprising a fragment of an amino acid sequence that is homologous to a human, mouse or rat polypeptide 1A sequence, wherein the polypeptide may be used to raise antibodies which are immunospecific for a polypeptide of the first aspect of the invention.

As used herein, the term "fragment", as applied to a polypeptide 1 A sequence or an amino acid sequence homologous to a human, mouse or rat polypeptide 1A sequence refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of exon 1 A amino acid sequence or an amino acid sequence homologous to a human, mouse or rat polypeptide 1A sequence respectively. The fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 10 or more (for example 12, 14, 16, 18, 20, 22, 24, 26 or more).

The functionally-equivalent polypeptides of the first aspect of the invention also include fusion proteins incorporating the polypeptides described above. For example, it is often advantageous to include one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production. Alternatively or additionally, the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).

Fusion proteins may also be useful to screen peptide libraries for inhibitors of the activity of the DMTl isoform 1A polypeptide. It may be useful to express a fusion protein that can be recognised by a commercially-available antibody. A fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein. By a "heterologous protein", we include a protein which, in nature, is not found in association with a polypeptide of the invention. Preferably, the functionally-equivalent polypeptide of the first aspect of the invention possesses activity characteristic of a naturally occurring DMTl isoform 1A polypeptide, preferably a non-mutated DMTl isoform 1A polypeptide. Preferably, the functionally- equivalent polypeptide of the first aspect of the invention possesses activity characteristic of a naturally occurring human DMTl isoform 1A polypeptide, preferably a non-mutated human DMTl isoform 1 A polypeptide.

Preferably, the above functionally equivalent polypeptides of the first aspect of the invention may be used to raise antibodies which are immunospecific for a polypeptide of the first aspect of the invention.

In one embodiment, the invention provides a polypeptide of the first aspect of the invention obtained from a nucleic acid sequence of the third aspect of the invention.

In a second aspect, the invention provides antibodies immunospecific for a polypeptide of the first aspect of the invention.

The polypeptides of the first aspect of the present invention or their immunogenic fragments (comprising at least one antigenic determinant) can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for a polypeptide of the first aspect of the invention. Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography. The antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader. An antibody which is "immunospecific" for a polypeptide of the first aspect of the invention means that the antibody has a substantially greater affinity for a polypeptide of the first aspect of the invention (e.g. the DMTl isoform 1 A polypeptide) than for other related polypeptides in the prior art, in particular polypeptides comprising the amino acid sequence encoded by exon IB forms of the DMTl gene, such as the DMTl isoform IB polypeptide whose amino acid sequence begins within exon 2. Preferably, the antibodies of the second aspect of the invention have substantially greater affinity for a polypeptide of the invention than a polypeptide comprising the amino acid sequence encoded by exon IB forms of the human, mouse or rat DMTl gene. Such reactivity can be determined by immunoprecipitation and Western blot analysis, using methods well known in the art.

By "substantially greater affinity" we mean that there is a measurable difference between the affinity of the antibody for a polypeptide of the first aspect of the invention compared to the affinity of the antibody for related polypeptides in the prior art, in particular polypeptides comprising the amino acid sequence encoded by exon IB forms of the DMTl gene.

Preferably, the antibody has an affinity for a polypeptide of the first aspect of the invention (e.g. the DMTl isoform 1A polypeptide) of at least 1 μm, 100 nm, 10 nm, lnm, 100 pM, 10 pM or lpM.

If polyclonal antibodies are desired, a selected mammal or suitable animal, such as mouse, rabbit, goat, horse, pig, chicken etc., may be immunised with a polypeptide of the first aspect of the invention or its fragment. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by a variety of methods, such as by immunoaffinity chromatography, using known procedures. The polypeptide used to immunise the animal can be derived by recombinant DNA technology, by purification of the natural protein or can be synthesized chemically. If desired, the polypeptide can be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin. The coupled polypeptide is then used to immunise the animal. Monoclonal antibodies immunospecific for a polypeptide of the first aspect of the invention, and to the fragments thereof, can also be readily produced by one skilled in the art using, e.g., hybridoma technology. The general methodology for making monoclonal antibodies by using hybridoma technology is well known. For example, immortal antibody-producing cell lines can be created by cell fusion, as well as by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al. Hybridoma Techniques (1980); Hammerling et al. Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al. Monoclonal Antibodies (1980); U.S. Pat. Nos. 4,341 ,761 ; 4,399,121 ; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491 ,632; and 4,493,890. Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties; i.e., for isotype, epitope, affinity, etc.

Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.

As used herein, the term "antibody" denotes not only the intact molecule, but also active fragments thereof, such as Fab, F(ab')₂ and Fv, which retain their immunospecificity for a DMTl exon 1A polypeptide of the first aspect of the invention. (See, e.g., Baldwin, R. W. et al. in Monoclonal Antibodies for Cancer Detection and Therapy (Academic Press 1985) for a description of the production of antibody fragments.). The term also contemplates chimeric antibodies that retain immunospecificity for a polypeptide of the first aspect of the invention. In particular, the antibody can include the variable regions or fragments of the variable regions which retain specificity for a polypeptide of the first aspect of the invention. The remainder of the antibody can be derived from the species in which the antibody will be used. The chimeric antibodies may, for example, be made by joining or fusing non-human variable regions to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)) and Neuberger et al (1988, 8th International Biotechnology Symposium Part 2, 792-799)). Thus, if the antibody is to be used in a human, the antibody can be "humanized" in order to reduce immunogenicity yet retain activity. For a description of chimeric antibodies, see, e.g., Winter, G. and Milstein, C. (1991) Nature 349:293-299; Jones et al. (1986) Nature 321 :522- 525; Riechmann et al. (1988) 332:323-327; and Carter et al. (1992) Proc. Natl. Acad. Sci. USA 89:4285-4289. In a further alternative, the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.

Purification of the antibodies of the second aspect of the invention, can be accomplished by conventional techniques such as affinity chromatography or phage display. The binding agent is preferably an antibody or antigen binding fragment thereof such a Fab, Fv, ScFv and Ab, but it may also be any other ligand which exhibits the preferential binding characteristic mentioned above. Affinity chromatography is described in Scopes, R. K. (1993) Protein Purification: principles and practice 3rd Ed. Springer- Verlag, New York, ISBN 0-387-44072-3, 3-540- 94072-3. (See chapters 7 and 9 in particular). Further information on the above affinity chromatography techniques and the immunoassay of antigen and antibody is provided by Roitt (1991) Essential Immunology 7th Ed. Blackwell Scientific Publications, London, ISBN 0-632- 02877-7 (see chapter 5 in particular).

Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., (1991) Nature 352, 624-628). Antibodies generated by the above techniques, whether polyclonal or monoclonal, have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In these applications, the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme. The present invention also provides a method of making an antibody of the second aspect of the invention wherein the method comprises injecting a polypeptide of the first aspect of the invention into an animal and collecting antibodies generated from the animal against the polypeptide.

In a third aspect, the invention provides an isolated nucleic acid molecule which encodes a polypeptide according to the first aspect of the invention, with the proviso that the nucleic acid molecule does not comprise the sequence of exon IB between exon 1A and exon 2. Preferably, the nucleic acid molecule does not comprise exon IB.

Preferably, the isolated nucleic acid molecule does not comprise a nucleic acid sequence homologous with exon IB between exon 1A and exon 2. Preferably, the nucleic acid molecule does not comprise a nucleic acid sequence homologous with exon IB.

Preferably, the isolated nucleic acid molecule does not comprise a nucleic acid sequence which is a fragment of exon IB between exon 1A and exon 2. Preferably, the nucleic acid molecule does not comprise a nucleic acid sequence which is a fragment of exon IB.

Preferably, the isolated nucleic acid molecule does not comprise a nucleic acid sequence which is a fragment of a nucleic acid sequence, which is homologous with exon IB, between exon 1 A of the DMTl gene and exon 2. Preferably, the nucleic acid molecule does not comprise a fragment of a nucleic acid sequence which is homologous with exon IB. By a nucleic acid sequence homologous with exon IB we include nucleic acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical over their entire length to a naturally occurring exon IB of a human, mouse or rat DMTl gene.

Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/).

By "a nucleic acid sequence which is a fragment of exon IB" we include a nucleic acid sequence which comprises at least n consecutive nucleotides from exon IB where n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40, 45, 50 or more). By "a nucleic acid sequence which is a fragment of a nucleic acid sequence, which is homologous with exon IB" we include a nucleic acid sequence which comprises at least n consecutive nucleotides from a nucleic acid sequence which is homologous with exon IB where n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40, 45, 50 or more).

Preferably, the nucleic acid sequence of the third aspect of the invention comprises exons 1A and 2 of a DMTl gene wherein the said exons are contiguous.

Preferably, the nucleic acid molecule comprises the nucleic acid sequence recited in SEQ ID NO:3 (human DMTl exon 1A), SEQ ID NO:4 (mouse DMTl exon 1A) or SEQ ID NO:6 (rat DMTl exon 1A).

Alternatively, the nucleic acid molecule may comprise a fragment of the nucleic acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6,. Preferably, the nucleic acid molecules of the invention comprise at least n consecutive nucleotides from SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6 where n is 10 or more (for example, 10, 15, 20, 25, 30, 35, 40, 50 or more).

Preferably, the nucleic acid molecule comprises one or more of exons 2 to 17 of the human DMTl gene (and / or a fragment of one or more of these exons). Preferably, the nucleic acid molecule comprises at least n consecutive nucleotides from one or more of exons 2 to 17 where, depending on the particular exon, n is 20 or more (for example, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 125, 150, 500, 1000, 1500, 2000 or more).

Preferably the nucleic acid molecule comprises a full complement of DMTl exons. Preferably the nucleic acid molecule comprises the following human DMTl exons:

(i) exon 1 A, exons 2 to 16 and exon 16A; or

(ii) exon 1 A, exons 2 to 16 and exon 17. Preferably, the nucleic acid molecule comprises a naturally occurring exon 1A (e.g. SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.6) and / or one or more of the following: a naturally occurring exon 2; a naturally occurring exon 3; a naturally occurring exon 4; a naturally occurring exon 5; a naturally occurring exon 6; a naturally occurring exon 7; a naturally occurring exon 8; a naturally occurring exon 9; a naturally occurring exon 10; a naturally occurring exon 1 1 ; a naturally occurring exon 12; a naturally occurring exon 13; a naturally occurring exon 14; a naturally occurring exon 15; a naturally occurring exon 16; a naturally occurring exon 16A; and a naturally occurring exon 17. Preferably the exon(s) is/are human, mouse or rat exons. By a "naturally occurring exon sequence" we include a DMTl exon which occurs in nature, for example the exons of the human DMTl gene which are disclosed in Lee et al (1988) and in Kishi & Tabuchi (1988) and naturally-occurring variants thereof, such as a naturally-occurring allelic variant. It should be noted that where a nucleic acid sequence or amino acid sequence is referred to herein as being naturally occurring we refer to the sequence of the nucleic acid or polypeptide as being naturally occurring and not to the nucleic acid or polypeptide per se being naturally occurring. Thus, a naturally occurring exon sequence may, for example, be made by chemical synthesis, provided that the nucleic acid sequence of the exon occurs in nature.

Preferably the nucleic acid molecule comprises a full complement of exons which exons are naturally occurring exons. Preferably, the nucleic acid molecule comprises one or more nucleic acid sequences which are at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical over their entire length with one or more naturally occurring exons of a DMTl gene (preferably the human DMTl gene). Preferably, the nucleic acid molecule comprises a nucleic acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical over its entire length with a naturally occurring exon 1 A, preferably a naturally occurring human, mouse or rat exon 1 A.

By an "isolated nucleic acid molecule" we include a nucleic acid molecule which is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or; a nucleic acid sequence, as it exists in nature, but linked to a polynucleotide other than that to which it is linked in nature. The "isolated nucleic acid molecule" may comprise the promoter and/or other expression-regulating sequences which normally govern its expression and it may comprise introns, or it may consist of the coding sequence only, for example a cDNA sequence. The term "isolated" does not denote the method by which the nucleic acid molecules are obtained or the level of purity of the preparations. Thus, such isolated species may be produced recombinantly, isolated directly from the cell or tissue of interest or produced synthetically based on the determined sequences. Preferred nucleic acid molecules according to the third aspect of the present invention include nucleic acid molecules that encode polypeptides which possess DMTl activity or nucleic acid molecules which hybridize under conditions of high stringency to a naturally occurring exon 1A nucleic acid sequence (e.g. a nucleic acid sequence comprising a nucleic acid sequence in which exon 1A is contiguous with exon 2) but not to: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB mRNA lacking exon 1A but comprising exon IB) under high stringency conditions; and or (ii) not to a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1A is contiguous with exon 2 under high stringency conditions.

The nucleic acid molecules according to the third aspect of the present invention include variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions, but which are nevertheless capable of encoding a polypeptide of the first aspect of the invention or which are capable of hybridizing under conditions of high stringency to a naturally occurring exon 1A nucleic acid sequence but not to: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB gene lacking exon 1A but comprising exon IB) under high stringency conditions; and/or (ii) not to a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1 A is contiguous with exon 2 under high stringency conditions.

The substitutions, deletions or insertions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.

In a fourth aspect of the invention, the invention provides an isolated nucleic acid sequence which comprises one or more of the following: a fragment of exon 1A; a fragment of a nucleic acid sequence in which exon 1A is contiguous with exon 2; a homolog of a fragment of exon 1A; or a homolog of a fragment of a nucleic acid sequence in which exon 1A is contiguous with exon 2, wherein the said fragment or homolog is capable of hybridizing under conditions of high stringency to a naturally occurring exon 1A nucleic acid sequence but not to: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB mRNA lacking exon 1 A but comprising exon IB) under high stringency conditions; and/or (ii) a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1 A is contiguous with exon 2 under high stringency conditions.

By a "fragment of exon 1A" we include a nucleic acid sequence which is a fragment of a naturally occurring exon 1A, wherein the fragment encodes a fragment of a polypeptide 1A sequence having at least n consecutive amino acids where, n is 5 or more (for example, 7, 9, 1 1 , 12, 13, 14, 15, 16). Preferably the fragment of exon 1A is a fragment of a human, mouse or rat exon 1A. Preferably, the naturally occurring exon 1A is a non-mutated human, mouse or rat exon 1 A.

By a "fragment of a nucleic acid sequence in which exon 1A is contiguous with exon 2" we include a nucleic acid sequence which is a fragment of a naturally occurring nucleic acid sequence in which exon 1 A is contiguous with exon 2, wherein the fragment encodes at least n consecutive amino acids of an exon 1A amino acid where, « is 5 or more (for example, 7, 9, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29). Preferably the fragment of the nucleic acid sequence in which exon 1A is contiguous with exon 2 is a fragment of a human, mouse or rat nucleic acid sequence. Preferably the fragment of the nucleic acid sequence in which exon 1A is contiguous with exon 2 is a fragment of a non- mutated human, mouse or rat nucleic acid sequence.

By a "homolog of a fragment of exon 1A" or "a homolog of a fragment of a nucleic acid sequence in which exon 1A is contiguous with exon 2" we refer to a nucleic acid sequence which is at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98%, 99% identical over its entire length with a fragment of exon 1A or a fragment of a nucleic acid sequence in which exon 1A is contiguous with exon 2 respectively.

The nucleic acid molecules of the present invention may encode a leader or secretory sequence, such as a sequence encoding a pro-, pre- or prepro- polypeptide sequence.

The nucleic acid molecules of the present invention may comprise non-coding sequences, including introns, non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), translation, RNA processing, RNA transport, RNA localisation and mRNA stability.

Preferably, the nucleic acid molecules of the present invention comprise one or more regulatory sequences which are operably linked to the coding sequence(s) of the nucleic acid molecule. Examples of regulatory sequences include promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Preferably, the nucleic acid molecules of the present invention comprise one or more non- coding sequences that are characteristic of DMTl genes. For example, the nucleic acid molecules of the present invention may comprise an iron responsive element in the 3' untranslated region.

"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual biological function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

The nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.

The nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the polypeptide. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences. Site-directed mutagenesis may also be used to insert new restriction sites, change codon preference, produce splice variants, introduce mutations and so forth. The nucleic acid molecules of the invention also include nucleic acid sequences which "correspond" to those nucleic acid molecules described above. By a nucleic acid molecule which corresponds to a nucleic acid molecule described above, we refer to nucleic acid molecules which have: (i) the same base sequence as the above nucleic acid molecules; or (ii) the same base sequence as the above nucleic acid molecules save for the thymine (T) residues being replaced by uracil (U) residues.

The nucleic acid molecules of the invention also include nucleic acid molecules which are fully complementary (i.e. 100% complementary) to the nucleic acid molecules described above. Such nucleic acid molecules may be useful for antisense or probing purposes. Preferred nucleic acid molecules according to the third aspect of the present invention include nucleic acid molecules that encode polypeptides which possess DMTl activity or nucleic acid molecules which hybridize under conditions of high stringency to a naturally occurring exon 1A nucleic acid sequence (e.g. a nucleic acid sequence comprising a nucleic acid sequence in which exon 1A is contiguous with exon 2) but not to: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB mRNA lacking exon 1A but comprising exon IB) under high stringency conditions; and/or (ii) not to a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1A is contiguous with exon 2 under high stringency conditions. Those skilled in the art will appreciate that antisense molecules, such as oligonucleotides, can be designed to recognise, hybridize under conditions of high stringency to, and prevent transcription of a naturally occurring exon 1A nucleic acid sequence. Preferably, the antisense molecule does not recognise, hybridize under conditions of high stringency to, and prevent transcription of: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB mRNA lacking exon 1A but comprising exon IB) under high stringency conditions; and or (ii) a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1A is contiguous with exon 2 under high stringency conditions.

With regard to antisense techinques see, for example, Cohen, J.S., Trends in Phar . Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al. Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).

Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA. Alternatively, the nucleic acid molecules of the present invention may be in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA or a combination thereof.

The nucleic acid molecules of the invention may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences or chemically. The nucleic acid molecules of the present invention may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

Double strand RNA molecules corresponding to exon 1A forms can be used to trigger RNAi (RNA interference) and mediate changes in DMTl expression. RNAi technology is discussed in Bosher and Labouesse (2000); and by Carthew (2001). Advances in this field in cultured cells have been made by the group of Tuscl (see Elbashir, et al. 2001) allowing this technique to be applied to any mammalian cultured cells

The term "nucleic acid molecule" also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA). The term "PNA", as used herein, refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine. The terminal lysine confers solubility to the composition. PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).

The invention also provides a method of detecting a nucleic acid molecule, which nucleic acid molecule preferably comprises a naturally occurring exon 1A or a naturally occurring nucleic acid sequence in which exon 1A is contiguous with exon 2, the method comprising the steps of: (a) contacting a nucleic acid sequence probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.

The term "hybridization" as used here refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al. Molecular Cloning; A Laboratory Manual, Third Edition (2001)). The inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al [supra]). A substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399- 407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-51 1).

"Stringency" refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ. High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C. Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]). Preferably, the conditions used for hybridization are those of high stringency.

As discussed additionally below in connection with assays that may be utilised according to the invention, a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding a DMTl isoform 1A polypeptide. In this regard, the following techniques, among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein. Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD). Preferably, the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

One method for isolating a nucleic acid molecule encoding a DMTl isoform 1A polypeptide, is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992). Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, exon 1A are preferred. Such probes may be labelled with an analytically-detectable reagent to facilitate their identification. Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.

When screening for full-length cDNAs, it is preferable to use libraries that have been size- selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non- transcribed regulatory regions.

In a fifth aspect, the invention provides a vector, such as a cloning or expression vector, that comprises a nucleic acid molecule of the third or fourth aspects of the invention. The vector may be, for example, a bacterial vector (eg a plasmid) or a viral vector (eg a bacteriophage), provided that such clones are in isolation from clones constituting a DNA library of the relevant chromosome.

In a sixth aspect, the invention provides a host cell transformed with a vector of the fifth aspect of the invention.

The host cells of the invention, which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.

In a seventh aspect, the invention provides methods of recombinantly producing polypeptides of the first aspect of the invention. The polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression". Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).

Generally, any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al, (supra). Generally, the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.

Examples of suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids. Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.

Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems. Cell-free translation systems can also be employed to produce the polypeptides of the invention.

Introduction of nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 2001 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.

The encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. Leader sequences can be removed by the bacterial host in post-translational processing.

In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions. Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site. For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines. In the baculovirus system, the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac" kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.

There are many plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30, 3861-3863 (1991). In particular, all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene. Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.

Examples of particularly suitable host cells for fungal expression include yeast cells (for example, S. cerevisiae) and Aspergillus cells. Any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex vims thymidine kinase (Wigler, M. et al. (1977) Cell 1 1 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt± cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1- 14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.

Although the presence or absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the relevant sequence is inserted within a marker gene sequence, transformed cells containing the appropriate sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

Alternatively, host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 121 1-1216).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end- labelling or PCR amplification using a labelled polynucleotide. Alternatively, the sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).

Suitable reporter molecules or labels, which may be used for ease of detection, include radionucleides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

The polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.

Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins. Examples of such purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif. 3: 263-281) while the thioredoxin or enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D.J. et al. (1993; DNA Cell Biol. 12:441-453). If the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If the polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

In an eighth aspect, the invention provides a compound which specifically affects the amount of DMTl isoform 1A polypeptide produced from a DMTl gene or which specifically affects the activity of the DMTl isoform 1A polypeptide. Preferably, the compound is a ligand which binds to the DMTl gene or to a polypeptide of the first aspect of the invention respectively.

By "specifically affects" we mean that the decrease or increase in the amount of DMTl isoform 1 A polypeptide produced from a DMTl gene or in the decrease or increase activity of the DMTl isoform 1 A polypeptide is substantially greater than the decrease or increase in the amount of DMTl IB polypeptide produced from a DMTl gene or the decrease or increase in the activity of a DMTl IB polypeptide when exposed to the same compound.

By "substantially greater" we mean that the absolute increase or decrease in the activity of the DMTl isoform 1 A polypeptide is at least 50%, 75%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% 1500% or 2000% greater than the absolute increase or decrease in the activity of the DMTl isoform IB polypeptide.

Preferably, the compound has no effect, or only a negligible effect, on the expression of the DMTl isoform IB gene or the activity of a DMTl isoform IB polypeptide whose amino acid sequence begins within exon 2. A compound of the eighth aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.

A ninth aspect of the invention provides screening methods that are capable of identifying a compound according to the eighth aspect of the invention.

The polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention. Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, aptamers, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).

Aptamer techniques are discussed in Brody, Willis et al. 1999; Brody and Gold 2000; Hesselberth, Robertson et al. 2000; and Toulme 2000. The aptamers of the invention are specific for a polypeptide of the first aspect of the invention, that is the aptamer has a substantially greater affinity for a polypeptide of the first aspect of the invention (e.g. the DMTl isoform 1A polypeptide) than for other related polypeptides in the prior art, in particular polypeptides comprising the amino acid sequence encoded by exon IB forms of the DMTl gene, such as the DMTl isoform IB polypeptide whose amino acid sequence begins within exon 2. Preferably, the aptamers have substantially greater affinity for a polypeptide of the invention than for a polypeptide comprising the amino acid sequence encoded by exon IB forms of the human, mouse or rat DMTl gene.

By "substantially greater affinity" we mean that there is a measurable difference between the affinity of the aptamer for a polypeptide of the first aspect of the invention compared to the affinity of the aptamer for related polypeptides in the prior art, in particular polypeptides comprising the amino acid sequence encoded by exon IB forms of the DMTl gene. Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it. Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.

The polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. In general, such screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound. Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.

Alternatively, simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor. In another embodiment, competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide. Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564). In this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed. One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. The polypeptides of the first aspect of the invention may be used to identify membrane-bound or soluble interaction partners, through standard binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative interaction partner (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids). The efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy. Binding assays may be used for the purification and cloning of the interaction partner, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its interaction partner. Standard methods for conducting screening assays are well understood in the art.

The invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, interaction partners, substrates, enzymes, that are described below. The invention includes the agonists, antagonists, ligands, interaction partners, substrates and enzymes, and other compounds which modulate the expression, activity or antigenicity of the polypeptide of the invention discovered by the methods that are described below.

In a tenth aspect, the invention provides a polypeptide of the first aspect of the invention, an antibody of the second aspect of the invention, a nucleic acid molecule of the third or fourth aspect of the invention, a vector of the fifth aspect of the invention, a host cell of the sixth aspect of the invention, a method of the seventh aspect of the invention, a compound of the eighth aspect of the invention, or an aptamer of the invention for use in medicine, in particular in therapy or diagnosis.

The aptamers of the invention are specific for a polypeptide of the first aspect of the invention, that is the aptamer has a substantially greater affinity for a polypeptide of the first aspect of the invention (e.g. the DMTl isoform 1A polypeptide) than for other related polypeptides in the prior art, in particular polypeptides comprising the amino acid sequence encoded by exon IB forms of the DMTl gene, such as the DMTl isoform IB polypeptide whose amino acid sequence begins within exon 2. Preferably, the aptamers have substantially greater affinity for a polypeptide of the invention than for a polypeptide comprising the amino acid sequence encoded by exon IB forms of the human, mouse or rat DMTl gene.

By "substantially greater affinity" we mean that there is a measurable difference between the affinity of the aptamer for a polypeptide of the first aspect of the invention compared to the affinity of the aptamer for related polypeptides in the prior art, in particular polypeptides comprising the amino acid sequence encoded by exon IB forms of the DMTl gene.

Preferably, the various moieties of the invention are for use in therapy or diagnosis of a disease in which iron metabolism plays a relevant role. Preferably, the various moieties of the invention are for use in therapy or diagnosis of hereditary haemochromatosis, Parkinson's disease, Alzheimer's disease, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis).

One aspect of the invention relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques. Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 1 17-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.

In one embodiment, this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease. The method may comprise the steps of: a) contacting a sample of tissue or fluid from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease. A further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue or fluid sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue or fluid sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease. To aid the detection of nucleic acid molecules in the above-described methods, an amplification step, for example using PCR, may be included.

Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures. The presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand. Such diagnostics are particularly useful for prenatal and neonatal testing.

Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single- strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)). For example, a sequencing primer may be used with double-stranded PCR product or a single- stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags. Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. Further, point mutations and other sequence variations, such as polymorphisms, can be detected as described above, for example, through the use of allele- specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.

DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, Proc. Natl. Acad. Sci. USA (1985) 85: 4397- 4401). In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane. Fluorescence in situ hybridization (FISH) is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).

In another embodiment of the invention, an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M. Chee et al, Science (1996), Vol 274, pp 610-613). In one embodiment, the array is prepared and used according to the methods described in PCT application WO95/1 1995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675- 1680); and Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93: 10614-10619). Oligonucleotide pairs may range from two to over one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251 1 16 (Baldeschweiler et al). In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.

In addition to the methods discussed above, diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays). This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex. Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression. Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.

Antibodies and aptamers which are immunospecific or specific respectively for a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by aberrant expression of the DMTl polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules known in the art may be used, several of which are described above. Aptamers may be employed in a similar manner.

Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal_studies, in clinical trials or in monitoring the treatment of an individual patient. A diagnostic kit of the present invention may comprise:

(a) a nucleic acid molecule of the present invention;

(b) a polypeptide of the present invention; or

(c) a ligand of the present invention. In one aspect of the invention, a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease. The kit may further comprise a third container holding an agent for digesting unhybridised RNA.

In an alternative aspect of the invention, a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.

To detect a polypeptide according to the invention, a diagnostic kit may comprise one or more antibodies or aptamers that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody or aptamer and the polypeptide.

Such kits will be of use in diagnosing a disease or susceptibility to disease for example, Hereditary Haemochomatosis, other iron disorders, Parkinson's, Alzheimer's, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis). In an eleventh aspect, the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a DMTl isoform 1 A polypeptide, or the activity of a DMTl isoform 1A polypeptide encoded thereby, from a biological sample (e.g. tissue or fluid) from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease. Such a method will preferably be carried out in vitro. Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.

Preferably, the disease is a disease in which iron metabolism plays a relevant role. Preferably, the disease is hereditary haemochromatosis, Parkinson's disease, Alzheimer's disease, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis). The antibodies or aptamers of the invention can be used as diagnostic reagents, to screen biological samples (e.g. tissues and fluids) for the presence or absence of the DMTl isoform 1A polypeptide, or for the presence or absence of aberrant DMTl isoform 1A polypeptides, allowing for identification of individuals with iron absorption diseases, as well for the identification of carriers of the disease and the determination of individuals likely to develop such disease.

For example, the presence of DMTl isoform 1A polypeptides can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, or enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the DMTl isoform 1A polypeptides and the antibodies described above.

A preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody of the second aspect of the invention, or an aptamer of the invention, with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex. A number of different such methods according to the eleventh aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient. The invention also provides kits that are useful in these methods for diagnosing disease.

In a twelfth aspect, the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, an antibody of the second aspect of the invention a nucleic acid molecule of the third or fourth aspect of the invention, a vector of the fifth aspect of the invention, a compound of the eighth aspect of the invention, or an aptamer of the invention in conjunction with a pharmaceutically-acceptable carrier. Preferably the pharmaceutical composition is effective in treating a disease involving aberrant iron metabolism. Preferably, the pharmaceutical composition is effective in treating hereditary haemochromatosis, Parkinson's disease, Alzheimer's disease, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis).

The invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, or as other immunogenic compositions, as outlined in detail below.

According to the terminology used herein, a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.

The pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention. The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

A pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent. Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

The pharmaceutical compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means. Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.

If the activity of the polypeptide of the invention is in excess in a particular disease state, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, interaction partners, or by inhibiting a second signal, and thereby alleviating the abnormal condition. Preferably, such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.

In another approach, soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, interaction partner, in question, may be administered. Typically, the polypeptide may be administered in the form of fragments that retain the relevant portions.

In an alternative approach, expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). The complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.

In addition, expression of the polypeptide of the invention may be prevented by using ribozymes specific to its encoding mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non- natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non- traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases. For treating abnormal conditions related to an under-expression of the polypeptide of the invention and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described below, to alleviate the abnormal condition. Alternatively, a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.

Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene. Gene therapy of the present invention can occur in vivo or ex vivo. Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.

The therapeutic gene is typically "packaged" for administration to a patient. Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66

(1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top.

Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479. For example, a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector. This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches,

(and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read,

BIOS Scientific Publishers Ltd). Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.

In a thirteenth aspect, the present invention provides a polypeptide of the first aspect of the invention, an antibody of the second aspect of the invention a nucleic acid molecule of the third or fourth aspect of the invention, a vector of the fifth aspect of the invention, a compound of the eighth aspect of the invention, or an aptamer of the present invention for use in the manufacture of a medicament for the diagnosis or treatment of a disease, preferably a disease related to iron metabolism.

Preferably, the disease is hereditary haemochromatosis, Parkinson's disease, Alzheimer's disease, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis).

In a fourteenth aspect, the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, an antibody of the second aspect of the invention, a nucleic acid molecule of the third or fourth aspect of the invention, a vector of the fifth aspect of the invention, a compound of the eighth aspect of the invention or an aptamer of the invention.

Preferably, the disease involves aberrant iron metabolism. Preferably, the disease is hereditary haemochromatosis, Parkinson's disease, Alzheimer's disease, Ischemia Reperfusion Injury or local inflammatory states (such as Rheumatoid Arthritis). For diseases involving aberrant expression of the novel isoform of DMTl, in which the expression and or activity a DMTl isoform 1 A polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist or, in the case of a nucleic acid molecule, nucleic acid construct or vector administered to a patient, it should encode an agonist.

Conversely, for diseases in which the expression and / or activity of a DMTl isoform 1A polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist or, in the case of a nucleic acid molecule, nucleic acid construct or vector administered to a patient, it should encode an antagonist. Examples of such antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies. In a fifteenth aspect, the invention provides transgenic or knockout non-human animals, particularly rodent animals, that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention, preferably a DMTl isoform 1A polypeptide. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals are very useful models for the study of disease and may also be used in screening regimes for the identification of compounds that are effective as modulators of the polypeptides of the present invention and compounds which are effective in the treatment or diagnosis of diseases.

It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and it is not intended that this terminology should limit the scope of the present invention. The extent of the invention is limited only by the terms of the appended claims.

Standard abbreviations for nucleotides and amino acids are used in this specification. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art.

Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following (and more recent editions of the following): DNA Cloning, Volumes I and II (D.N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription and Translation (B.D. Hames & S.J. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. 1987, Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.); and Handbook of Experimental Immunology, Volumes 1-1V (D.M. Weir and C. C. Blackwell eds. 1986).

Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Identification of an alternative exon 1. Sequence of 5' region of the human DMTl gene. The alternative exon, namely exon 1 A is located upstream of the previously determined exon 1, annotated exon IB in this figure. The capital letters correspond to the exonic sequences.

Figure 2: Revised genomic organization of human DMTl . PA: promoter of the exon 1 A, PB: promoter of exon 1.

Figure 3: Sequence of the alternative 5' isoforms of human (A) and mouse (B) DMTl . The corresponding deduced amino acid sequence of the each isoform is represented. The vertical bars represent the boundaries between exon 1 A or B and exon2.

Figure 4: Iron regulation of the different isoforms of DMTl in human cell lines. RT-PCR experiment using total RNA from different cell line: HeLa (lane 1 to 3), 293 (lane 4 to 6), Caco2 (lane 7 to 9)C, treated with Hemin (H: lane 2, 5, 8); with desferrioxamine (D: lane 3,6, 9) or untreated (C: lane 1, 4, 7). Figure 5: Distribution of 5' variant of DMTl in mouse tissue. RT-PCR was performed using total RNA from wt mouse tissue (lane 1 to 17), from mouse macrophage cell line (line 18 and 19), from experimental mouse with control diet (lane 20) or iron deficient diet (lane 21). Dl, D2 and D3 samples correspond respectively to the first, the second and the third centimetre of the duodenum. Figure 6: Western blot of mouse duodenal extracts from mouse fed with a control diet (lane 1 , 3, 5) or an iron deficient diet (lane 2, 4, 6). Upper panel: Detection using an antibody raised against the lA-peptide (CELK). This anti-lApeptide antibody was pre-incubated without any peptide (lane 1 and 2), or with an unrelated peptide (KPSQ, lane 3 and 4), or with the peptide used to raise the antibody (CELK, lane 5 and 6). Lower panel: Detection using an anti-UlA antibody as loading control.

EXAMPLES

Example 1

1. Materials and Methods Cell culture and Iron treatment

CaCo2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μM non-essential aminoacids, 1 mM sodium pyruvate, 100 Unit/1 penicillin and 100 Unit/1 streptomycin. HeLa cells and 293 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 Unit/1 penicillin and 100 Unit/1 streptomycin. For iron regulation experiments, cells were incubated for 10 to 12 hours in medium supplemented with 100 μM Hemin or 100 μM Desferrioxamine.

5'RACE (5' Rapid Amplification of cDNA Ends)

Amplification of 5' ends of human DMTl Total RNA was isolated from CaCo2 cells treated with Desferrioxamine using the RNA clean reagent (Hybaid) according to the manufacturer's instructions. To amplify the 5' end of the human DMTl mRNA, we used the Gene Racer kit (Invitrogen). 5 μg of total RNA from Caco2 cells treated with Desferrioxamine were prepared according the manufacturer's protocol. The full-length mRNA ligated to Gene Racer oligo was reverse transcribed using Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) and a gene specific primer 5'CACGGGTGGCTTCTTCTGTCAGCAG3'. The amplification of 5' ends of the cDNA was performed using the Advantage 2 polymerase Mix (Clontech) with the Gene Racer 5' Primer and a DMTl specific primer. The cycling parameters were: 1 cycle at 95°C for 1 min, 25 cycles at 95°C for 30 sec and lmin 30sec at 68°C, and 1 cycle 2min at 68°C. A nested PCR was performed using the Gene Racer 5'Nested Primer and a second DMTl specific primer 5'TTCTTCTGTCAGCAGGCCTTTAGAGATGC3'. The product of the second amplification was purified on gel, extracted and cloned into pCR4-TOPO (Invitrogen). The relevant clones were sequenced using DNA sequencing kit (USB).

Amplification of 5' ends of mouse DMTl Total RNA from mouse duodenum was prepared using the TRIzol Reagent (GibcoBRL) according to the manufacturer's instruction. The cDNA was synthesized from 5 μg total RNA using Random Primers according to the Gene Racer kit 's instruction. The first PCR amplification was performed using the Gene Racer 5' Primer and the mouse DMTl specific 5'CTAGGTAGGCAATGCTCATAAGAAAGCCAGG3' and 35 cycles of the settings described previously. The second amplification was performed for 30 cycles with the Gene Racer 5'Nested Primer and a second mouse DMTl specific primer 5'CATAAGAAAGCCAGGCCCCGTGAACGCC3'. The products of PCR were processed as described for the 5'RACE of human DMTl. RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction)

Total RNA was isolated using the RNA clean reagent (Hybaid). The first strand of cDNA was obtained by reverse transcription from 5 μg of total RNA using 1 μg of oligodT primers and the Superscript II RT kit (Gibco BRL) according to manufacturer's instruction. This step is followed by Rnase H treatment to eliminate RNA from the RNA:DNA duplex. The Polymerase Chain Reactions were carried out from l/20^th of the cDNA mix as template using the AmpliTaq DNA Polymerase (Perkin-Elmer Biosystem). The amplification of the cDNA were performed at 94°C for 30sec, 55°C for 40sec and 72°C for 40 sec for 16 to 30 cycles depending of the concentration of cDNA and the sets of primers used, listed in Table 1. The PCR products were loaded on ethidium bromide stained gels, scanned and quantified using the Fuji FLA2000 Fluorlmager. The reported quantification for mRNA levels are normalised with respect to Gapdh levels. cDNA Name* 5' primer 3' primer hGapdh CCATGGAGAAGGCTGGGG CAAAGTTGTCATGGATGACC hDMTl

1A-16A GGAGCTGGCATTGGGAAAGTC TGGCTTCTTCTGTCAGCAG

1A-17 GGAGCTGGCATTGGGAAAGTC CTGAGCTGTCAATCCCAGATG

1B-16A GGAGCTGGCATTGGGAAAGTC TGGCTTCTTCTGTCAGCAG

1B-17 GGAGCTGGCATTGGGAAAGTC CTGAGCTGTCAATCCCAGATG mDMTl

16A CTGCTGAGCGAAGATACCAG CTCAGGAGCTTAGGTCAGAAG

17 CGCCCAGATTTTACACAGTG AAGCTTCACTACCTGCACAC

1A-3 GTACTCCTCTGCATATATAGAGG CTAGGTAGGCAATGCTCATAAGAAAGCCAGG

1B-3 CAATCACGGGAGGGCAGGAG CTAGGTAGGCAATGCTCATAAGAAAGCCAGG Table 1 : Sets of primers used in this study. * The name of the primers sets referred to the corresponding exon number.

2: Identification of a novel exon in human DMTl gene

To determine the precise 5' part of the human DMTl mRNA, a 5'Rapid Amplification of cDNA Ends (RACE) was performed using total RNA from CaCo-2 cells treated with Desferrioxamine as a template. The sequencing of the PCR product revealed the presence of two forms of DMTl cDNA, which differ in the 5' region. One isoform corresponds to the published DMTl cDNA and the second has a different and unrelated 5' sequence. The comparison of the two alternative sequences with the human genomic sequence of DMTl has shown that the second 5' sequence corresponds to the usage of an alternative exon 1. This novel exon has been mapped at 1 ,9 kb upstream the previously determined exon 1 (figure 1). This observation leads to a revised genomic organization of the human DMTl gene (figure 2). Due to their respective localisation on genomic sequence, the novel exon 1 has been named exonl A and the previously described first exon, exon IB.

Interestingly, the alternative isoform of the DMTl mRNA, containing the exon 1 A, possesses an open reading frame encoding for 29 amino acids in frame with the DMTl protein sequence, leading to an extended DMTl protein (figure 3A). This additional amino acid sequence does not share any similarity with any known protein or domain.

3. The alternative 5' isoform is conserved in mouse

To determine whether the presence of this alternative exon is conserved in mouse, a 5'RACE was performed using total RNA from mouse duodenum. Using the same strategy, an alternative 5' variant of DMTl mRNA in mouse was identified (figure 3B). The 5' nucleotide sequence of the two isoforms doesn't share any similarity with each other or with the human sequence. However the deduced amino sequence of the 5' sequence of the new mouse isoform is 66% similar to the human extended N-terminal sequence. 4. The expression of the exon 1A is iron regulated

Due to the presence of this alternative exon 1 , the DMTl gene can generate four forms of mRNA which differ by the 5' part, due to the usage of the exon 1 A or IB, and by the 3' region, due to alternative splicing of exon 16A or 17. To determine which isoforms are present and iron regulated, we have investigated their expression in different human cell lines and in different iron condition. Three cell lines, HeLa, 293 and Caco2, were treated with Hemin (an iron donor), desferrioxamine (an iron chelator) or untreated (control).To monitor the change in mRNA expression, quantitative RT-PCR was performed using a specific set of primers for each isoforms (figure 4). This experiment shows that the exon IB is expressed in all cell lines but it is poorly iron regulated. In contrast, the exon 1 A is strongly expressed in the Caco2 cells and shows a strong upregulation in iron deficiency (reflected by the desferal treatment).

5. The expression of the exon 1 is tissue specific

To identify the expression pattern of the exon 1 A, RT-PCR experiments were performed using total RNA from different mouse tissue and organs (figure 5). This experiment shows that the distribution of the isoform 1A and IB is different. Whereas the exon IB containing mRNA is ubiquitously expressed, the expression of the exon 1A containing mRNA shows a restrictive distribution in the intestinal tract, mainly the duodenum, and in the kidney. Interestingly, in previous observation, these two tissues have shown the strongest upregulation in dietary iron deficiency.

In conclusion, we have identified an alternative exon 1 in the human DMTl gene and its homolog in mice. This novel exon is expressed in a tissue specific manner, mainly in the duodenum, which is the main tissue responsible for the iron absorption at the body level. Furthermore, the expression of the novel exon is regulated by cellular iron concentration. Interestingly, the novel human exon contains an open reading frame that encodes for an extra 29 amino-acid peptide, in the N terminal part of the DMTl protein. Although our studies have indicated that the novel exon is expressed mainly in the duodenum, the 1A isoform may also play a role in extra intestinal tissues, such as, but not limited to, the brain, the endothelium, the kidney and the joints.

Example 2

Protocols

Animals

Experiments were performed with the C57BL6 inbred mouse strain. For this study, weaned animals (about four week old) were fed for four weeks with a control diet (C1000) or an iron deficient diet (C1038) (Altromin, GmBH, Germany). Efficiency of iron deprivation was ascertained by comparison of serum iron levels (29.3 ± 6.8 μmol/1 receiving the control and 5.0 ± 1.8 μmol/1 receiving the iron deficient diet) and of hemoglobin levels (15.0 ± 1.0 g/dl and 11.9 ± 0.7 g/dl, respectively).

Antibody Production and Purification

Rabbit polyclonal sera were raised against synthetic peptides which sequence (CELKSYSKSTDPQVS) corresponds to the 15^th to 28^th amino acid of the mouse DMT1- 1A additional peptide (SIGMA). Crude serum was affinity purified on GST-mouse 1A peptide (amino acid 1-30 from mouse peptide 1A sequence) fusion protein covalently linked to glutathion agarose beads.

Preparation of duodenal extracts

The 1^st cm of duodenum was removed after death from mouse with a control or an iron defiecient diet and immediately frozen into liquid nitrogen. Samples kept frozen were homogenized on ice in lysis buffer containing lOmM TRisHCl pH8, 150mM NaCl, ImMEDTA, 1% Nonidet P40, 0,1 % SDS supplemented by a cocktail of protease inhibitor Complete™ from Roche. The lysate were incubated 20 min at 4°C and centrifuged at 13 OOOg, 4°C. The protein concentration of the supernatant (= duodenal extract) was determined by Bradford assay (BioRad).

Immunoblot

20 μg of total protein of duodenal extract from control mouse or dietary iron deficient mouse were separated on 10% SDS-PAGE and transferred to PVDF membranes. The blot were then incubated 1H at room temperature with affinity purified anti-mDMTl-lApeptide diluted 1:50 in blocking solution (PBS containing 0,1% tween and 3% low fat milk). For blocking peptide experiment the 1^st antibody was pre-incubated 1H at room temperature with lμg/ml of peptide (1A peptide corresponds to the sequence CELKSYSKSTDPQVS , the unrelated peptide corresponds to the sequence KPSQSQVLRGMFVPSC which is present in an internal loop of DMTl (positions 230-245 of previously published DMTl protein sequence, Ace Nos:P49282). After incubation with the primary antibody and several washes in blocking solution, the membranes were incubated with peroxidase labeled anti-rabbit secondary antibody for 1H at room temperature, and the signal was visualized by Chemiluminescence Reagent Plus from Perkin Elmer according to the manufacturer's instructions. Results

To determine whether the novel isoform of DMTl containing the exon 1A could encode for an extended protein, rabbit polyclonal antibody was raised against a peptide sequence corresponding to the 15^th to 28^th amino acid of the mouse DMT1-1A additional predicted peptide. This antibody was incubated with a membrane containing duodenal extract from control mouse or dietary iron deficient mouse (figure 6, lane 1 and 2). To assess the specificity of the antiserum, peptide blocking experiment were performed. This experiment shows, first of all that the antiserum recognize specifically a signal from 80 to 90 kDa which corresponds to different glycosylated forms of DMTl (Canonne-Hergaux et al., 1999) in iron deficient mouse extract. This observation indicates that the additional peptide encoded by the 1A isoform of DMTl mRNA is indeed expressed and so that the 1A isoforms of DMTl mRNA give rise to extended versions of the protein. Secondly, this experiment shows that the level of expression of DMTl protein is very low in duodenal extract from control mouse whereas it is very high in duodenal extract from iron deficient mouse. This result suggests that the 1A isoform of DMTl is highly regulated by iron levels.

REFERENCES

I . Fleming, M.D., Trenor, C.C., 3rd, Su, M.A., Foernzler, D., Beier, D.R., Dietrich, W.F. and Andrews, N.C. (1997) Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet, 16, 383-386. 2. Gunshin, H., Mackenzie, B., Berger, UN., Gunshin, Y., Romero, M.F., Boron, W.F.,

Nussberger, S., Gollan, J.L. and Hediger, M.A. (1997) Cloning and charterization of a mammalian proton-coupled metal-ion transporter. Nature, 388, 482-488.

3. Kishi, F. and Tabuchi, M. (1998) Human natural resistance-associated macrophage protein 2: gene cloning and protein identification. Biochem Biophys Res Commun, 251 , 775-783.

4. Lee, P.., Gelbart, T., West, C, Halloran, C. and Beutler, E. (1998) The human Nramρ2 gene: characterisation of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis, 24, 199-215.

5. Canonne-Hergaux, F., S. Gruenheid, et al. (1999). "Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron." Blood 93(12): 4406-17.

6. Bosher, J. M. and M. Labouesse (2000). "RNA interference: genetic wand and genetic watchdog." Nat Cell Biol 2(2): E31-6.

7. Carthew, R. W. (2001). "Gene silencing by double-stranded RNA." Curr Opin Cell Biol 13(2): 244-8.

8. Elbashir, S. M., J. Harborth, et al. (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells." Nature 41 1(6836): 494-8.

9. Brody, E. N. and L. Gold (2000). "Aptamers as therapeutic and diagnostic agents." J Biotechnol 74(1): 5-13. 10. Brody, E. N., M. C. Willis, et al. (1999). "The use of aptamers in large arrays for molecular diagnostics." Mol Diagn 4(4): 381-8.

I I . Hesselberth, J., M. P. Robertson, et al. (2000). "In vitro selection of nucleic acids for diagnostic applications." J Biotechnol 74(1): 15-25.

12 . Toulme, J. J. (2000). "Aptamers: selected oligonucleotides for therapy." Curr Opin Mol Ther 2(3): 318-24. 3. Canonne-Hergaux, F., Gruenheid, S., Ponka, P., and Gros, P. (1999). Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron. Blood 93, 4406-17.

Claims

1. An isolated polypeptide, which polypeptide is: i) a polypeptide comprising a polypeptide 1 A sequence that consists of an amino acid sequence encoded by exon 1 A of a naturally occurring DMTl gene and a portion of exon 2 of the said naturally occurring DMTl gene, the portion of exon 2 of the DMTl gene being that which is located upstream of, and not including, the ATG start codon used in the IB isoform; or ii) a functional equivalent of (i).

2. An isolated polypeptide according to claim 1 , wherein the polypeptide 1A sequence is a human, mouse or rat polypeptide 1 A sequence.

3. An isolated polypeptide according to claim 2, wherein the polypeptide 1A sequence is the sequence set forth in SEQ ID NO:l , SEQ ID NO:2 or SEQ ID NO:5.

4. An isolated polypeptide according to any one of claims 1 to 3, wherein the isolated polypeptide is a DMTl isoform 1 A polypeptide truncated at its C-terminus.

5. An isolated polypeptide according to any one of claims 1 to 4, wherein the polypeptide is a DMTl isoform 1 A polypeptide.

6. An isolated polypeptide according to claim 5, wherein the isolated polypeptide is a human, mouse or rat DMTl isoform 1 A polypeptide.

7. An antibody which is immunospecific for a polypeptide according to any one of claims 1 to 6.

8. An antibody according to claim 7, wherein the antibody is a monoclonal antibody.

9. An isolated nucleic acid molecule which encodes a polypeptide according to any one of claims 1 to 6, with the proviso that the nucleic acid molecule does not comprise the sequence of DMTl exon IB between a DMTl exon 1 A and a DMTl exon 2.

10. An isolated nucleic acid molecule according to claim 9, wherein the nucleic acid molecule comprises exons 1 A and 2 of a DMTl gene wherein the said exons are contiguous.

1 1. An isolated nucleic acid molecule according to claim 9 or claim 10, wherein the nucleic acid molecule comprises the nucleic acid sequence recited in SEQ ID NO:3 (human DMTl exon 1 A), SEQ ID NO:4 (mouse DMTl exon 1 A) or SEQ ID NO:6 (rat DMTl exon 1 A).

12. An isolated nucleic acid molecule according to any one of claims 9 to 11, wherein the nucleic acid molecule comprises one or more of exons 2 to 17 of the human DMTl gene.

13. An isolated nucleic acid molecule according to any one of claims 9 to 1 1 , wherein the nucleic acid molecule comprises a full complement of exons of the mouse or rat DMTl gene.

14. An isolated nucleic acid sequence which comprises one or more of the following: a fragment of exon 1A; a fragment of a nucleic acid sequence in which exon 1A is contiguous with exon 2, which fragment encodes at least 5 consecutive amino acids of an exon 1 A amino acid; a homolog of a fragment of exon 1 A; or a homolog of a fragment of a nucleic acid sequence in which exon 1 A is contiguous with exon 2, wherein the said fragment or homolog is capable of hybridizing under conditions of high stringency to a naturally occurring exon 1 A nucleic acid sequence but not to: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB mRNA lacking exon 1A but comprising exon IB) under high stringency conditions; and/or (ii) a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1 A is contiguous with exon 2 under high stringency conditions.

15. An isolated nucleic acid molecule which corresponds to a nucleic acid molecule according to any of claims 9 to 14.

16. An isolated nucleic acid molecule which is fully complementary to a nucleic acid molecule according to any of claims 9 to 15.

17. An isolated nucleic acid molecule according to claims 15 or 16 wherein the nucleic acid molecule is an antisense molecule which can recognise, hybridize under conditions of high stringency to, and prevent transcription of a naturally occurring exon 1A nucleic acid sequence, with the proviso that the antisense molecule does not recognise, hybridize under conditions of high stringency to, and prevent transcription of: (i) a naturally occurring nucleic acid sequence which lacks exon 1A (e.g. a DMTl isoform IB mRNA lacking exon 1A but comprising exon IB) under high stringency conditions; and/or (ii) a naturally occurring nucleic acid sequence which does not comprise a nucleic acid sequence in which exon 1 A is contiguous with exon 2 under high stringency conditions.

18. A vector which comprises a nucleic acid molecule according to any one of claims 9 to 17.

19. A host cell transformed with a vector according to claim 18.

20. A method of recombinantly producing a polypeptide according to any one of claims 1 to 6, the method comprising culturing a cell according to claim 19.

21. A compound which specifically affects the amount of DMTl isoform 1A polypeptide produced from a DMTl gene or which specifically affects the activity of a DMTl isoform 1 A polypeptide.

22. A compound according to claim 21 wherein the compound is a ligand.

23. A screening method that is capable of identifying a compound according to claim 21 or claim 22.

24. A screening method according to claim 23 wherein the method comprises detecting the effect of an added test compound on the production of mRNA encoding a polypeptide according to any one of claims 1 to 6.

25. A screening method according to claim 24 wherein the mRNA encodes a DMTl isoform 1 A polypeptide.

26. A screening method according to claim 23 wherein the method utilises an ELISA that measures secreted or cell-associated levels of DMTl isoform 1A polypeptide using a monoclonal or polyclonal antibody.

27. An aptamer which has a substantially greater affinity for a polypeptide according to any one of claims 1 to 6 than for a DMTl isoform IB polypeptide.

28. A polypeptide according to any one of claims 1 to 6, an antibody according to claim 7 or 8, a nucleic acid molecule according to any one of claims 9 to 17, a vector according to claim

18, a host cell according to claim 19, a compound according to claim 21 or 22, or an aptamer according to claim 27 for use in medicine.

29. A method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a DMTl isoform 1A polypeptide, or the activity of a DMTl isoform 1A polypeptide encoded thereby, from a biological sample (e.g. tissue or fluid) from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.

30. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 6, an antibody according to claim 7 or 8, a nucleic acid molecule according to any one of claims 9 to 17, a vector according to claim 18, a host cell according to claim 19, a compound according to claim 21 or 22, or an aptamer according to claim 27 in conjunction with a pharmaceutically-acceptable carrier.

31. The use of a polypeptide according to any one of claims 1 to 6, an antibody according to claim 7 or 8, a nucleic acid molecule according to any one of claims 9 to 17, a vector according to claim 18, a host cell according to claim 19, a compound according to claim 21 or 22, or an aptamer according to claim 27 in the manufacture of a medicament for the diagnosis or treatment of a disease.

32. A method of treating a disease in a patient comprising administering to the patient a polypeptide according to any one of claims 1 to 6, an antibody according to claim 7 or 8, a nucleic acid molecule according to any one of claims 9 to 17, a vector according to claim 18, a host cell according to claim 19, a compound according to claim 21 or 22, or an aptamer according to claim 27.

33. A method according to claim 29 or 32, or the use of claim 31 wherein the disease is hereditary haemochromatosis, Parkinson's disease, Alzheimer's disease, Ischemia Reperfusion Injury or an inflammatory disease, such as Rheumatoid Arthritis.

34. A transgenic or knockout non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide according to any one of claims 1 to 6.