CA2333853A1 - Mucins - Google Patents

Abstract

prflated MUC nucleic acids are provided which correspond to a Mucin gene located on human chromosome 7q22, or on a mammalian chromosome structurally or functionally equivalent thereto, which Mucin gene is normally predominantly expressed in the colon. Also provided are diagnostic and therapeutic uses of isolated MUC nucleic acids, MUC polypeptides encoded thereby and anti-MUC mA b.

Description

TITLE
"MUCINS"
f=IELD OF THE INVENTION
THIS INVENTION relates generally to nucleic acids corresponding to mammalian Mucin genes, and to polypeptides encoded thereby. More particularly, the present invention provides isolated nucleic acids which correspond to Mucin regulatory genes that are predominantly expressed in the colon. These Mucin genes are associated with disease conditions including colorectal cancer, breast cancer, cystic fibrosis, respiratory diseases, inflammatory bowel disease, ulcerative colitis and Crohn's disease and/or any other conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins. In particular, the present invention provides methods for the diagnosis and therapy of the abovementioned disease conditions.
BACN;GROUND OF THE INVENTION
The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly the case in cancer research. However, despite the effectiveness of this powerful technology, progress has been slow in developing effective recombinant DNA-derived therapeutic or diagnostic agents for cancers. One difficulty has been a lack of understanding of many cancers and other disease conditions. Regulatory genes are an imporlrant component of these complex regulatory mechanisms.
Cancer suppressor genes, for example, are regulators of cell growth and differentiation (Weinberg et al., 1995, Ann. NY Acad. Sci. 758 331 ). The paradigm for their role in cancer is that they are traps-acting and recessive at the cellular level; loss of one homologue has no effect on cell function and homozygous inactivation is required for carcinogenesis (Cavenee et al., 1983, Nature 305 779).
Colorectal cancers contribute to a major proportion of the mortality and morbidity associated with cancer development. There is a particular need, therefore, to understand the complex regulatory mechanisms associated with colorectal cancers as well as cancers in anatomically adjacent regions.
The epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues. They are characterised by a central polymorphic tandem repeat structure, which comprises most of the protein backbone, and .a large number of O-linked carbohydrate side chains (Gum et al., 1995, Biochem. Soc. Traps. 23 795). The complex structure and large size of these molecules makes it difficult to characterise them using classical biochemical techniques. The genes are also difficult to clone because of their large size and the presence of GC-rich tandem repeats.
Ten mucin genes havE: been identified; MUC3, MUC4, MUCSAC, MUCSB, MUC6 and MUC8 have been partially cloned and full-length cDNA clones are available for MUC1, MUC2, MUC7 and MUC9.
Mucins .are known to contribute to pathology in a number of epithelial diseases including cystic fibrosis (CF), inflammatory bowel disease (IBD) and adenocarcinomas. Gastrointestinal mucins which have been described to date include: the transmembrane mucins MUC1 and MUC4; the gel-forming mucins MUC2, MUCSAC and MUC6; and MUC3 which has an unclear structure and function.
As used herein, Mucin genes or isolated nucleic acids corresponding thereto will be expressed in italicized form as MUC. Mucin polypeptides will be expressed as MUC.
Immunohistochemical staining and Western blotting analysis with mature MUC1-specific antibodies revealed that MUC1 became ectopically expressed in colorectal tumours and levels were significantly higher in primary tumours of patients with metastases. Experimentally increased expression of gel-forming mucins resulted in increased metastasis in colon cancer cells in xenograft metastasis models (Ho et al., 1995, Int. J.
Oncol. 7 913). Northern blot analysis has been employed to investigate expression of MUC9, MUC2, MUC3 and MUC4 in paired normal and colonic tumour tissues and in nine colorectal cancer (CRC) cell lines (Ogata et al., 1992, Cancer Res. 52 5971 ). MUCH and MUC4 were present in colonic mucosa with similar expression levels in carcinomas, but occasionally elevated levels of MUC4 were apparent. Levels of MUC2 and MUC3 were decreased by varying degrees in the tumours of most patients. There was no apparent correlation between the expression of any mucin gene and the site, stage or histological type of tumour. All four mucin genes were expressed at low levels or not at all in the nine CRC cell lines under investigation; MUCH transcripts were detected in COL0205, MUC2 and MUC4 probes hybridised weakly to all nine cell lines, and MUC3 expression was observed in five of the lines. Using a combination of in situ hybridisation and immunohistochemistry, Chang et a! (Chang et al., 1994, Gastroenterology 107 ~'.8) also found MUC2 and MUC3 were downregulated in CRC. A more recent in situ hybridisation study found expression of MUC2 and MUC3 mRNA was markedly reduced in poorly, moderately and well-differentiated colorectal tumours but preserved in mucinous carcinomas (Weiss et ai., 1996, ,J. Histochem. Cytochem. 44 1161 ). It is noted that MUC3 is located on human chromosome 7q22, or an equivalent location on other mammalian chromosomes, and is primarily expressed under normal conditions in the small intestine (Shekels et al., 1998, Biochem J. 330 1301 ).
OBJECT OF THE INVENTION
The present inventors have realized that the Mucins constitute an incomplete family of genes and gene products implicated in a variety of disease conditions. Surprisingly, the present inventors have identified novel Mucin genes located on human chromosome 7q22, and isolated novel nucleic acids corresponding thereto. Furthermore, the present inventors have found that these; novel Mucin genes are predominantly expressed in the colon, and may be involved in cancer of the large bowel, cystic fibrosis, breast cancer, inflammatory bowel disease, ulcerative colitis respiratory diseases and Crohn's disease and/or any ather conditions associated with WO 00/04142 PCT/AlJ99/00579 aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
It is therefore an object of the invention to provide novel Mucin genes and isolated nucleic acids corresponding thereto.
SUMMARY OF THE INVENTION
The present invention is broadly directed to an isolated MUC
nucleic acid which corresponds to a MUC gene located on mammalian chromosome 7q22, or on a mammalian chromosome structurally or functionally equivalent thereto, which MUC gene is normally predominantly expressed in the colon.
In a first aspect, the MUC gene of the present invention is MUC11.. Accordingly, "a MUC11 nucleic acid" means an isolated nucleic acid of the invention which corresponds to the MUC11 gene.
PreferabVy, the isolated MUC11 nucleic acid comprises a nucleotide sequence Encoding an amino acid sequence which comprises SGLSEESTTSHSSPGSTHTTLSPASTTT (SEQ ID NO: 1 ).
More preferably, the isolated MUC11 nucleic acid comprises a nucleotide sequence encoding the amino acid sequence according to SEQ
ID N0:3.
Even more preferably, the isolated MUC11 nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 2.
In a second aspect, the MUC gene of the present invention is MUC12. Accordingly, ".a MUC12 nucleic acid" means an isolated nucleic acid of the invention which corresponds to the MUC12 gene.
Preferat~ly, the isolated MUC12 nucleic acid comprises a nucleotide sequence encoding an amino acid sequence which comprises SGLSQESTTFHSSPGSTETTLAPASTTT (SEQ ID N0: 4).
More preferably, the isolated MUC12 nucleic acid comprises a nucleotide sequence encoding the amino acid sequence according to SEQ
ID N0:6.
Even more preferably, the isolated MUC12 nucleic acid comprises a nucleotide sequence according to SEQ ID NO: 5.
In a third aspect, the present invention resides in an isolated MUC polypeptide.
In one embodiment, the isolated MUC polypeptide has an 5 amino acid sequence according to SEQ 1D NO: 3, hereinafter referred to as a "MUC11 polypeptide".
In another embodiment, the isolated MUC polypeptide has an amino acid sequence according to SEQ ID N0:6, hereinafter referred to as a "MUC12 polypeptide".
In a fourtlh aspect, the present invention resides in an antibody specific for a MUC poiypeptide (hereinafter referred to as an anti-MUC
antibody).
Preferably, the anti-MUC antibody is selected from the group consisting of:-(i) an anti-MUC11 IgM monoclonal antibody hereinafter referred to as M11.9; and (ii) an anti-MUC12 IgM monoclonal antibody hereinafter referred to as M12.15.
In a fifth aspect, the present invention resides in methods of detecting a MUC gene, a MUC gene transcript or a MUC polypeptide. The fifth aspect extends to methods for detecting a polymorphism, deletion, mutation, truncation or expansion in a MUC gene, a MUC gene transcript or a MUC polypeptide, or detecting a level of expression thereof. One embodiment of the fifth aspect is directed to use of an isolated MUC nucleic acid to determine whether a mammal has a disease condition, or a predisposition thereto. Another embodiment is directed to use of an isolated MUC polypeptide to determine whether a mammal has a disease condition, or a predisposition thE:reto.
In a sixth aspect, the present invention provides a method of gene therapy of a disease condition in a mammal, said method including administering to said mammal a gene therapy construct which includes an isolated MUC nucleic acid as hereinbefore defined, to thereby alleviate one or more symptoms of ;>aid disease condition in said mammal.
fn a seventh aspect, the present invention provides a method of treating a disease condition in a mammal, said method comprising the step of administering to said mammal a pharmaceutically effective amount of a MUC polypeptide or an anti-MUC antibody.
In an eigth aspect, the present invention resides in a pharmaceutical composition comprising a MUC polypeptide or anti-MUC
antibody, together with a pharmaceutically acceptable carrier andlor diluent.
Preferably, the mammal is a human.
As used herein, the "disease condition" is associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
Preferably, the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis, asthma and chronic bronchitis.
More preferably, the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD) and breast cancer (BC).
As used herein, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(A): Autoradiograph of a differential display gel showing amplified products from RNA isolated from matched normal colon (N) and primary colorectal tumor (P) tissues. Differentially expressed bands dd29 (MUC12) and dd34 (MUC11) are arrowedl.

FIG. 1(B): Northern blot analysis of total RNA from patient 101 hybridized with the dd29 probe to detect a MUC12 gene transcript (mRNA). Signal corresponding to 18S ribosomal RNA is shown as a loading control.
FIG. 1{C): Northern blot analysis of RNA from patient 112 hybridized with the dd34 probe to detect a MUC11 gene transcript (mRNA) .
Signal corresponding to 18S ribosomal RNA is shown as a loading control.
FIG. 1(D): Multiplex semi-quantitative RT-PCR showing amplification of MUC12 mRNA transcripts from matched normal colonic mucosa and primary tumor # 40, normal mucosa from patient # 81 and six colorectal cancer cell lines. Amplification of ~2 microglok~ulin ((i2 MG) is included as a measure of total RNA.
FIG. 1(E): Multiplex semi-quantitative RT-PCR showing amplification of MUC11 nnRNA transcripts in matched normal colonic mucosa and primary tumors of patients # 40, 164, and 97 and six colorectal cancer cell lines. Amplification of (i2-microglobulin ((3z-MG) its included as a measure of total RNA.
FIG. 1 (F): Multiplex: semi-quantitative RT-PCR showing amplification of MUC12 mRNA transcripts from matched normal colonic mucosa and primary tumors # 346, 84, 128, 97 and 316 and from five unpaired Dukes' stage D tumors (M) # 93, 361, 107, 357 andl 367. Amplification of (32-microglobulin (~ -~VIG) is included as a measure of total RNA.
FIG. 1(G): Multiplex semi-quantitative RT-PCR showing MUC11 mRNA
transcripts in matched normal colonic mucosa and primary tumors of patients # 110, 346, 84, 128, and 348 and from five unpaired Dukes' stage D tumors (M) # 93, 107, 361, 367 and 357. Amplification of pz-microglobulin (biz MG) is included as a measure of total RNA. Ma denotes molecular size markers in FIG 1 D-G.

FIG. 2: Predicted amino acid sequence of MUC92. Numbering of amino acids is given on the right. The consensus sequence of the degenerate tandem repeat structure is shown at the top.
The two cysteine-rich EGF-like domains are double underlined, a potential coiled-coil domain is in bold, the hydrophobic domain singly underlined and potential N-glycosylation sites shaded. The stop codon is denoted by an asterisk.
F1G. 3: Amino acid sequence alignment of the carboxyl termini of MUC12, hMUC3 (amino acids 1-366), mMuc3 (Shekels et al., 1998, supra; amino acids 637-1015), rMuc3 (Gum et al., 1991, supra; K.hatri et al., 1997, Biochem. Biophys. Acta 1326 7;
amino acids 356-447 and 1-379 respectively), hMUC4 (Moniaux et al., 1998, Biochem. J. 338 1998; amino acids 861-1156) and rMuc4 (Sheng et al., 1992, J. Biol. Chem. 267 16341; amino acids 451-744). Light shading demonstrates identity with MUC12 and dark shading highlights all cysteine residues. Hyphens indicate gaps inserted to optimize the alignment.
FlG.4: Predicted amino acid sequence of MUC11 showing the degenerate tandem repeat structure. The consensus sequenG~ is shown at the top and amino acids not consistent with this aequence are shown in bold. Hyphens indicate gaps placed in order to optimize the amino acid alignment. A
potential N-glycosylation site is shaded.
FIG. 5: mRNA tissue distribution of the 7q22 mucin gene family. Only those tissues showing a positive signal by Northern blot analysis are represented in the histogram. Sixteen tissues of neural origin, heart, aorta, skeletal muscle, bladder, stomach, testis, ovary, spleen, pituitary gland, adrenal gland, thyroid gland, salivary gland and mammary gland were negative for mucin nnRNA expression. Expression was quantified by densitometry and is shown as a proportion of the tissue showing highest expression.
FIG.6: Domain organization of the C-termini of human MUC12, hMUC3, the rodent Muc3 mucins and the rat and human MUC4 mucins. l'he relative size of domains is accurate except that the N-glycasylated domain adjacent to the mucin domain in MUC4 is shown at approximately one fifth of its actual size. Only the beginning of the large mucin domains are shown.
FIG. 7: Alignment of the first extracellular EGF-like domain of MUC12 with human EGF-like growth factors. Dark shading highlights identical amino acids and light shading indicates conservative amino acid substitutions.
FIG. 8: Schematic representation of MUC 11 cloning (A) and MUC 12 cloning (B).
FIG. 9: Normal colonic expression patterns of MUC11 (A, B) and MUC12 (C) poiypeptides as determined by anti-MUC mAb M11.9 and M12.15 immunostaining, respectively. (D) shows MUC 11 gene transcript (mRNA) expression detected by in sifu hybridization in normal colonic epithelium and loss of expression in CRC (top right).
FIG. 10: Expression of MUC11 and MUC12 mRNA in normal colon as detectedl by RT-PCR. Cytokeratin 20, (CIC20) a colonic epithelia) marker, was employed as a loading control. 'RC' denotes right colon, 'TC' the transverse colon, 'LC' the left colon, ';iC' sigmoid colon; 'CA' refers to the caecum and 'R' denotes the rectum.
FIG.11: Expression of MUC11 and MUC12 mRNA in CRC cell lines as detected by RT-PCR. The loading control is (iZ microglobulin (B2MG) and 'M' denotes the molecular weight marker.
FIG. 12: Expression of MUC11 and MUC12 mRNA in IBD as detected by RT-PC;R. Cytokeratin 20 (CK20) a colonic epithelial marker, was empNoyed as a loading control. 'N' denotes tissues which appear macroscopically normal and 'D' refers to tissues reported t.o have IBD. 'CA' refers to the caecum, 'CO' the colon, 5 'LC' the 4eft colon, 'TC' the transverse colon, 'RS' the recto-sigmoid colon, 'Sf the small intestine, 'IL'denotes the ileum and 'IP' an ileal pouch.
F1G. 13: Expression of MUC11 and MUC12 mRNA in BC as detected by RT-PCR. The loading control is (i2 microglobulin denoted by 10 B2MG and the molecular weight marker is denoted by 'M'. The positive control was normal colonic cDNA from patient 164.
FIG 14: Northern blot analysis of MUC11 expression in normal colon (N) and primary CRC (P) of six patients, assessed using a probe corresponding to dd34. The position of ribosomal RNAs are indicated, and signal from 18S ribosomal RNA was used as a loading control.
DETAILED DESCF:IPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated in part on the identification of novel MUC11 and MUC12 genes which are normally predominantly expressed in the colon. The isolated MUC nucleic acids and MUC genes of the invention may be useful in treatment and diagnosis of disease conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins. Such disease conditions include but are not limited to cancer of the large bowel (CRC), cystic fibrosis {CF), inflammatory bowel disease (IBD), respiratory diseases such as asthma and chronic bronchitis>, breast cancer (BC), ulcerative colitis and Crohn's disease.
The present invention is particularly directed to cancers of the large bowel, which includes the colon, rectum and anal canal, such as CRC, although it extends t.o biochemically, physiologically andlor genetically related cancers in other parts of the gastrointestinal tract.

The MUC; genes are, for example, down-regulated in CRC.
By "predominantly expressed" is meant that a MUC gene transcript or MUC polypeptide encoded by said MUC gene is expressed in the colon at a level greater than in any other organ.
By "associated with" is meant that the disease condition displays symptoms consistent with aberrant Mucin expression, altered properties of mucus or Epithelial inflammation involving Mucins. The disease association may be merely correlative or may reflect a causative role of Mucins in the disease condition.
The term "cancer" is used in its broadest sense to include malignant tumours, carcinomas and sarcomas.
In light of the foregoing, it will be appreciated that a MUC
nucleic acid "corresponds to" a MUC gene by being an isolated nucleic acid derived from said MUC gene, or a portion thereof. Thus it will be understood that said gene has components including amino acid coding sequences and non-coding sequences. Non-coding sequences include, for example, introns and regulatory sequences which include a promoter, translation initiation and termination sequences and a polyadenylation sequence, for example. The isolated MUC nucleic acid may therefore correspond to some or all of the aforementioned components of the corresponding MUC gene.
It should be noted that MUC terminology has recently undergone revision. In particular, MUC12 was formerly known as dd 29 or MUC10. Also, MUC11 was formerly known as dd 34. Therefore, with this in mind, should the term "MUC10" or "dd29" be encountered herein, it should in all cases be taken to mean MUC12.
It will also be understood that a MUC polypeptide is encoded by an isolated MUC nucleic acid or by a MUC gene as hereinbefore defined.
Isolated MUC nucleic acids of the invention may be in DNA
(e.g. cDNA or genomic DNA), RNA {e.g. mRNA) or hybrid DNA:RNA form, eithre in double-stranded or single-stranded form. For example, single-stranded MUC nucleic acids include nucleic acids having sequences complementary to the nucleotide sequences of SEQ ID N0:2 and SEQ ID
N0:5.
In one embodiment, the isolated MUC nucleic acid of the invention comprises a nucleotide sequence having at least 60% identity to the nucleotide sequence according to SEQ 1D N0:2, or a nucleotide sequence capable of hybridizing thereto under at least low stringency conditions.
In another embodiment, the isolated MUC nucleic acid of the invention comprises a nucleotide sequence having at least 60% identity to the nucleotide sequence according to SEQ ID N0:5, or a nucleotide sequence capable of hybridizing thereto under at least low stringency conditions.
According to these embodiments, it is preferable that the nucleotide sequence has at least 75% identity.
More preferably, the nucleotide sequence has at least 90%
sequence identity.
The terra "identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, such as but not limited to the Geneworl~s program (Intelligenetics). For this purpose, BLAST
family programs may also be useful (Altschul et al., 1997, Nucl. Acids Res.
3389, which is herein incorporated by reference). A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which 25 is herein incorporated by reference.
According to these embodiments, it is preferable that the nucleotide sequence is capable of hybridizing under medium stringency conditions.
More preferably, the nucleotide sequence is capable of hybridizing under high stringency conditions Reference herein to low stringency conditions includes and encompasses from at least about 1 % v/v to at least about 15% vlv formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C.
Low stringency conditions also include 1 % Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2xSSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM
EDTA, 40 mM NaHP04 (pH 7.2), 5% SDS for washing at room temperature.
Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridisation at 42°C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42°C.
Medium stringency conditions also include 1 % Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2 x SSC, 0.1 % SDS; or (ii) 0.5% BSA, 1 mM
EDTA, 40 mM NaHP0,4 (pH 7.2), 5% SDS for washing at 42°C.
High stringency includes and encompasses from at least about 31 % vlv to at least about 50% v/v formamide and from at least about 0.01 M
to at least about 0.15 M salt for hybridisation at 42°C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42°C.
High stringency also includes 1 % BSA, 1 mM EDTA, 0.5 M
NaHP04 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 0.2 x SSC, 0.1 SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHP04 (pH 7.2), 1 % SDS for washing at a temperature in excess of 65°C.
In general, washing is carried out at Tm = 69.3 + 0.41 (G + C) % _ -12°C. However, 'the Tm of a duplex DNA decreases by 1 °C
with every increase of 1 % in the number of mismatched based pairs.
Although the MUC genes and isolated MUC nucleic acids of the present invention are exemplified in relation to the human mammalian species, the present invention extends to orthologs in non-human mammals such as in primates, laboratory test animals (e.g. mice, rates, rabbits, guinea pigs, hamsters), companion animals (e.g. dogs, cats), livestock animals (e.g.

sheep, pigs, horses, donkeys, cows) and captive wild animals (e.g. deer, fox).
In light o~f the foregoing, the term "MUC homologsr is used to encompass MUC orthologs, isolated nucleic acids which hybridize to MUC
nucleic acids of the invention and isolated nucleic acids which display at least 60% sequence identity to isolated MUC nucleic acids.
It will also be appreciated that MUC homologs encompass single or multiple nuclE:otide substitutions, deletions andlor additions to the isolated MUC nucleic acids of the invention, inclusive of mutants, fragments, parts, portions and segments of the nucleotide sequences of the invention.
The isolated MUC nucleic acids of the present invention and homologs thereof therefore include oligonucleotides, primers (such as for PCR), antisense sequences, molecules suitable for use in co-suppression and fusion nucleic acid molecules. Ribozymes are also contemplated by the present invention. It will be understood that probes, primers and antisense sequences correspond to distinct portions of isolated MUC nucleic acids of the invention, in that they contain nucleotide sequences based on said distinct portions of an isolated MUC nucleic acid sequence. Such probe and primer sequences may be based on a MUC sequence of the invention by being identical thereto, or by being degenerate with respect thereto.
As used herein, "oligonucleotides" are nucleic acids which comprise a contiguous sequence of no more than seventy (70) nucleotides, whereas "polynucleotides" are nucleic acids which comprise a contiguous sequence of more than seventy (70) nucleotides. A "probe" may be an oligonucleotide or a polynucleotide, either double-stranded or single-stranded, for use in hybridization techniques such as Northern blotting, Southern blotting or in situ hybridization. The skilled person will realize that in situ hybridization also includes Fluorescence In Situ Hybridization (FISH), which is used for determining chromosomal localization. In situ hybridization techniques applicable to the present invention will be described in detail hereinafter.
A "primer" is a nucleic acid (usually an oligonucleotide) capable WO 00/04142 PCT/ALl99/00579 of annealing to a nucleic acid template under appropriate conditions of ionic strength and temperai:ure, which annealed primer can be extended in a template-dependent fashion by a suitable nucleic acid polymerase (for example Taq polymerase or SequenaseT""~. It will therefore be understood 5 that primers of the invention may be useful for PCR, sequencing, RACE, primer extension and the like.
In use, isolated MUC nucleic acids, probes and primers may be modified such as by end-labeling with ~zP-ATP and T4 polynucleotide kinase or by random primed labeling with 32P-dCTP and DNA polymerase.
70 Biotinylation is also contemplated, as is modification with phosphorothiorates, fluorochromes, digoxigenin, enzymes and peptides, for example.
It is contemplated that diagnostic methods may be employed which utilize isolated M~JC nucleic acids of the present invention, or portions thereof such as probes and PCR primers. Also, diagnostic methods 15 employing MUC polypeptides will be discussed in more detail hereinafter.
Diagnostic methods may include detection of MUC genes, transcripts and/or polypeptides in samples such as fecal specimens and/or in colonic biopsies, analysis of serum MUC levels in patients with epithelial diseases including cancers, breast tissue biopsy samples or in respiratory mucus samples from patients suffering from CF, asthma or chronic bronchitis.
The diagnostic methods of the present invention may therefore be applicable to determining whether an individual has a disease condition associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins, or a predisposition to said disease. It will be appreciated that "predispositionn as used herein refers to an increased probability that an individual will contract the disease.
However, it will also be appreciated that the diagnostic methods may also indicate whether an individual actually suffers from the disease, assist in assessing the severity of disease, a prognosis of the likely course of disease and appropriate treatments for the disease. Thus, the diagnostic methods of the invention may be useful whether or not the individual suffers from one or 1fi more symptoms of the disease.
The present invention therefore contemplates methods of detecting MUC genes and MUC gene transcripts (e.g. mRNA), such as involving hybridization techniques (for example, by Northern or Southern blotting or in situ hybridization) or polynucleotide sequence amplification techniques (for example RT-PCR). Such methods may detect:-(i) a polymorphism, deletion, mutation, expansion, and/or truncation in a MUC gene or MUC gene transcript; and (ii) a relative level of expression of a MUC gene transcript (an mRNA transcript derived from a MUC gene).
Such methods of detection facilitate determination of whether said MUC gene is aberrantly-expressed as an indication of a disease condition or a predisposition thereto. Also, MUC gene poiymorphisms, deletions, mutations, truncations or deletions may be detected which indicate a disease condition or a predisposition thereto.
It will be appreciated, for example, that measurement of a relative level of expression of a MUC gene transcript facilitates diagnostic assessment of whether MUC gene expression is downregulated and thereby indicative of CRC.
Although PCR is the preferred nucleic acid sequence amplification technique, It will be appreciated that there are a variety of polynucleotide sequence amplification techniques other than PCR, which include rolling circlE; amplification {RCA) and strand displacement amplification (SDA). With regard to RCA, reference is made to W097/19193 which is herein incorporated by reference. With regard to SDA, reference is made to U.S. Patent No. 5455166, which is herein incorporated by reference.
Detailed PCR methods are provided hereinafter, although the skilled person is also rE:ferred to Chapter 15 of CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which is herein incorporated by reference, for a detailed discussion and examples of PCR methods.

WO 00/04142 PCT/AU99/00579 _ It will also be understood that PCR includes within its scope RT-PCR and multiplex PCR as will be described in detail hereinafter. Such methods may be used for qualitative or semi-quantitative analysis. PCR-based Restriction Fragment Length Palymorphism (PCR-RFLP) methods are also contemplated, which methods are useful when a polymorphism, deletion mutation, truncation and/or expansion either introduces or removes one or more restriction endonuclease sites in a MUC gene.
The skilled person will appreciate that Northern, Southern and in situ hybridization methods involve formation of a hybrid nucleic acid comprising a MUC gene or mRNA transcript and a corresponding isolated MUC nucleic acid or portion thereof.
RNA isolation and Northern hybridization methods are described in detail herein, although the skilled person is also referred to Chapter 4 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds Ausubei et ai., (John Wiley & Sons), which is herein incorporated by reference.
Furthermore, Southern hybridization methods are described in detail herein, although the skilled person is also referred to sections 2.9A-B
and 2.10 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds Ausubel et al., (John Wiley & Sons), which is herein incorporated by reference.
Also, determining whether a MUC gene or MUC gene transcript includes a polymorphism, mutation, deletion, truncation andlor expansion can be performed using rr~ethods such as PCR-RFLP analysis, Single Strand Conformational Polymorhpism (SSCP) analysis and Denaturing Gradient Gel Electrophoresis (DGGE). These techniques have become well known in the art of mutation detection. A non-limiting example of DGGE is provided in Folde & Loskoot, 1994, Hum. Mut. 3 83, which is herein incorporated by reference. A non-limiting example of specific allele detection by PCR-RFLP
and SSCP is provided in Lappalainen ef al., 1995, Genomics 27 274, which is herein incorporated by reference.

It is proposed that mutations in MUC11 or MUC12 genes are associated with bowel cancers (CRC), CF, BC, IBD, chronic bronchitis, asthma, ulcerative colitis and/or Crohn's disease. These are examples of disease conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
The isolated MUC nucleic acids now provide a means for genetic screening of the abovementioned disease conditions in human and other mammalian species. Genetic screening may be conducted by determining full expression or full-length transcript production by Northern blot, cloning and sequencing ~ of the MUC genes or identifying mutations by oligonucleotide hybridisation or by direct sequencing of PCR amplification products of the MUC genes. In addition, the present invention extends to nucleic acid molecules having translation-terminating mutations leading to truncation mutants. The detection of truncation mutants imay be important for genetic analysis of people with, for example, cancer of the large bowel or with a propensity to develop large bowel cancer, determined on, for example, hereditary grounds.
Truncated MUC polypeptides may also be useful in developing therapeutic agents such as antagonists or for developing antibodies.
Truncational mutants may be readily detected by a direct protein truncation test. In essence, DNA fragments including PCR amplification products or corresponding mRNA molecules are subjected to in vitro translation and optionally also transcription and the translation products assayed by, for example, SDS-PAGE or by differential antibody binding assays. This assay may also be employed to screen for agents capable of inducing truncation mutations or for agents acting as antagonists for truncation mutant-inducing agents.
Alternatively, MUC polypetides may be assayed by, for example, by antibody screening such as in an ELISA.
Thus, it will be appreciated that the present invention contemplates isolated MUC polypeptides, and also:-(i) polypeptides which comprise an amino acid sequence having at least 60% identity to a MUC polypeptide amino acid sequence, preferably at least 75% identity thereto, or more preferably at least 90% identity thereto;
and (ii) polypeptides encoded by MUC homologs.
Such polypeptides are hereinafter referred to as "MUC
homologs".
The MUt; polypeptide homologs of the invention include amino acid substitution(s), deletions) and/or additions) to a MUC polypeptide sequence. Particular examples include antigenic fragments and analogues useful in immunoassays and as therapeutic agents as well as other fragments carrying B cell andlor T cell linear or conformational epitopes.
Additions to the amino acid sequence include fusion partners in the form of peptides or polypeptides, which create a MUC fusion polypeptide.
Fusion polypeptides include the MUC polypeptide(s) together with fusion partners such as HIS6, glutathione-s-transferase (GST), thioredoxin (TR) and maltose binding protein (MBP). Fusion partners greatly assist recombinant synthetic polypeptide purification by virtue of each fusion partner affording affiniity purification by a specific affinity matrix.
Preferably, the fusion polypeptide also includes a protease-specific cleavage site, so that the fusion partner may be cleaved and removed following purification to leave a substantially unmodified MUC polypeptide.
The usE: of fusion partners for purification of recombinant expressed polypeptides is well known in the art. Indeed, there are a variety of commercial sources. applicable to fusion partners and purification systems such as the QlAexpressT"" (HIS)s system, the Pharmacia GST purification system and the New (England Biolabs MBP system.
Also within the scope of fusion partners are "epitope tags".
Such tags are well known in the art and include c-myc, influenza hemagglutinin and FLAG tags.
Furthermore, Green Fluorescent Protein (GFP) is a well known z0 fusion partner applicable to MUC polypeptides of the invention. A particularly useful application of GFP fusion partners is in the visible identification of cells or tissues which express a GFP-MUC fusion polypeptide of the invention. Identification may be performed by flow cytometry or fluorescence microscopy, as are well known in the art.
The MUC polypeptides and MUC homologs of the invention may be in recombinant form of may be chemically synthesized, as is well known in the art. Chemical synthesis is preferably suited to production of MUC peptides. As used herein, "peptides" have no more than fifty (50) contiguous amino acids.
Preferably, MUC polypeptides are in recombinant form.
In order to produce recombinant MUC polypeptides, isolated MUC nucleic acids of the present invention may be ligated into an expression vector to form an expression construct capable of directing expression of said MUC nucleic acid in a prokaryotic cell (for example, E. col~~ or in a eukaryotic cell (for example, yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
Suitably, the expression vector comprises one or more regulatory elements which direct expression of the nucleic acid ligated in said expression construct. Such regulatory sequences include promoters, enhancers, splice donor/acceptor sites, polyadenylation sequences, translation initiation (Kozak sequences) and translation termination signals.
Suitable promoters may be constitutive (for example, CMV- or SV40-derived promoters) or inducible (for example, Zn responsive metallothionein promoters) or repressible (tet-repressible promoters).
Exemplary methods useful for recombinant protein expression and purification, including fusion polypeptides, can be found in Chapters 16 of CURRENT PROTOCOLS fN MOLECULAR BIOLOGY (Eds. Ausubel et al.;
John Wiley & Sons Inc., 1997 Edition) and Chapters 5 and 6 of CURRENT
PROTOCOLS IN PROTEIN SCIENCE (Eds. Coligan et al.; John Wiley &
Sons Inc., 1997 Edition) which are herein incorporated by reference.

"Analogues" of the MUC polypeptides of the invention contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues. Such chemical analogues may be useful in providing stable means for diagnostic purposes or for producing agonists or antagonists or for producing stable molecules for use in natural product screening.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4;
amidination with methylacetimidate; acylation with acetic anhydride;
carbamoylation of amiino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, malefic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pl-~.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyi halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methyiheptanoic acid, 2-thienyl alanine andlor D-isomers of amino acids. A list of unnatural amino acids, contemplated herein us shown in Table 1. Crosslinkers can be used, for example, to stabilise tertiary conformation, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CHZ)~ spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COON). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and Na-methylamino acids, introduction of double bonds between Ca and Cs atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C i:erminus.
The present invention further contemplates chemical analogues of the polypeptides of the invention capable of acting as antagonists or agonists thereof, or which can act as functional analogues thereof. Chemical analogues may not necessarily be derived from the polypeptides of the invention, but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of MUC poypeptides. Chemical analogues may be chemically synthesised or may be detecked following, for example, natural product screening. Useful sources for screening for natural products include coral, reefs, sea beds, river beds, plants, microorganisms and aqua and antarctic environments.
Still anather aspect of the present invention is directed to antibodies specific for MUC polypeptides andlor homologs thereof.
In one embodiment, the anti-MUC antibody is M11.9.
In another embodiment, the anti-MUC antibody is M12.15.
A detailed method of anti-MUC antibody preparation is provided hereinafter.
In this regard, it will be understood that anti-MUC polypeptide antibodies may be produced by immunization with MUC polypeptides or MUC
peptides.
In particular, it is also likely that naturally-occurring anti-MUC
antibodies may well have naturally arisen against MUC polypeptides.
In light of the foregoing, it will be appreciated that "anti-MUC
antibody" as used herein is an antibody specific for, or at least binds to, a MUC polypeptide, irrespective of how the anti-MUC antibody was produced.
The anti-MUC antibodies of the present invention may be useful as therapeutic or diagnostic agents.
For example, a MUC polypeptide or homolog can be used to screen for naturally occurring anti-MUC antibodies. These may occur, for example in some autoimmune diseases. Alternatively, anti-MUC antibodies can be used to screen for MUC polypeptides. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of endogenous MUC polypeptide levels may be important for diagnosis of large bowel cancer or a predisposition to large bowel cancers or for monitoring certain therapeutic protocols. This knowledge may also be important in other epithelial cancers such as cancer of the breast.
Anti-MUC antibodies of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing cancer development or cancer cell apoptosis or monitoring the program of a therapeutic regimum.
For example, anti-MUC antibodies can be used to screen for endogenous MUC polypeptides. The latter would be important, for example, as a means for screening for levels of the MUC polypeptide in a cell extract or other biological fluid or purifying the MUC polypeptide made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonaf or fragments of antibodies or synthetic antibodies) directed to~ the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of the MUC polypeptide.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The rnethods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by infection of a suitable laboratory animal with an effective amount of a MUC polypeptid~, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
5 The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the horniogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation 10 can be done by techniques which are well known to those who are skilled in the art.
The present invention contemplates a method for detecting a MUC polypeptide in a protein extract obtained from a mammal, said method including the step of farming a complex between an anti-MUC antibody and 15 a MUC polypeptide, and then detecting said complex.
The presence of a MUC polypeptide may be determined in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both 20 single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of 25 variations of the sandwich assay technique exist and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized to a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by measurement of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the protein extract might be a cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and gastrointestinal fluid. T'he extract is, therefore, generally a biological sample.
In the typical forward sandwich assay, a first antibody having specificity for MUC or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surtace is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-finking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient {e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from 4°C to 3T°C) to allow binding of any subunit present in the antibody. Following the incubation period, the solid phase complex is washed and dried and incubated with a second antibody which is specific for a portion of the antigen (i.e. MUC). The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to MUC.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody.
Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorochromes or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, such as via glutaraldehyde or periodate amongst other means. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample. The term "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
Also, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state of excitability in the molecule, followed by eneossion of the light at a characteristic colour visually detectable with a light microscope.
As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of the appropriate wavelength and the fluorescence observed indicates the presence of the antigen of interest. Immunofluorescene and EIA techniques are both very well established in the art. Other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
The MUC; genes of the present invention are likely to function in cell adhesion, signal transduction, growth regulation, epithelial cell protection andlor immunological reactions. The classical gel-forming mucins function in protecting and lubricating epithelial tissues (particularly those of the respiratory and gastrointestinal tracts) by forming a layer of viscoelastic gel. These new mucins, MUC11 and MUC12, show structural similarity to MUC1. MUC1 can be secreted, but unlike the classical mucins, it is primarily a type I transmembrane protein that interacts and complexes with other adhesion molecules, and is involved in signal transduction. MUC12 has an EGF growth factor-like domain, is likely to be a transmembrane protein and has a putative tyrosine phosphorylation site that may participate in intracellular signalling. It is hypothesised that loss of MUC12 may be associated with poor prognosis in CRC.

The isolated MUC nucleic acids of the present invention are, therefore, considered in one embodiment, to correspond to cancer suppressor genes. Suppression may mean total inhibition of any development of large bowel cancer or a limitation of the severity of or an amelioration of the condition resulting from a large bowel cancer. The MUC
nucleic acids of the present invention are also considered in another embodiment to be capable of modulating disease conditions such as CRC, BC, IBD, CF, asthma, chronic bronchitis, ulcerative colitis and/or Crohn's disease Cystic fibrosis (CF) is an inherited disease of epithelial cell chloride ion transport that affects multiple organ systems. It is the most common cause of severe, progressive lung disease and exocrine pancreatic insufficiency in childhood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene located on chromosome 7q22 encodes a large single chain protein that forms a chloride channel. Virtually all of the morbidity and mortality associated with mutations in the CFTR gene causing cystic fibrosis arise from respiratory disease due to chronic infection and mucus obstruction. The precise mechanism of mucus accumulation in cystic fibrosis is controversial. Data suggest that CFTR malfunction may trigger mucin secretion and alter mucus properties, andlor bacterial infection triggers the hypersecretion of mucin in CF patients. The gene of the present invention is expressed in the colon, pancreas, small intestine, and lung, all tissues where mucus obstruction occurs. Accordingly, aberrant expression of the genes may contribute to cystic fibrosis.
Aberrant mucin expression is also a recognised component of IBD. Inflammatory bowel disease is characterised by considerable alterations in glycosylation, sialyation and sulphation of glycoproteins. It is unclear whether the changes in mucus production are a cause or response to the disease. Susceptibility genes for inflammatory bowel disease have been localised to chromosomes 3, 12 and 7q22. Accordingly, the MUC genes of the present invention are considered candidates for susceptibility genes for IBD. Up or down regulation, or altered secretion of one of these mucins may influence the quality of colonic mucus and therefore the pathology of these diseases. Certain inherited forms of these genes may indicate a predisposition to IBD.
5 The identification of MUC genes and isolated MUC nucleic acids permits the generation of a range of therapeutic methods and compositions. Such therapeutics may modulate MUC gene expression and the activity of MUC polypeptides. Modulators contemplated by the present invention includes agonists and antagonists of MUC gene expression.
10 Antagonists of MUC: gene expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter activity or interfere with negative mechanisms. Agonists of MUC include molecules which overcome any negative regulatory mechanism. Antagonists of MUC poiypeptides include antibodies and 15 inhibitor peptide fragments. Another class of therapeutics may be designed to mimic or block intracellular signal transduction by MUC polypeptides.
In accordance with the present invention, it is proposed that MUC functions as a suppressor of cancer development in the large bowel.
Hereditary cancers arise with loss of the wild-type gene. In addition, 20 germline mutations underlying large bowel cancer are inactivated for the MUC genes and, therefore, hereditary cancers have no functional copy of the gene. Furthermore, sporadic large bowel cancers arise with somatic loss of both copies of the gene. The present invention extends to the use of modulating levels of expression of MUC genes or their translation products 25 in the context of cancers related thereto.
Thus, the present invention contemplates a method of gene therapy of a mammal. .Such a method utilizes a gene therapy construct which includes an isolated MUC nucleic acid ligated into a gene therapy vector which provides one or more regulatory sequences that direct expression of 30 said nucleic acid in said mammal.
Such regulatory sequences may include a promoter, an enhancer, a polyadenylation sequence, splice donor/acceptor sequences and translation termination and intiation sequences.
Typically, gene therapy vectors are derived from viral DNA
sequences such as adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses. Suitable gene therapy vectors currently available to the skilled person may be found in Robbins et al., 1998, Trends Biotechnol. 16 35, for example, which is herein incorporated by reference.
If "anti-sense" therapy is contemplated, then one or more selected portions of a MUC nucleic acid may be oriented 3'-~5' in the gene therapy vector.
Administration of the gene therapy construct to said mammal, preferably a human, rnay include delivery via direct oral intake, systemic injection, or delivery to,elected tissues) or cells, or indirectly via delivery to cells isolated from the mammal or a compatible donor. An example of the latter approach would be stem-cell therapy, wherein isolated stem cells having potential for growth and differentiation are transfected with the vector comprising the MUC nucleic acid. The stem-cells are cultured for a period and then transferred to the mammal being treated.
Delivery of said gene therapy construct to cells or tissues of said mammal or said compatible donor may be facilitated by microprojectile bombardment, liposome mediated transfection (e.g. lipofectin or lipofectamine), electroporation, calcium phosphate or DEAE-dextran-mediated transfection, for example. A discussion of suitable delivery methods may be found in Chapter 9 of CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY (Eds. Ausubel et al.; John Wiley & Sons lnc., 1997 Edition), for example, which is herein incorporated by reference.
For example, a MUC nucleic acid may be introduced into a cell to enhance the ability of that cell to survive, conversely, MUC antisense sequences such as 3'-~ 5' oligonucleotides may be introduced to decrease the survival capacity of any cell expressing an endogenous MUC gene.
In this regard, increased MUC expression or activity is important in conditions of repressing cancer growth and/or development.
Decreased MUC expression or activity may be important, for example, in the treatment of cystic fibrosis or the treatment of inflammatory bowel disease.
Accordingly, the present invention contemplates a pharmaceutical composition comprising a MUC polypeptide or a derivative thereof or a modulatar of MUC gene expression or activity, inclusive of anti MUC antibodies. These components are referred to herein as the "active ingredients", and are suitably provided in combination with one or more pharmaceutically-acceptable carriers andlor diluents.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like) or suitable mixtures thereof as well as vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated 'with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of ac,~tive compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will bE: obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 Ng and 2000 mg of active ingredient.
The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds) may be incorporated into sustained-release preparations and formulations.
Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosagE~ unit form for ease of administration and uniformity of dosage. Dosage unit firm as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in 'which bodily health is impaired.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 Ng to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 Ng to about 2000 mg/mt. of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. It is WO 00/04142 PCT/AU99/005'79 also convenient to represent the effective amounts of active ingredients as an amount per kg body weight. For example, the present invention encompasses effective amounts for 0.005 Ng/kg body weight at 2000 mg/kg body weight.
5 The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating MUC gene expression or MUC polypeptide activity. The vector may, for example, be a viral vector.
10 From the foregoing, it is apparant that therapeutic methods and compositions of the invention are useful in the treatment of disease conditions associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins.
Preferably, the disease condition is selected from the group 15 consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis, asthma and chronic bronchitis.
More preferably, the disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), 20 inflammatory bowel disease (IBD) and breast cancer (BC). although not limited thereto. The therapeutic methods of the invention may therefore be used to alleviate one or more symptoms of diseases or be used as prophylactic treatments to prevent, or reduce the likelihood of, said symptoms from occurring.
25 The present invention is further described by the following non-limiting Examples.
EXAMPLES
EXAMPLE 1: Tissue Specimens Tissue specimens were collected from patients undergoing 30 surgery (Dukes' A n=5; Dukes' B n=5, Dukes C n=5, Dukes' D n=5). Colonic specimens were obtained from patients undergoing either colectomy or partial hepatectomy far colorectal carcinoma. Samples of normal colonic mucosa, primary colon cancer, liver metastases (if present) and adjacent normal liver were rapidly excised from operative specimens, snap-frozen in liquid nitrogen and stored at -70°C until use. Care was taken to exclude normal mucosal tissue from tumour samples. functional tissue specimens from four tumours of each Dukes' stage were randomly selected for in situ hybridisation. Tissues were fixed for 24-48 hours in 10% v/v buffered formalin, dehydrated in ethanol, cleaned in chloroform and embedded in parraffin wax. Biopsy specimens of normal colonic epithelium from four 10' distinct regions of the colon were collected via colonoscopy from each of three healthy individuals undergoing routine colonoscopic screening.
Similarly, intestinal biopsies were obtained via colonoscopy from ten patients with inflammatory bowel disease. Specimens were snap frozen and stored at -70°C until RNA was extracted as per Example 3 below.
EXAMPLE 2: Cell Lines and Culture Seven human colonic tumour lines were obtained: LIM1215, LIM2405, LIM1863, LIM1899 (Ludwig Institute, Melbourne, Australia), HT29 (ATCC HTB38), SW480 (ATCC CCL 228) and SW620 (ATCC CCL 227).
LIM1215 and SW620 are each derived from CRC metastases. Cell lines were maintained in RPMI 1640 with 10% v/v fetal calf serum, 2 mM glutamate, 25 mM HEPES, 60 mg/ml penicillin G and 100 mglml streptomycin sulfate and incubated in 5% vlv COZ and 95% v/v air at 37°C. Cultures were passaged twice weekly using standard techniques. The following breast carcinoma lines were included in this study: KPL-1 (a gift of Dr Junichi Kurebayashi, Suzuki, Japan), MA11 (a gift of Dr Philip Rye, Oslo, Norway), BT 20, DU4475, MCF-7, MDA-MB-453, SK-Br-3, T47D, UACC-893, ZR-75-1 and ZR-75-30 (ATCC, Rockville, MD), and MDA-MB-435 and MDA-MB-468 (a gift of Dr. Janet Price, MD Anderson Cancer Center, Houston, TX). All breast cancer cell cultures were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum and 0.006% penicillin and 0.01 % streptomycin with the following exceptions: DU-4475 in RPMI-1640 with 20% FCS, KPL-1 was maintained in DMEM with 5% FCS, MA11 in 1:1 Ham's F12:RPM1-1640 with 10% FCS, SK-Br-3 in McCoy's medium with 15% FCS, and UACG-893 in RPMI-1640 with 15% FCS.
EXAMPLE 3: RNA Extraction Total RNA was isolated by the method of Chomczynski and Sacchi (Chomczynski et al., 1987, Anal. Biochem. 162 156). Cells were resuspended in RNA. extraction buffer (4 M guanidinium isothiocyanate containing 25 mM sodium citrate, pH7.0, 0.5 % w/v sodium lauroyl sarcosine (SLS) and 0.1 M 2-mercaptoethanol). Tissue samples were homogenised in RNA extraction buffer. Extracted RNA was dissolved in RNase free water and the concentration and purity determined by spectrophotometry at 260 and 280 nm (Sambrook et al., Molecular Cloning, A Laboratory Manual. 2nd Ed.
Cold Spring Harbour Laboratory Press. Cold Spring Harbour, NY, 1989). The integrity of the RNA was assessed by denaturing agarose gel electrophoresis and samples transferred to HYBOND N {Amersham, Bucks, England) membrane by capillary blotting.
EXAMPLE 4: DNA Seauencing Approximately 500 ng of DNA were employed in a cycle sequencing reaction with 2.5 pmol of primer and 4 N1 of Dye terminator or dRhodamine reaction mix (DNA Cycle Sequencing Kits, Perkin Elmer, Norwalk, CT,) in a total volume of 10 NI. Reaction mixes contained Amplitaq DNA polymerise, dNTPs and fluorescently labelled dideoxynucleotides (dye terminators). Cycling reactions were as follows: 25 cycles of denaturation at 96°C (30 s), primer annealing at 50°C (15 s) and extension at 60°C (4 min).
Unincorporated nucleotides were removed by ethanol precipitation. The reactions were analysed on a Model 373A automated DNA sequencer (Applied Biosystems) run by technical staff in the core sequencing facility of the Queensland Institute of Medical Research.
EXAMPLE 5: Identification by Differential Disptay of Two cDNAs ~ncodina Mucins Downreaulated in Colorectal cancer WO 00/04142 PC'T/AU99/00579 The differential display method was devised from the original technique described by Liang & Pardee, 1992, Science 257 967. Total RNA
was isolated by the method as described previously. Reverse transcription was carried out using one of four anchored primers, T,2MG, T,2MC, T,2MA
and T~2MT (Operon Technologies Inc., Alameda, CA) and Superscript RNAse H- reverse transcriptase (Gibco BRL, Gaithersburg, MD). One arbitrary 10mer primer (Operon Technologies Inc.) was selected at random to be employed in a PCR with the appropriate anchored primer. Two patients, 101 and 112, were analysed simultaneously and duplicates of two separate reverse transcription reactions electrophoresed on each gel. Gels were put down wet and autoradiographed for 1-3 days. DNA was removed from gel slices by boiling and reamplified by PCR. Bands were then cloned into pGEM-T {Promega Corporation, Madison, WI) and sequenced. Sequences were analysed by multiple sequence similarity searches using BLAST
algorithms (Altshcul et al., 1990, supra) accessed through the National Centre of Biotechnology Information (NCBI; http:llwww.ncbi.nlm.nih.gov).
Differential display was performed on RNA from paired normal colonic mucosa and primary colorectal cancers. Using a PCR primer combination of T~2MG and 10mer 5'-ACTTCGCCAC-3' (SEQ ID N0:7), bands dd29 (MUC12) and dd34 (MUC91) were both amplified from normal colonic mucosal RNA of two patients and were consistently downregulated in the tumors from these patients in multiple PCR reactions (FIG. 1A).
Following reamplification PCR, discrete bands of approximately 720 by for dd29 and 530 by for dd34 were isolated and cloned into pGEM-T. Sequence analysis showed that both cDNAs were novel, with no match in any database accessed through the NCBI. Repetitive segments typical of mucin tandem repeats were observed in dd34.
EXAMPLE 6: Northern Blot Analysis Northern blot analysis was performed on paired normal and tumor total RNA extracted from the same patients employed in the differential display experiment. dd29 (MUC12) and dd34 (MUC11) were random primer-labeled using a Megaprime DNA labeling system (Amersham, Aylesbury, UK) and hybridization performed at 65°C in buffer containing 7% SDS, 0.26 M
Na2HP04, 1 mM EDTA, 1 % BSA.
Northern blot analyses of dd29 (FIG. 1 B) and dd34 (FIG. 1 C) with colonic total RNA used for the differential display reactions revealed a polydisperse signal beginning near the top of the gel for RNA isolated from normal colonic mucosa and no signal in tumor-derived RNA. Probe dd29 showed some cross-hybridization to ribosomal RNA. Polydispersity of signal is a hallmark of mucin RNA blots due to shearing of very high molecular weight transcripts.
EXAMPLE 7: Multiplex Semi-guantitative RT PCR
Multiplex semi-quantitative RT-PCR was pertormed on total RNA isolated from six colorectal cancer cell lines and from paired normal colonic mucosa and tumor colorectal cancer tissues from 20 patients, eve of each Dukes' stage. Informed consent was obtained from each subject after approval by the appropriate hospital Ethics Committee. PCR products were quantitated relative to a [iz-microglobulin cDNA amplification control using densitometry. First strand cDNA synthesis was accomplished using 1 pg of total RNA. PCR amplification of cDNA was pertormed in a total volume of 25 pl containing 1 NI of the first strand cDNA synthesis reaction products, 2.5 pl 10x Taq polymerise buffer (25 mM TAPS (tris-[hydroxymethylJ-methyl-amino-propane-sulfonic acid, ;>odium salt) pH 9:3, 50 mM KCI), 2 mM dNTPs, 25 mM
MgCl2, 20 pmol each of the forward and reverse primers, and 2.5 U Taq polymerise. Gene-specific forward and reverse primers for MUC92 and MUCH were designed to produce PCR products of 510 by and 169 by respectively. Primers for [32-microglobulin generated a PCR product of 247 by (Gussow et al., 1987, J. Immunol. 139 3132). Primers were:
MUC12F1; 5'-TGAAGGGCGACAATCTTCCTC-3' (SEQ ID N0:8);
MUC12R1; 5'-TACACGAGGCTCTTGGCGATGTTG-3' (SEQ ID N0:9);
MUC11 F1; 5'-CAGGC;GTCAGTCAGGAATCTACAG-3' (SECI ID N0:10);
MUC11 R1; 5'-GAGGCTGTGGTGTTGTCAGGTAAG-3' (SEQ ID N0:11 );

~i-21 F; 5'-TGAATTGCTATGTGTCTGGGT-3' (SEQ ID N0:12);
~i-21 R; 5'-CCTCCATGATGCTGCTTACAT-3' (SEQ ID N0:13);
MUC12TOTF1;5'-AGCCAACCAGGCTCAGCTCT-3' (SEQ ID N0:14); and MUC12TOTR1;5'-GC~'CACACAGTGGATGCTACC-3' (SEQ ID N0:15}.
5 After an initial denaturation step of 94°C for 5 minutes, the amplification conditions were: 21 cycles of denaturation at 94°C (30 s) for MUC12, (24 cycles of denaturation at 94°C (30 s) for MUC11);
annealing at 60°C (30 s) and extension at 72°C (30 s). PCR products were electrophoresed on 1.2% 1x TBE gels and photographed.
10 Due to the polydisperse signals obtained by :Northern analysis, expression of MUC19 and MUC12 was examined in a range of colorectal cancer cell lines and tissue mRNAs by multiplex semi-quantitative RT-PCR.
dd29 was not expressE:d in any of six colorectal cancer cell lines examined (FIG. 1 D). In contrast, dd34 showed a different pattern of expression, with 15 HT29; LIM1215, LIM1899, LIM1863 lines revealing very faint PCR products, and SW620 and SW480 lines showing relatively high levels of expression {FIG.1 E). For tumor tissue-derived RNA, downregulation was defined as amplified band intensity less than 30% of that observed from paired normal colon tissue. dd29 was found to be downregulated or absent in 6115 {40%) 20 tumors with paired normal samples, and at low levels in 3/5 (60%) Dukes' stage D samples (where normal colon was not available for comparison) (FIG.1 F). dd34 was downregulated in the tumors of 12!15 (80%) paired samples and expressed at low levels in 415 (80%) Dukes' stage D samples.
One of five Dukes' stage D samples showed relatively high levels of 25 expression of dd34 (FIG. 1 G). Significantly, 13115 (87%) colorectal cancers showed downregulation of at least one of these mucin genes, with 5115 (33%) showing downregulation of both genes.
EXAMPLE 8: Differential Tissue Distribution of MUC11, MUC12 and MUC3 mRNAs 30 A human RNA "master blot" (Clontech, Palo Alto, CA, catalogue number 7770-1 ) with RNA from 50 different tissues and controls was used to WO 00/04142. PCT/AU99/00579 examine mucin gene expression. DNA fragments encoding dd29, dd34 and MUC3 (Genbank Accession No. M55405, a gift from Dr. Sandra Gendler, Mayo Clinic, Scotsdale, Arizona) were excised from vector and radiolabeled as described above. Hybridization was performed as per the manufacturer's instructions. The master blot was reprobed with a radiolabeled ~3-actin cDNA
as a loading control.
Analysis of the tissue distribution of MUC11, MUC12 and MUC3 transcripts in RNA isolated from 50 different normal tissues showed a distinct pattern of expression for each gene (FiG. 5). MUC12 and MUC11 showed highest expression in colon but had different patterns in other organs, mainly restricted to those of epithelial type. MUC11 had a wider epithelial distribution than MUC12 which was restricted to expression in the colon, and weakly in the pancreas, prostate and uterus. Consistent with published findings (Van Klinken et al., 1997, Biochem. Biophys. Res. Comm. 238 143), MUC3 was found to be predominantly expressed in the small intestine and at much lower levels in the colon. Interestingly, it was also present in the thymus.
EXAMPLE 9: Extendin4 the Seyuences of dd29 and dd34 The strategy employed in the cloning of_MUC11 and MUC12 is shown in FIGS. 8A and 8B respectively.
9.1 Library ~~creenin4.
A hgt11 human fetal brain 5'-STRETCH PLUS cDNA library (Clontech, Palo Alto, C;A) was screened using radiolabeled dd29 and dd34.
~ DNA was extracted and inserts were excised, cloned into pBSK- and sequenced.
9.2 PCR to extend the seqruence of dd34 by IinkinQ clones 2 and li5 Screening of the fetal brain library with clone dd34, yielded two new cDNA clones: clone 2 (1043 bp) and clone li5 (1045 bp). Clone dd34 was a perfect match to the middle of the larger clone 2. cDNA from clone 1i5, however, was highly homologous but not identical to the cDNA from clone dd34. To ascertain whether these partial cDNAs arose from a single mRNA

transcript, RT-PCR was carried out using combinations of forward and reverse primers specific for each cDNA in an attempt to link them. RT-PCR
was performed on total RNA extracted from normal colon in a stringent touchdown PCR using high fidelity DyNAzyme DNA polymerase (Finnzymes, Espoo, Finland). Primer combination MUC11 F1 and IiSR {5'-GGGAACACTGTGGTTTCAGTTGAG-3'; SEQ ID N0:16) yielded a PCR
product of 2 kb demonstrating that these two cDNAs were derived from a single transcript. This product was cloned into pGEM-T and sequenced.
9.3 PCR library screening - to extend seguence of dd29 ~ Forward and reverse primers for dd29 (dd29F1 and dd29R1 ) were used in combination with a T7 vector-derived primer in a stringent touchdown PCR to screen an ulcerative colitis (UC) plasmid library (a gift from Dr. Jonathon Fawcett, Queensland Institute of Medical Research, Brisbane, Australia). Amplified products were purified, cloned into pGEM-T
and sequenced.
EXAMPLE 10: Seguence Analysis of dd29 (MUC~2) The sequence of dd29 revealed that it was amplified as a result of priming of random 'I Omer at both ends of the PCR product and that it did not contain a 3' untranslated region (3'-UTR) or poly A tail. Screening of an UC cDNA library with ~dd29-specific primers extended the sequence 840 by in the 5' direction and 800 by in the 3' direction to the poly A tail (Genbank Accession Number AF147790). To confirm contiguous cDNA sequence, primers MUC12TOTF'1 and MUC12TOTR1 were designed to produce an expected PCR product of 1532 bp; primers corresponded to bases 230-250 and 1742-1762, respectively, in SEQ ID N0:6. In a stringent touchdown PCR
amplification procedure an intense discrete product of the expected size was identified from normal colonic cDNA and cDNA from the Caco-2 colonic cancer cell line. This reaction confirmed the reported MUC12 cDNA
sequence.
Conceptual translation of the composite MUC92 cDNA reveals the presence of serinelthreonine and proline-rich degenerate tandem repeats (FIG. 2) consistent with this protein being a member of the epithelial mucin family. The deduced 28 amino acid tandem repeat structure is shown in FIG.
2. Following the mucin-repeat domain, MUC12 contains two cysteine-rich EGF-like domains separated by a 150 amino acid non-mucin-like sequence (amino acids 261-410) containing five N-glycosylation sites and a potential coiled-coil domain. The second cysteine-rich EGF-like domain is immediately followed by a putative transmembrane domain containing 26 hydrophobic or uncharged amino acids, and a cytoplasmic tail of 75 amino acids at the carboxyl terminus.
Sequence alignment of MUC12, human MUC3 (hMUC3), rat Muc3 (rMuc3), mouse Muc3 (mMuc3), human MUC4 (hMUC4) and rMu~ is shown in FIG. 3. When aligned by the transmembrane amino acid sequences, MUC12 was found to have areas of significant homology to rMuc3, mMuc3 and hMUC3, including perfect conservation of eight cysteine residues in the second EGF-like domain. With inclusion of three small gaps, each of these cysteines also align with those in rat and human MUC4.
Interestingly, all six mucins contain a conserved EGF-like sequence of Cx(5)GPxCxCx(9)GExC. Furthermore, there is some (4 out of 8) conservation of the cysteine residues between MUC12 and the human and rodent MUC3 and MUC4 mucins in the first EGF-like domain.
EXAMPLE 11: SeQUence Analysis of dd34 (MUC99) Clone dd34 (544 bp) was also obtained as a result of priming of random 10mers at both ends of the PCR product. Screening of a ~gt11 human fetal brain library yielded two positive plaques which hybridized to dd34, clone li5 (1045 bp) and clone 2 (1043 bp). These two clones represented opposite ends of a 2.8 kb partial MUC9 9 cDNA sequence (Genbank Accession Number AF147791 ), the finking of which was established by PCR (see Methods). Conceptual translation of the MUC91 composite is shown in FIG. 4. The entire 957 amino acid sequence consisted of serine, threonine and proline-rich tandem repeats of 28 amino acids in length, consistent with it being derived from a large epithelial mucin. The deduced tandem repeat structure and consensus repeat sequence for MUC11 is shown in FIIG. 4.
EXAMPLE 12: Chromosomal Localization of MUC11 and MUC12 DNA fragments excised from dd29 (720 bp) and dd34 (530 bp}
were nick translated with biotin-14-dATP and hybridized in situ at a final concentration of 10 ng/N1 to metaphases from two normal males. The fluorescence in situ hybridization (FISH) method was modified from that previously described (fallen et al., 1990, Ann. Genet. 33 219) in that chromosomes were stained before analysis with both propidium iodide as counterstain and DAPI for chromosome identification. Images of metaphase preparations were captured by a cooled CCD camera using the CyroVision Ultra image collection and enhancement system (Applied Imaging Int Ltd, Sunderland, U.K.).
Twenty metaphases from a normal male were examined for hybridization to dd29 and dd34 probes. For both genes, all of the metaphases showed strong signal on one or both chromatids of chromosome 7, at band 7q22 (data not shown). A similar result was obtained using metaphases from a second normal male.
EXAMPLE 13: Production of monoclonal antibodies reactive with MUC11 and 12 The following peptides were conjugated to keyhole limpet haemocyanin (KLH} with the heterobifunctional cross-linking agent m-maleimidobenzoyl-N-hydroxysuccinimide ester using standard techniques (Harlow, E. & Lane, D. Antibodies: A .Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which is herein incorporated by reference):-MUC11: CFHSRPASTHTTLFTED (SEQ ID NO: 17); corresponding to part of the degenerate tandem repeat region, specifically amino acid residues 690-705 deduced from the partial cDNA MUC11 clone, 'with an N-terminal cysteine residue added for conjugation);

MUC12: TYRNF1-EKMNDASSQEC (SEQ ID NO: 18); corresponding to part of 'the N- glycosylated region, specifically amino acid residue:. 286-302 deduced from the partial cDNA MUC12 clone, with a C-terminal cysteine residue added for 5 conjugation).
One Balblc mouse was immunised with each KLH-conjugated peptide as per the following protocol:-Day 0: KLH-conjugated peptide was diluted to 100 NglmL in phosphate buffered saline (PBS) and mixed with an equal 10 volume of complete Freund's adjuvant (CFA). Each mouse was injected intra-peritonealy with 0.5 mL of this mixture.
Day 14: Each mouse was immunised as above but peptide was mixed with incomplete Freund's adjuvant (IFA).
Day 33: Each mouse was immunised as on day 14.
15 Day 43: Each mouse was bled from the tail to assess antibody production by ELISA (see below).
Day 53: Each mouse was injected intra-venously with 100 NL of peptide at 100 Ng/mL in PBS without adjuvant, and with 700 NL mixed with IFA intra-peritonealy.
20 Day 56: Mice were euthanased, and the spleen removed for fusion with myeioma cells.
Splenocytes were fused to Ag8 mouse myeloma cells at a ratio of 5:1 with polyethylene glycol using established methods (Harlow & Lane, supra).
25 Specific antibody producing clones were screened by a solid phase antigen antibody capture ELISA with the immunizing peptides bound to polystyrene assay plates using established methods (Harlow & Lane, supra). Positive clones were expanded, retested for specific antibody production and recloned by limiting dilution. Clones were further tested for 30 reactivity with paraffin embedded normal colonic mucosa.
93.1 MUC19 and MUC92 reactive hvbridomas Two hybridomas, one reacting with each of MUC11 and MUC12 peptides and with paraffin embedded colonic sections are described in Table 2.
13.2 Immunohistochemical detection of MUC11 and MUC12 in normal colonic epithelium using antibodies M11.9 and M12.15 Paraffin sections (4 Nm) of normal colonic epithelium were dewaxed with xylene, hydrated in a graded series of ethanol to water and treated with 0.1 U/mL neuraminidase (Boehringer, Germany) in 50 mM Na acetate, 150 mM NaCI, 100 mM CaCl2 buffer, pH 5.5 for 1 hr at room termperature to remove sialic acid groups. Sections were then treated with 1 % H20z, 0.1 % NaN3; in Tris buffered saline (TBS) for 10 min to quench endogenous peroxidase activity, and non-specific protein binding blocked with 4% skim milk in TBS for 15 min. Monoclonal antibodies M11.9 and M12.15 were semi-purified by PEG precipitation and diluted to 5-50 Ng/mL
in TBS/50% non-immune goat serum and incubated for 2 hours overnight at room temperature. Sections were washed once with 1 % TX-100 in TBS for 5 min and then twice in TBS for 5 min. Sections were incubated for 30 min at room temperature with pre-diluted biotinylated goat anti-mouse immunoglobulins (Zymed, USA) and then washed as above. Sections were then incubated for 15 min at room temperature with pre-diluted streptavidin-conjugated horseradish peroxidase (Zymed Laboratories) and then washed as above. Peroxidase activity was detected using 10 mg/mL 0.05%
diaminobenzidine, 0.03% HZOZ in Tris saline, pH 7.6. Sections were counterstained with haematoxylin, dehydrated with ethanol, cleared with xylene and mounted in DePeX.
M11.9 reacts strongly with colonic epithelium, primarily with columnar cells of the surface epithelium (see FIG. 9A). Both goblet and columnar cells deep in the crypts are not stained by this antibody (see FIG.
9A). In surface epithelial columnar cells M11.9 reacted with the perinuclear cytoplasm, lateral cell membranes and most strongly as granular staining in the subapical cytoplasm (FIG. 9B). This localisation suggests reactivity with precursor in the rough endoplasmic reticulum (perinuclear staining), reactivity with mature mucin on the lateral membranes at columnar cell junctions with other cells, and reactivity with processed mature mucin in granules for apical secretion or incorporation into the apical cell membrane. This pattern of reactivity is distinct from that seen for other known mucin core proteins.
M12.15 also reacts strongly with colonic epithelium, and like M11.9 it reacts primarily with columnar cells of the surface epithelium (see FIG. 9C). However, M12.15 gave a more diffuse cytoplasmic staining pattern than that seen with M11.9, although, like M11.9, the strongest staining was in the apical cytoplasm. , Imrnunohistochemistry in normal colonic mucosa with these antibodies demonstrates protein expression of the MUC11 and MUC12 gene, supporting the mRNA studies. The co-expression of MUC11 and MUC12 in normal colon is also consistent with the RT-PCR data showing similar levels of relative expression of these two mucin genes in different regions of the intestinal tract.
EXAMPLE 14: Expression of MUC11 by in situ hybridization 74.9 Methods Optimisation of conditions for in situ hybridisation, outlined below, was based upon published techniques (Rex & Scotting, 1994, Biochemica 3 24, which is herein incorporated by reference). Riboprobes were made by in vifro transcription of DNA with SP6 and T7 RNA
polymerases and incorporation of a digoxigenin-labelled uridine triphosphate (DIG-UTP). The orientation of inserts in pGEM-T was established by sequencing. Insert in the antisense direction and thus complementary to RNA
template was the hybridisation probe and insert in the sense direction was used as a negative control. 1 mg of purified linearised plasmid pGEM-T was labelled in the presence of 1/10 volume 10 x transcription buffer, 1/10 volume 10 x NTP mix (1 mM ATP, CTP, GTP, 0.65 mM UTP, 0.35 mM DIG-UTP), 10U RNase inhibitor and 40U of either SP6 or T7 RNA polymerase. The reaction was carried oust at 37°C for 2 hours and terminated by addition of 2 NI of 0.2M EDTA. Probes were ethanol precipitated with 1 /11 volume 4M LiCI
and placed at -20°C for 2 hours. They were then centrifuged at 12,000 g for 30 min at 4°C. Pellets were washed with 70% ethanol, air-dried for 10 min and resuspended in 100m1 of RNase-free water.
Paraffin-embedded functional tissue specimens were sectioned at 4 Nm onto sterile water and affixed to Vectabond-treated slides (Vector Laboratories). Sections were dewaxed in xylene, rehydrated and then incubated for 5 min in 0.2 N HCI. HCI treatment contributes to an improvement in the signal to noise ratio by extraction of proteins and partial hydrolysis of target sequences. Slides were washed in sterile water for 5 min, followed by 5 min in I'BT (PBS and 0.1 % Tween 20). Sections were then incubated in proteinase K (5 mg/ml) at 37°C for 15 min and washed briefly in 3 x PBT. They were fixed in 4% paraformaldehyde for exactly 20 min and prehybridised for 4 hours at 70°C in hybridisation buffer (50%
formamide, 5 x SSC, 1 % SDS, 500 mg/mL tRNA, 50 mg/mL heparin). Denatured probe (0.5 mg/section) was added to hybridation buffer and sections hybridised overnight at 70°C.
Sections were washed in 2 x wash solution 1 (50% formamide, 5 x SSC, 1% SDS) at E35°C followed by 2 x washes in wash solution 2 (50%
formamide, 2 x SSC) also at 65°C. Sections were then incubated with anti-digoxygenin-AP antibody at 1/2000 in PBS overnight at 40°C.
Excess antibody was removed by 3 x 20 minute washes in PBT.
Sections were then washed 2 x 20 minute in NTMT buffer (100 mM Tris, (pH
9.5), 50 mM MgCl2, 100 mM NaCI, 0.1 % Tween 20, 2 mM levamisole).
Hybridisation was viisualised with NBT and BCIP overnight at room temperature. The reaction was stopped by immersion of slides into 1 x TE
and sections lightly counterstained in eosin. Sections were then dehydrated through ethanols of iincreasing concentration to xylene and mounted in DePeX. Slides were photographed within 3 days due to fading of the signal with time.
14. 2 Detection of MUC 9 9 mRNA b y in situ hybridization Intense signal for MUC11 was observed in the columnar cells of the surface epithelium in all specimens of the normal colon. However, it was not possible to conclusively identify positive signal in the goblet cells of the colonic epithelium. Transcripts for MUC91 were not detected in adjacent carcinoma of several functional tissue specimens (an example is shown in FIG. 9D), thus confirming the findings of the differential display and Northern blot analyses.
EXAMPLE 15: E~rpression of MUC~1 and MUC92 in normal colon by RT PCR
The results of RT-PCR experiments to determine the expression patterns of MUC19 and MUC12 genes in normal colonic epithelium are shown FIG. 10.
MUC17 and MUC92 are predominantly expressed in the colon, although the data in FIG. 10 show that in fact their levels of expression vary within the colon. In this regard, a progressive increase (3-4 fold) in the expression of both MUC99 and MUC12 was seen from the right colon to the rectum.
EXAMPLE 16: Expression of MUC1~ and MUC~2 in CRC by RT
PCR and Northern hybridization The expression patterns of MUC19 and MUC92 in CRC were investigated by RT-PCR, and the results are shown in FIG. 11. After 40 rounds of amplification, MUC97 expression was observed in all CRC cell lines under investigation. Similarly, MUC92 expression was observed in all cell lines, although two cell lines, SW620 and SW116 revealed low levels of expression.
These observations, together with the downregulation data, show that although these genes are downregulated in CRCs, they are still detectable in CRC cell lines. In contrast to the normal gastrointestinal tract and IBD tissues, the expression of MUC19 and MUC92 in CRCs and in CRC
cell lines show patterns of expression distinct form each other.
Referring to FIG. 14, the results of Northern analysis with a dd34 (MUC19) probe showed that in nucleic acid extracts obtained from colonic tissue of four (4) of the (6) CRC patients tested, the level of MUC91 mRNA expression was lower relative to normal colonic tissue from the same patients. Similarly, MUC12 mRNA was downregulated in three (3) of five (5) 5 CRC patients (data not shown).
Such quantitative (e.g. downregulation of these genes and differential downregulation expression patterns of MUC1 ~ and MUC92) and also qualitative changes of these genes, e.g. mutations, could be used for diagnostic and prognostic testing in CRC.
10 EXAMPLE 17: Expression of MUC19 and MUC~2 in !BD by RT PCR
The expression patterns of MUC11 and MUC12 in IBD were investigated by RT-PCR, and the results are shown in FIG. 12. Cytokeratin 20, (CK20) a colonic epithelial marker, was employed as a loading control due to the variable epithelial content of IBD tissues. 'N' denotes tissues 15 which appear macroscopically normal and 'D' refers to tissues reported to have IBD. 'CA' refers to the caecum, 'CO' the colon, 'LC' the left colon, 'TC' the transverse colon, ''RS' the recto-sigmoid colon, 'SI' the small intestine, 'IL'denotes the ileum and 'IP' an ileal pouch.
Two patients, patient 1 and patient 4, show 3-4 fold upregulated 20 expression of MUC~7 and MUC92 in diseased tissues, compared with the same intestinal region observed in the 3 normal controls. Patient 6, who has a history of severe ulcerative colitis in the right colon, also revealed approximately 3-fold upregulated expression of MUCH 9 and MUC12 compared to the right colon observed in the normal controls.
25 There is coordinate regulation of Mucin expression in the normal gastrointestinal tract as well as in IBD tissues and upregulation of both Mucin genes wars observed in 3110 patients. Given the documented quantitative changes in the expression of MUCH and MUC92, their expression levels may form the basis of useful diagnostic and prognostic 30 testing for this disease. Qualitative changes in these genes, eg. mutations may also be useful markers for IBD.

EXAMPLE 18: Expression of MUC11 and MUC12 in BC by RT PCR
The expression patterns of MUC11 and MUC92 in BC tissue were investigated by Rl'-PCR, and the results are shown in FIG. 13. After 40 rounds of amplification, MUC99 expression was identified in all breast cancer cell tines under investigation; at low levels in BT-20, DU4475, MDA-MB-435 and ZR-75-30 cell lines and at higher levels in the remaining nine cell lines.
Eight of the cell lines .showed MUC91 expression higher than the normal colonic cDNA positive control. MUC19 is clearly highly expressed by most breast cancers and may impact upon the behaviour of the breast cancer cells. MUC91 may also be secreted by breast cancers and detection in serum could form the basis of diagnostic and prognostic testing for breast cancer.
MUC92 expression was only readily identifiable in one breast cancer cell line, MCF7, although faint bands were observed for BT20, KPL-1 and MA11 cell fines.
EXAMPLE 19: Experimental Summary Differential display has been used to identify two partial cDNAs, which encode novel colonic mucin-like proteins. Expression of both cDNAs, designated MUC11 and MUC12 by the Human Nomenclature Committee, was commonly downregulated in colorectal cancers.
MUC97 and MUC92 were mapped by FISH to chromosome band 7q22. The location of another mucin gene, MUC3, at 7q22, suggests the identification of a new cluster of mucin genes at this locus.
Interestingly, four genes encoding gel-forming mucins are found in a cluster on chromosome 11 and these genes appear to have originated from a common ancestral gene. WhilE; the mucin cDNAs mapped to 7q22 most likely represent separate genes, it is also possible that they are produced as a result of alternative mRNA splicing from a single, large mucin gene. Northern blot analysis for MUC91, MUC12 and MUC3 shows that these encode large transcripts, estimated to be greater than 12 kb.
Multiple tissue RNA analysis showed no cross-reactivity between MUC11, MUC92 or MUC3. MUC91 and MUC92 showed predominant expression in the colon, while MUC3 was predominantly expressed in the small intestine and at very low levels in the colon. This expression pattern constitutes an important point of distinction between MUC17 and MUC92 genes of the present invention and MUC3. Furthermore, the sequences of MU'C19 and MUC12 are not homologous with any other human mucin genes, but show some degree of similarity within their variable tandem repeat regions to each other (71 % aver 653 bp). However, their clear differential expression patterns in normal and tumor tissues as well as tumor cell lines, show that they are distinct from each other, and from MUC3.
While both MUC11 and MUC12 contain variable repeat regions typical of mucins, MUC12 is putatively a transmembrane mucin with features suggesting an involvement in growth regulation, a largely unrecognized function in human mucins. MUC12 is only the fourth human membrane-anchored epithelial mucin to be described to date, along with MUC1, MUC3 and MUC4. MUC1 has been shown to be involved in cell signaling via multiple tyrosine phosphorylation sites on its highly conserved cytoplasmic tail (Zrihan-Licht et al., 1994, FEES Lett. 356 130). At its carboxyl terminus, MUC12 possesses a cytoplasmic tail containing a YNNF sequence (amino acids 557-560 in FIG. 2) which is similar to motifs recognized by SH2 domain-containing proteins (Songyang et al., Mol. Cell. Biol. 14 2777), suggesting that MUC12, like MUC1, may be involved in signal transduction.
The deduced amino acid sequence of the partial MUC9 9 cDNA
was composed entirely of serinelthreonine-rich tandem repeats. There is a similarity between the tandem repeat consensus sequences of MUC11 (FIG.
4) and MUC12 (FIG. 2) and these also show limited homology to the MUC3 repeat (ITTETTSHS'T'PSFTSS). These similarities are consistent with evolution from a common ancestral gene. MUC11 is more widely expressed than MUC12 and MUC3 however, with RNA detected in gastrointestinal, respiratory, reproductive and urinary tracts, and unexpectedly in the liver and thymus.
The physiological roles of MUC99 and MUC92 in colonic WO 00/04142 PCT/AU99/00579 _ epithelium are unknown. MUC11 and MUC12 are commonly downregulated in colorectal cancer suggesting they may play a role in epithelial cell growth modulation and/or differentiation. At present, it is not possible to comment on whether downregulation of these genes is related to stage of tumor progression, as only ;?0 patients were analyzed in this study. However, downregulation appears to be so frequent, that it may be an early event in tumorigenesis. Given the co-localization of the MUC11 and MUC12 genes on chromosome 7q22, it is possible that their expression is co-ordinately regulated and hence they are simultaneously downregulated in a large proportion of colorectal cancers. The effect of downregulation of these mucins on normal colonic epithelial cells could be substantial. Mucins are believed to protect epithelial cells from attack by pathogenic organisms and from mechanical and chemical damage. Therefore, reduced expression of these mucins could expose colonic epithelial cells to the harsh environment of the intestinal lumen. /Furthermore, loss of a transmembrane mucin such as MUC12 may also contribute to loss of critical cell signaling.
The location of these two novel mucin genes on chromosome 7q22 may have significance for two non-malignant epithelial diseases where aberrant mucin expre:>sion and/or function is a recognized component of pathology, namely, inflammatory bowel disease and cystic fibrosis.
Susceptibility genes for inflammatory bowel disease have been located to chromosomes 3, 12 and 7q22 (Satsangi et al., 1996, Nature Genet. 14 199).
Thus, MUC11 and MUC12 must be considered candidates for involvement in inflammatory bowel disease given their chromosomal localization, expression in normal colon, and the documented alterations in mucins in this disease (Rhodes, 1997, QJM 90 79). Mucins may also play a role in cystic fibrosis as patients wifh the same CFTR gene mutation do not demonstrate exactly the same phenotype in terms of mucus obstruction. The existence of modifier genes has been postulated and mucin genes are obvious candidates (Harris & Reid, 1997, J. Med. Genet. 35 82). A murine Mucin gene that shows C-terminal homology with MUC12 has recently been shown to be a major constituent of obstructive mucus in the gastrointestinal tract of mice with CF (Parmley et al:, 1998, J. Clin. Invest 102 1798).
The CFTR gene lies in the adjacent chromosome band (7q31 ) to the MUC3, MUC99 and MUC92 genes. While the significance of these findings is not clear, MUC19 and MUC92, which are expressed in many of the tissues affected by cystic fibrosis, should be considered as candidate modifier genes involved in the aetiology of this disease.
Mucins are encoded by large genes which have proved difficult to clone by conventional methods due to the repetitive nature of their tandem repeat regions. Hereinbefore, the present inventors have unexpectedly identified by differential display two partial cDNAs which represent novel mucin genes that are predominantly expressed in colonic epithelium, both of which are downregulated in colorectal cancer. In this regard, MUCH and MUC92 differ from the other mucin gene located on chromosome 7q22, MUC3. These findings together with the sequence homology between the MUC12 EGF-like domain and EGF receptor-binding growth factors, suggest MUC11 and MUC12 may function as growth regulators in colonic epithelium.
Downregulation of these two novel mucin genes could be an important and previously unrecognized step in colorectal carcinogenesis.
Those skilled in the ark will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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M11.9 MUC11 IgM ++++ Reacts with paraffin embedded tissue, reactivity enhanced by pre-treatment of sections with neuraminidase which removes sialic acid rou s.
M12.15 MUC12 IgM ++++ Reacts with paraffin embedded tissue, reactivity enhanced by pre-treatment of sections with neuraminidase which removes sialic acid rou s.

SEQUENCE LISTING
<110> The Council of the Queensland Institute of Medical The Corporation of the: Trustees of the order of th <120> MUCINS
<130> mucinqimr <140>
<141>
<150> PP4708 <151> 1998-07-16 <160> 18 <170> PatentIn Ver. 2.0 <210> 1 <211> 28 <212> PRT
<213> Homo sapiens <220>
<221> REPEAT
<222> (1)..(28) <223> MUC11 consensus tandem repeat sequence <400> 1 Ser Gly Leu Ser Glu Glu Ser Thr Thr Ser His Ser Ser Pro Gly Ser Thr His Thr Thr Leu Ser Pro ,Ala Ser Thr Thr Thr <210> 2 <211> 2872 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (1)..(2871) <400> 2 agg aac agg ccg cac aca aca gca ttc cct ggc agt acc acc atg cca 48 Arg Asn Arg Pro His Thr Thr Al.a Phe Pro Gly Ser Thr Thr Met Pro ggc gtc agt cag gaa tct aca get tcc cac agc agc cca ggc tcc aca 96 Gly Val Ser Gln Glu Ser Thr Al.a Ser His Ser Ser Pro Gly Ser Thr gac aca aca ctg tcc cct ggc agt acc aca gca tca tcc ctt ggt cca 144 Asp Thr Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Pro gaa tct act acc ttc cac agc ggc cca ggc tcc act gaa aca aca ctc 192 Glu Ser Thr Thr Phe His Ser Gly Pro Gly Ser Thr Glu Thr Thr Leu tta cct gac aac acc aca gcc i:.cc ggc ctc ctt gaa gca tct acg ccc 240 Leu Pro Asp Asn Thr Thr Ala Ser Gly Leu Leu Glu Ala Ser Thr Pro gtc cac agc agc act gga tcg cca cac aca aca ctg tcc cct gcc ggc 288 Val His Ser Ser Thr Gly Ser ;Pro His Thr Thr Leu Ser Pro Ala Gly tct aca acc cgt cag gga gaa tct acc acc ttc cag agc tgg cct aac 336 Ser Thr Thr Arg Gln Gly Glu ;5er Thr Thr Phe Gln Ser Trp Pro Asn tcg aag gac act acc cct gca cct cct act acc aca tca gcc ttt gtt 384 Ser Lys Asp Thr Thr Pro Ala Pro Pro Thr Thr Thr Ser Ala Phe Val gag cta tct aca acc tcc cac ggc agc ccg agc tca act cca aca acc 432 Glu Leu Ser Thr Thr Ser His Gly Ser Pro Ser Sex Thr Pro Thr Thr cac ttt tct gcc agc tcc aca acc ttg ggc cgt agt gag gaa tcg aca 480 His Phe Ser Ala Ser ser Thr Thr Leu Gly Arg Ser Glu Glu Ser Thr aca gtc cac agc agc cca gtt gca act gca aca aca ccc tcg cct gcc 528 Thr Val His Ser Ser Pro Val Ala Thr Ala Thr Thr Pro Ser Pro Ala cgc tcc aca acc tca ggc ctc gtt gaa gaa tct acg acc tac cac agc 576 Arg Ser Thr Thr Ser Gly Leu Val Glu Glu Ser Thr Thr Tyr His Ser agc ccg ggc tca act caa aca atg cac ttc cct gaa agc gac aca act 624 Ser Pro Gly Ser Thr Gln Thr M:et His Phe Pro Glu Ser Asp Thr Thr tca ggc cgt ggt gaa gaa tca aca act tcc cac agc agc aca aca cac 672 Ser Gly Arg Gly Glu Glu Ser Thr Thr Ser His Ser Ser Thr Thr His aca ata tct tca get cct agc acc aca tct gcc ctt gtt gaa gaa cct 720 Thr Ile Set Ser Ala Pro Ser Thr Thr Ser Ala Leu Val Glu Glu Pro acc agc tac cac agc agc ccg ggc tca act gca aca aca cac ttc cct 768 Thr Ser Tyr His Ser Ser Pro Gly Ser Thr Ala Thr Thr His Phe Pro gac agc tcc aca acc tca ggc cgt agt gag gaa tca aca gca tcc cac 816 Asp Ser Ser Thr Thr Ser Gly A.rg Ser Glu Glu Ser Thr Ala Ser His agc aac caa gac gca acg gga aca ata gtc cta cct gcc cgc tcc aca 869 Ser Asn Gln Asp Ala Thr Gly Thr Ile Val Leu Pro Ala Arg Ser Thr acc tca gtt ctt ctt gga gaa t.ct acg acc tca ccc atc agt tca ggc 912 Thr Ser Val Leu Leu Gly Glu S'~er Thr Thr Ser Pro Ile Ser Ser Gly tca atg gaa acg aca gcg tta c:cc ggc agt acc aca acg cca ggc ctc 960 Ser Met Glu Thr Thr Ala Leu Pro Gly Ser Thr Thr Thr Pro Gly Leu agt gag aaa tct acc act ttc c:ac agt agc ccc aga tca cca gcc aca 1008 Ser Glu Lys Ser Thr Thr Phe His Ser Ser Pro Arg Ser Pro Ala Thr aca ctc tca cct gcc agc acg aca agc tca ggc gtc agt gaa gaa tcc 1056 Thr Leu Ser Pro Ala Ser Thr '.Chr Ser Ser Gly Val Ser Glu Glu Ser acc acc tcc cac agc cga cca ggc tca acg cac aca aca gca ttc cct 1104 Thr Thr Ser His Ser Arg Pro Gly Ser Thr His Thr Thr Ala Phe Pro gac agc acc acc acg cca ggc ctc agt cgg cat tct aca act tcc cac 1152 Asp Ser Thr Thr Thr Pro Gly Leu Ser Arg His Ser Thr Thr Ser His agc agc cca ggc tca acg gat aca aca ctg tta cct gcc agc acc acc 1200 Ser Ser Pro Gly Ser Thr Asp Thr Thr Leu Leu Pro Ala Ser Thr Thr acc tca ggc ccc agt cag gaa tca aca act tcc cac agc agc cca ggt 1248 Thr Ser Gly Pro Ser Gln Glu Ser Thr Thr Ser His Ser Ser Pro Gly tca act gac aca gca ctg tcc cct ggc agt acc aca gcc tta tcc ttt 1296 Ser Thr Asp Thr Ala Leu Ser Pro Gly Ser Thr Thr Ala Leu Ser Phe ggt caa gaa tct aca acc ttc cac agc agc cca ggc tcc act cac aca 1344 Gly Gln Glu Ser Thr Thr Phe His Ser Ser Pro Gly Ser Thr His Thr aca ctc ttc cct gac agc acc aca agc tca ggc atc gtt gaa gca tct 1392 Thr Leu Phe Pro Asp Ser Thr Thr 5er Ser Gly Ile Val Glu Rla Ser aca cgc gtc cac agc agc act ggc tca cca cgc aca aca ctg tcc cct 1440 Thr Arg Val His Ser Ser Thr Gly Ser Pro Arg Thr Thr Leu Ser Pro gcc agc tcc aca agc cct gga ctt cag gga gaa tct acc gcc ttc cag 1488 Ala Ser Ser Thr Ser Pro Gly Leu Gln Gly Glu Ser Thr Rla Phe Gln acc cac cca gcc tca act cac acg acg cct tca act cct agc acc gca 1536 Thr His Pro Ala Ser Thr His Thr Thr Pro Ser Thr Pro Ser Thr Ala aca gcc cct gtt gaa gaa tct aca acc tac cac cgc agc cca agc tcg 1584 Thr Ala Pro Val Glu Glu Ser Thr Thr Tyr His Arg Ser Pro Ser Ser act cca aca aca cac ttc cct gcc agc tcc aca act tcg ggc cac agt 1632 Thr Pro Thr Thr His Phe Pro Ala Ser 5er Thr Thr Ser Gly His Ser gag aaa tca aca ata ttc cac agc agc cca gat gca agt gga aca aca 1680 Glu Lys Ser Thr Ile Phe His Ser Ser Pro Asp Ala Ser Gly Thr Thr ccc tca tct gcc cac tcc aca acc tca ggt cgt gga gaa tct aca acc 1728 Pro Ser Ser Ala His Ser Thr Thr Ser Gly Arg Gly Glu Ser Thr Thr tca cgc atc agt cca ggc tca, act gaa ata aca acg tta cct ggc agt 17'76 Ser Arg Ile Ser Pro Gly Ser Thr Glu Ile Thr Thr Leu Pro Gly Ser acc aca aca cca ggc ctc agt gag gca tct acc acc ttc tac agc agc 1824 Thr Thr Thr Pro Gly Leu Ser Glu Ala Ser Thr Thr Phe Tyr Ser Ser ccc aga tca cca acc aca aca ctc tca cct gcc agc atg aca agc cta 1872 Pro Arg Ser Pro Thr Thr Thr Leu Ser Pro Ala Ser Met Thr Ser Leu ggc gtc ggt gaa gaa tcc acc acc tcc cgt agc caa cca ggt tct act 1920 Gly Val Gly Glu Glu Ser Thr Thr Ser Arg Ser Gln Pro Gly Ser Thr cac tca aca gtg tca cct gcc agc acc acc acg cca ggc ctc agt gag 1968 His Ser Thr Val Ser Pro Ala Ser Thr Thr Thr Pro Gly Leu Ser Glu gaa tct acc acc gtc tac agc agc agc cca ggc tca act gaa acc aca 2016 Glu Ser Thr Thr Val Tyr Ser 5er Ser Pro Gly Ser Thr Glu Thr Thr gtg ttc cct cgc agc acc aca acc tca gtt cgt ggt gaa gag cct aca 2064 Val Phe Pro Arg Ser Thr Thr Thr Ser Val Arg Gly Glu Glu Pro Thr acc ttc cac agc cgg cca gcc tca act cac aca aca ctg ttc act gag 2112 Thr Phe His Ser Arg Pro Ala 5er Thr His Thr Thr Leu Phe Thr Glu gac agc acc acc tcg ggc ctc act gaa gaa tct aca gcc ttc ccc ggc 2160 Asp Ser Thr Thr Ser Gly Leu Thr Glu Glu Ser Thr Ala Phe Pro Gly agc cca gcc tcc acc caa aca ggg tta cct gcc aca ctc aca acc gca 2208 Ser Pro Ala Ser Thr Gln Thr Gly Leu Pro Ala Thr Leu Thr Thr Ala gac ctc ggt gag gaa tca act acc ttt ccc agc agc tca ggc tca act 2256 Asp Leu Gly Glu Glu Ser Thr Thr Phe Pro Ser Ser Ser Gly Ser Thr gga aca aca ctc tca cct gcc cgc tcc acc acc tct ggc ctc gtt gga 2304 Gly Thr Thr Leu Ser Pro Ala Arg Ser Thr Thr Ser Gly Leu Val Gly gaa tcc aca ccc tca cgc ctc agt cca agc tca acc gaa aca aca act 2352 Glu Ser Thr Pro Ser Arg Leu Ser Pro Ser Ser Thr Glu Thr Thr Thr tta ccc ggc agt ccc aca aca cca agc ctc agt gag aaa tca acc acc 2400 Leu Pro Gly Ser Pro Thr Thr Pro Ser Leu Ser Glu Lys Ser Thr Thr ttc tac act agc ccc aga tca cca gat gca aca ctc tca cct gca acc 2448 Phe Tyr Thr Ser Pro Arg Ser Pro Asp Rla Thr Leu Ser Pro Ala Thr aca aca agc tca ggc gtc agc gaa gaa tcc agc aca tcc cac agt caa 2496 Thr Thr Ser Ser Gly Val Ser Glu Glu Ser Ser Thr Ser His Ser Gln cca ggc tca acg cac aca aca gcg ttc cct gac agc acc acc acc tca 2544 Pro Gly Ser Thr His Thr Thr Ala Phe Pro Asp Ser Thr Thr Thr Ser ggc ctc agt cag gaa cct aaa act tcc cac agc agc caa ggc tca aca 2592 Gly Leu Ser Gln Glu Pro Lys Thr Ser His Ser Ser Gln Gly Ser Thr gag gca aca ctg tcc cct ggc agt acc aca gcc tca tcc ctt ggt caa 2640 Glu Ala Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Gln caa tct aca acc ttc cac agc agc cca ggc gac act gaa acc aca ctc 2688 Gln Ser Thr Thr Phe His Ser Ser Pro Gly Asp Thr Glu Thr Thr Leu tta cct gat gac acc ata acc tca ggc ctc gtg gag gca tct aca ccc 2736 Leu Pro Asp Asp Thr Ile Thr Ser Gly Leu Val Glu Ala Ser Thr.Pro acc cac agc agc act ggc tcg cta cac aca aca ctg acc cct gcc agc 2789 Thr His Ser Ser Thr Gly Ser Leu His Thr Thr Leu Thr Pro Ala Ser tcc aca agc get ggc ctt cag gaa gaa tct act act ttc cag agc tgg 2832 Ser Thr Ser Ala Gly Leu Gln Glu Glu Ser Thr Thr Phe Gln Ser Trp cca agc tca agt gac aca aca cct tca cct ccc ggc ccg g 2872 Pro Ser Ser Ser Asp Thr Thr Pro Ser Pro Pro Gly Pro <210> 3 <211> 957 <212> PRT
<213> Homo Sapiens <900> 3 Arg Asn Arg Pro His Thr Thr .Ala Phe Pro Gly Ser Thr Thr Met Pro Gly Val Ser Gln Glu Ser Thr.Ala Ser His Ser Ser Pro Gly Ser Thr Asp Thr Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Pro Glu Ser Thr Thr Phe His Ser Gly Pro Gly Ser Thr Glu Thr Thr Leu Leu Pro Asp Asn Thr Thr Ala Ser Gly Leu Leu Glu Ala Ser Thr Pro Val His Ser Ser Thr Gly Ser Pro His Thr Thr Leu Ser Pro Ala Gly Ser Thr Thr Arg Gln Gly Glu Ser Thr Thr Phe Gln Ser Trp Pro Asn Ser Lys Asp Thr Thr Pro Ala Pro Pro Thr Thr Thr Ser Ala Phe Val Glu Leu Ser Thr Thr Ser His Gly Ser Pro Ser Ser Thr Pro Thr Thr His Phe Ser Ala 5er Ser Thr Thr Leu Gly Arg Ser Glu Glu Ser Thr Thr Val His Ser Ser Pro Val Ala Thr Ala Thr Thr Pro Ser Pro Ala Arg Ser Thr Thr Ser Gly Leu Val Glu Glu Ser Thr Thr Tyr His Ser Ser Pro Gly Ser Thr Gln Thr Met His Phe Pro Glu Ser Asp Thr Thr Ser Gly Arg Gly Glu Glu Ser Thr Thr Ser His Ser Ser Thr Thr His Thr Ile Ser Ser Ala Pro Ser Thr Thr Ser Ala Leu Val Glu Glu Pro Thr Ser Tyr His Ser Ser Pro Gly Ser Thr Ala Thr Thr His Phe Pro Asp Ser Ser Thr Thr Ser Gly Arg Ser Glu Glu Ser Thr Ala Ser His Ser Asn Gln Asp Ala Thr Gly Thr Ile Val Leu Pro Ala Arg Ser Thr Thr Ser Val Leu Leu Gly Glu Ser Thr Thr Ser Pro Ile Ser Ser Gly Ser Met Glu Thr Thr Ala Leu Pro Gly Ser Thr Thr Thr Pro Gly Leu Ser Glu Lys Ser Thr Thr Phe His Ser Ser Pro Arg Ser Pro Ala Thr Thr Leu Ser Pro Ala Ser Thr Thr Ser Ser Gly Val Ser Glu Glu Ser Thr Thr Ser His Ser Arg Pro Gly Ser Thr His Thr Thr Ala Phe Pro Asp Ser Thr Thr Thr Pro Gly Leu Ser Arg His Ser Thr Thr Ser His Ser Ser Pro Gly Ser Thr Asp Thr Thr Leu Leu Pro Ala Ser Thr Thr Thr Ser Gly Pro Ser Gln Glu Ser Thr Thr Ser His Ser Ser Pro Gly Ser Thr Asp Thr Ala Leu Ser Pro Gly Ser Thr Thr Ala Leu Ser Phe Gly Gln Glu Ser Thr Thr Phe His Ser Ser Pro Gly Ser Thr His Thr Thr Leu Phe Pro Asp Ser Thr Thr Ser Ser Gly Ile Val Glu Ala Ser Thr Arg Val His Ser Ser Thr Gly Ser Pro Arg Thr Thr Leu Ser Pro Ala Ser Ser Thr Ser Pro Gly Leu Gln Gly Glu Ser Thr Ala Phe Gln Thr His Pro Ala Ser Thr His Thr Thr Pro Ser Thr Pro Ser Thr Ala Thr Ala Pro Val Glu Glu Ser Thr Thr Tyr His Arg Ser Pro Ser Ser Thr Pro Thr Thr His Phe Pro Ala Ser Ser Thr Thr Ser Gly His Ser Glu Lys Ser Thr Ile Phe His Ser Ser Pro Asp Ala Ser Gly Thr Thr Pro Ser Ser Ala His Ser Thr Thr Ser Gly Arg Gly Glu Ser Thr Thr Ser Arg Ile Ser Pro Gly Ser Thr Glu Ile Thr Thr Leu Pro Gly Ser Thr Thr Thr Pro Gly Leu Ser Glu Ala Ser Thr Thr Phe Tyr Ser Ser Pro Arg Ser Pro Thr Thr Thr Leu Ser Pro Ala Ser Met Thr Ser Leu Gly Val Gly Glu Glu Ser Thr Thr Ser Arg Ser Gln Pro Gly Ser Thr His Ser Thr Val Ser Pro Ala Ser Thr Thr Thr Pro Gly Leu Ser Glu Glu Ser Thr Thr Val Tyr Ser Ser Ser Pro Gly Ser Thr Glu Thr Thr Val Phe Pro Arg Ser Thr Thr Thr Ser Val Arg G1y Glu Glu Pro Thr Thr Phe His Ser Arg Pro Ala Ser Thr His Thr Thr Leu Phe Thr Glu Asp Ser Thr Thr Ser Gly Leu Thr Glu Glu Ser Thr Ala Phe Pro Gly Ser Pro Ala Ser Thr Gln Thr Gly Leu Pro Ala Thr Leu Thr Thr Ala Asp Leu Gly Glu Glu Ser Thr Thr Phe Pro Ser Ser Ser Gly Sex Thr Gly Thr Thr Leu Ser Pro Ala Arg Ser Thr Thr Ser Gly Leu Val Gly Glu Ser Thr Pro Ser Arg Leu Ser Pro Ser Ser Thr G1u Thr Thr Thr Leu Pro Gly Ser Pro Thr Thr Pro Ser Leu Ser Glu Lys Ser Thr Thr Phe Tyr Thr Ser Pro Arg Ser Pro Asp Ala Thr Leu Ser Pro Ala Thr Thr Thr Ser Ser Gly Val Ser Glu Glu Ser Ser Thr Ser His Ser Gln Pro Gly Ser Thr His Thr Thr Ala Phe Pro Asp Ser Thr Thr Thr Ser Gly Leu Ser Gln Glu Pro Lys Thr Ser His Ser Ser Gln Gly Ser Thr Glu Ala Thr Leu Ser Pro Gly Ser Thr Thr Ala Ser Ser Leu Gly Gln Gln Ser Thr Thr Phe His Ser Ser Pro Gly Asp Thr Glu Thr Thr Leu Leu Pro Asp Asp Thr Ile Thr Ser Gly Leu Val Glu Ala Ser Thr Pro Thr His Ser Ser Thr Gly Ser Leu His Thr Thr Leu Thr Pro Ala Ser Ser Thr Ser Ala Gly Leu G1n Glu Glu Ser Thr Thr Phe Gln Ser Trp Pro Ser Ser Ser Asp Thr Thr Pro Ser Pro Pro Gly Pro <210> 4 <211> 28 <212> PRT
<213> Homo sapiens <220>
<221> REPEAT
<222> (1)..(281 <223> MUC12 consensus tandenn repeat sequence <400> 4 Ser Gly Leu Ser Gln Glu Ser Thr Thr Phe His Ser Ser Pro Gly Ser Thr Glu Thr Thr Leu Ser Pro Ala Ser Thr Thr Thr <210> 5 <211> 2095 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (3)..(1757) <400> 5 as aca ctc tca cct gcc agc atg aga agc tcc agc atc agt gga gaa 47 Thr Leu Ser Pro Ala Ser f4et Arg Ser Ser Ser Ile Ser Gly Glu ccc acc agc ttg tat agc caa gca gag tca aca cac aca aca gcg ttc 95 Pro Thr Ser Leu Tyr Ser Gln Ala Glu Ser Thr His Thr Thr Ala Phe cct gcc agc acc acc acc tca ggc ctc agt cag gaa tca aca act ttc 143 Pro Ala Ser Thr Thr Thr Ser Gly Leu Ser Gln Glu Ser Thr Thr Phe cac agt aag cca ggc tca act gag aca aca ctg tcc cct ggc agc atc 191 His Ser Lys Pro Gly Ser Thr Glu Thr Thr Leu Ser Pro Gly Ser Ile aca act tca tct ttt get caa gaa ttt acc acc cct cat agc caa cca 239 Thr Thr Ser Ser Phe Ala Gln Glu Phe Thr Thr Pro His Ser Gln Pro ggc tca get ctg tca aca gtg tca cct gcc agc acc aca gtg cca ggc 287 Gly Ser Ala Leu Ser Thr Val Ser Pro Ala Ser Thr Thr Val Pro Gly ctt agt gag gaa tct acc acc ttc tac agc agc c<:a ggc tca act gaa 335 Leu Ser Glu Glu Ser Thr Thr Phe Tyr Ser Ser Pro Gly Ser Thr Glu acc aca gcg ttt tct cac agc aac aca atg tcc att cat agt caa caa 383 Thr Thr Ala Phe Ser His Ser Asn Thr Met Ser Ile His Ser Gln Gln tct aca ccc ttc cct gac agc cca ggc ttc act cac aca gtg tta cct 431 Ser Thr Pro Phe Pro Asp Ser Pro Gly Phe Thr His Thr Val Leu Pro gcc acc ctc aca acc aca gac att ggt cag gaa tca aca gcc ttc cac 479 Ala Thr Leu Thr Thr Thr Asp Ile Gly Gln Glu Ser Thr Ala Phe His agc agc tca gac gca act gga aca aca ccc tta cct gcc cgc tcc aca 527 Ser Ser Ser Asp Ala Thr Gly Thr Thr Pro Leu Pro Ala Arg Ser Thr gcc tca gac ctt gtt gga gaa cct aca act ttc tac atc agc cca tcc 575 Ala Ser Asp Leu Val Gly Glu Pro Thr Thr Phe Tyr Ile Ser Pro Ser cct act tac aca aca ctc ttt cct gcg agt tcc agc aca tca ggc ctc 623 Pro Thr Tyr Thr Thr Leu Phe Pro Ala Ser Ser Ser Thr Ser Gly Leu act gag gaa tct acc acc ttc cac acc agt cca agc ttc act tct aca 671.
Thr Glu Glu Ser Thr Thr Phe His Thr Ser Pro Ser Phe Thr Ser Thr att gtg tct act gaa agc ctg gaa acc tta gca cca ggg ttg tgc cag 719 Ile Val Ser Thr Glu Ser Leu Glu Thr Leu Ala Pro Gly Leu Cys Gln gaa gga caa att tgg aat gga aaa caa tgc gtc tgt ccc caa ggc tac 767 Glu Gly Gln Ile Trp Asn Gly Lys Gln Cys Val Cys Pro Gln Gly Tyr gtt ggt tac cag tgc ttg tcc cct ctg gaa tcc ttc cct gta gaa acc 815 Val Gly Tyr Gln Cys Leu Ser Pro Leu Glu Ser Phe Pro Val Glu Thr ccg gaa aaa ctc aac gcc act tta ggt atg aca gtg aaa gtg act tac 863 Pro Glu Lys Leu Asn Ala Thr Leu Gly Met Thr Val Lys Val Thr Tyr aga aat ttc aca gaa aag atg aat gac gca tcc tcc cag gaa tac cag 911 Arg Asn Phe Thr Glu Lys Met Asn Asp Ala Ser Ser Gln Glu Tyr Gln aac ttc agt acc ctc ttc aag aat cgg atg gat gtc gtt ttg aag ggc 959 Asn Phe Ser Thr Leu Phe Lys Asn Arg Met Asp Val Val Leu Lys Gly gac aat ctt cct cag tat aga ggg gtg aac att cgg aga ttg ctc aac 1007 Asp Asn Leu Pro Gln Tyr Arg Gly Val Asn Ile Rrg Arg Leu Leu Asn ggt agc atc gtg gtc aag aac gat gtc atc ctg gag gca gac tac act 1055 Gly Ser Ile Val Val Lys Asn Asp Val Ile Leu Glu Ala Asp Tyr Thr tta gag tat gag gaa ctg ttt gaa aac ctg gca gag att gta aag gcc 1103 Leu Glu Tyr Glu Glu Leu Phe Glu Asn Leu Ala Glu Ile Val Lys Ala aag att atg aat gaa act aga aca act ctt ctt gat cct gat tcc tgc 1151 Lys Ile Met Asn Glu Thr Arg Thr Thr Leu Leu Asp Pro Asp Ser Cys aga aag gcc ata ctg tgc tat agt gaa gag gac act ttc gtg gat tca 1199 Arg Lys Ala Ile Leu Cys Tyr 5er Glu Glu Asp Thr Phe Val Asp Ser tcg gtg act ccg ggc ttt gac ttc cag gag caa tgc acc cag aag get 1297 Ser Val Thr Pro Gly Phe Asp Phe Gln Glu Gln Cys Thr Gln Lys Ala gcc gaa gga tat acc cag ttc tac tat gtg gat gtc ttg gat ggg aag 1295 Ala Glu Gly Tyr Thr Gln Phe Tyr Tyr Val Asp Val Leu Asp Gly Lys ctg gcc tgt gtg aac aag tgc acc aaa gga acg aag tcg caa atg aac 1343 Leu Ala Cys Val Asn Lys Cys Thr Lys Gly Thr Lys Ser Gln Met Asn tgt aac ctg ggc aca tgt cag~ ctg caa cgc agt ggc ccc cgc tgc ctg 1391 Cys Asn Leu Gly Thr Cys Gln. Leu Gln Arg Ser Gly Pro Arg Cys Leu tgc cca aat acg aac aca cac: tgg tac tgg gga gag acc tgt gaa ttc 1439 Cys Pro Asn Thr Asn Thr His Trp Tyr Trp Gly Glu Thr Cys Glu Phe aac atc gcc aag agc ctc gtc~ tat ggg atc gtg ggg get gtg atg gcg 1487 Asn Ile Ala Lys Ser Leu Val Tyr Gly Ile Val Gly Ala Val Met Ala gtg ctg ctg ctc gca ttg atc atc cta atc atc tta ttc agc cta tcc 1535 Val Leu Leu Leu Ala Leu Ile Ile Leu Ile Ile Leu Phe Ser Leu Ser cag aga aaa cgg cac agg gaa cag tat gat gtg cct caa gag tgg cga 1583 Gln Arg Lys Arg His Arg Glu Gln Tyr Asp Val Pro Gln Glu Trp Arg aag gaa ggc acc cct ggc atc ttc cag aag acg gcc atc tgg gaa gac 1631 Lys Glu Gly Thr Pro Gly Ile Phe Gln Lys Thr Ala Ile Trp Glu Asp cag aat ctg agg gag agc aga ttc ggc ctt gag aac gcc tac aac aac 16?9 Gln Asn Leu Arg Glu Ser Arg Phe Gly Leu Glu Asn Ala Tyr Asn Asn ttc cgg ccc acc ctg gag act gtt gac tct ggc aca gag ctc cac atc 1727 Phe Arg Pro Thr Leu Glu Thr Val Asp Ser Gly Thr Glu Leu His Ile cag agg ccg gag atg gta gca tcc act gtg tgagccaacg ggggcctccc 1777 Gln Arg Pro Glu Met Val Ala Ser Thr Val accctcatct agctctgttc aggagagctg caaacacaga gcccaccaca agcctccggg 1837 gcgggtcaag aggagaccga agtcaggccc tgaagccggt cctgctctga gctgacagac 1897 ttggccagtc ccctgcctgt gctcctgctg gggaaggctg ggggctgtaa gcctctccat 1957 ccgggagctt ccagactccc agaagcctcg gcacccctgt ctcctcctgg gtggctcccc 2017 actctggaat ttccctacca ataaaagcaa atctgaaagc tcaaaaaaaa aaaaaaaaaa 2077 aaaaaaaaaa aaaaaaaa 2095 <210>6 <211>585 <212>PRT

<213>Homo Sapiens <400> 6 Thr Leu Ser Pro Ala Ser Met Arg Ser Ser Ser I1e Ser Gly Glu Pro 1 5 i0 15 WO 00/04142 PCT/AU99/00579 _ Thr Ser Leu Tyr Ser Gln Ala Glu Ser Thr His Thr Thr Ala Phe Pro Ala Ser Thr Thr Thr Ser Gly Leu Ser Gln Glu Ser Thr Thr Phe His Ser Lys Pro Gly Ser Thr Glu Thr Thr Leu Ser Pro Gly Ser Ile Thr Thr Ser Ser Phe Ala Gln Glu Phe Thr Thr Pro His ser Gln Pro Gly Ser Ala Leu Ser Thr Val Ser Pro Ala Ser Thr Thr Val Pro Gly Leu Ser Glu Glu Ser Thr Thr Phe Tyr Ser Ser Pro Gly Ser Thr Glu Thr Thr Ala Phe Ser His Ser Asn Thr Met Ser Ile His Ser Gln Gln Ser Thr Pro Phe Pro Asp Ser Pro Gly Phe Thr His Thr Val Leu Pro Ala Thr Leu Thr Thr Thr Asp Ile Gly Gln Glu Ser Thr Ala Phe His Ser Ser Ser Asp Ala Thr Gly Thr Thr Pro Leu Pro Ala Arg Ser Thr Ala Ser Asp Leu Val Gly Glu Pro Thr Thr Phe Tyr Ile Ser Pro Ser Pro Thr Tyr Thr Thr Leu Phe Pro Ala Ser Ser Ser Thr Ser Gly Leu Thr Glu Glu Ser Thr Thr Phe His Thr Ser Pro Ser Phe Thr Ser Thr Ile Val Ser Thr Glu Ser Leu Glu Thr Leu Ala Pro Gly Leu Cys Gln Glu Gly Gln Ile Trp Asn Gly Lys Gln Cys Val Cys Pro Gln Gly Tyr Val Gly Tyr Gln Cys Leu Ser Pro Leu Glu Ser Phe Pro Val Glu Thr Pro Glu Lys Leu Asn Ala Thr Leu Gly Met Thr Val Lys Val Thr Tyr Arg Asn Phe Thr Glu Lys Met Asn Asp Ala Ser Ser Gln Glu Tyr Gln Asn Phe Ser Thr Leu Phe Lys Asn Arg Met Asp Val Val Leu Lys Gly Asp Asn Leu Pro Gln Tyr Arg Gly Val Asn Ile Arg Arg Leu Leu Asn Gly Ser Ile Val Val Lys Asn Asp Val Ile Leu Glu Ala Asp Tyr Thr Leu Glu Tyr Glu Glu Leu Phe Glu Asn Leu Ala Glu Ile Val Lys Ala Lys Ile Met Asn Glu Thr Arg Thr Thr Leu Leu Asp Pro Asp Ser Cys Arg Lys Ala Ile Leu Cys Tyr ser Glu Glu Asp Thr Phe Val Asp Ser Ser Val Thr Pro Gly Phe Asp Phe Gln Glu Gln Cys Thr Gln Lys Ala Ala Glu Gly Tyr Thr Gln Phe Tyr Tyr Val Asp Val Leu Asp Gly Lys Leu 42p 425 430 Ala Cys Val Asn Lys Cys Thr Lys Gly Thr Lys Ser Gln Met Asn Cys Asn Leu Gly Thr Cys Gln Leu Gln Arg Ser Gly Pro Arg Cys Leu Cys Pro Asn Thr Asn Thr His Trp Tyr Trp Gly Glu Thr Cys Glu Phe Asn Ile Ala Lys Ser Leu Val Tyr Gly Ile Val Gly Ala Val Met Ala Val Leu Leu Leu Ala Leu Ile Ile Leu Ile Ile Leu Phe Ser Leu Ser Gln Arg Lys Arg His Arg Glu Gln. Tyr Asp Val Pro Gln Glu Trp Rrg Lys Glu Gly Thr Pro Gly Ile Phe Gln Lys Thr Ala Ile Trp Glu Asp Gln Rsn Leu Arg Glu Ser Arg Phe Gly Leu Glu Asn Ala Tyr Asn Asn Phe Arg Pro Thr Leu Glu Thr Val Asp Ser Gly Thr Glu Leu His Ile Gln Arg Pro Glu Met Val Ala Ser Thr Val <210> 7 <211> 10 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Random lOmer PCR primer <400> 7 acttcgccac 10 <210> 8 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:MUC 12 PCR
forward primer <400> B
tgaagggcga caatcttcct c 21 <210> 9 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:MUCl2 reverse PCR primer <400> 9 tacacgaggc tcttggcgat gttg 24 <210> 10 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Mucll forward PCR primer <400> 10 caggcgtcag tcaggaatct acag 24 <210> 11 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Mucll reverse PCR primer <400> 11 gaggctgtgg tgttgtcagg taag 24 <210> 12 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:
Beta2-microglobulin forward PCR primer <400> 12 tgaattgcta tgtgtctggg t 21 <210> 13 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Beta 2-microglobulin reverse PCR primer <400> 13 cctccatgat gctgcttaca t 21 <210> 14 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: MUC12 forward primer for verification of contiguous sequence <400> 14 agccaaccag gctcagctct 20 <210> 15 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: MUC12 reverse primer for verification of contiguous sequence <400> 15 gctcacacag tggatgctac c 21 <210> 16 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Clone Ii5 reverse PCR primer <900> 16 gggaacactg tggtttcagt tgag 24 <210>17 <211>17 <212>PRT

<213>Homo Sapiens <220>
<221> PEPTIDE
<222> (1)..(17) <223> MUC11 immunizing peptide <400> 17 Cys Phe His Ser Arg Pro Ala Ser Thr His Thr Thr Leu Phe Thr Glu ~P
<210>18 <211>17 <212>PRT

<213>Homo Sapiens <220>
<221> PEPTIDE
<222> (1)..(17) <223> MUC12 immunizing peptide <400> 18 Thr Tyr Arg Asn Phe Thr Glu Lys Met Asn Asp Ala Ser Ser Gln Glu Cys

Claims (32)

62

1. An isolated MUC nucleic acid corresponding to a MUC gene located on human chromosome 7q22, or a mammalian chromosome structurally or functionally equivalent thereto, which MUC gene is normally predominantly expressed in the colon.

2. The isolated MUC nucleic acid of Claim 1 which comprises a nucleotide sequence encoding an amino acid sequence which comprises SGLSEESTTSHSSPGSTHTTLSPASTTT(SEQ ID NO:1).

3. The isolated MUC nucleic acid of Claim 2, wherein the nucleic acid comprises a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO:3.

4. The isolated MUC nucleic acid of Claim 2, wherein the nucleic acid includes a sequence of nucleotides according to SEQ ID NO: 2.

5. The isolated MUC nucleic acid of Claim 1, wherein the nucleic acid which comprises a nucleotide sequence which encodes an amino acid sequence whuch comprises SGLSQESTTFHSSPGSTETTLAPASTTT (SEQ
ID NO: 4).

6. The isolated MUC nucleic acid of Claim 5, wherein the nucleic acid comprises a nucleotide sequence which encodes an amino acid sequence according to SEQ ID NO:6.

7. The isolated MUC nucleic acid of Claim 5, wherein the nucleic acid includes a sequence of nucleotides according to SEQ ID NO: 5.

8. A MUC nucleic acid homolog which hybridizes to the isolated MUC nucleic acid of any one of Claims 2-7 under conditions of at least low stringency.

9. A MUC nucleic acid homolog which has at least 60%
nucleotide sequence identity with the isolated MUC nucleic acid of any one of Claims 2-7.

10. An isolated MUC polypeptide having an amino acid sequence according to SEQ ID NO: 3.

11. An isolated MUC polypeptide having an amino acid sequence according to SEQ ID NO: 6.

12. An isolated MUC polypeptide homolog which has at least 60%
amino acid identity with the MUC polypeptide of Claim 10 or Claim 11.

13. An antibody specific for the MUC polypeptide or MUC
polypeptide homolog of any one of Claims 10-12.

14. An antibody according to Claim 13 which is a monoclonal antibody.

15. A monoclonal antibody according to Claim 14, which monoclonal antibody is selected from the group consisting of M11.9 and M12.15.

16. A method of detecting the MUC polypeptide of Claim 10 or Claim 11, including the steps of:
(i) obtaining a sample from said mammal;
(ii) forming a complex between said MUC polypeptide, if present in said sample, and an anti-MUC polypeptide antibody; and (iii) detecting said MUC polypeptide in said complex.

17. The method of Claim 17, wherein the antibody is selected from the group consisting of M11.9 and M12.15.

18. A method of detecting a MUC gene or a MUC gene transcript including the steps of:
(i) obtaining a nucleic acid extract from said mammal;
(ii) forming a hybrid nucleic acid comprising a MUC gene or a MUC gene transcript if present in said sample, and a corresponding isolated MUC nucleic acid according to Claim 1, or a portion thereof; and (iii) detecting said hybrid nucleic acid.

19. The method of Claim 18, wherein the isolated MUC nucleic acid has a nucleotide sequence selected from the group consisting of SEQ
ID NO:2 and SEQ ID NO:5.

20. A method of detecting a MUC gene or a MUC gene transcript including the steps of:
(i) obtaining a nucleic acid extract from said mammal;
(ii) using one or more primers, each having a nucleotide sequence corresponding to a distinct portion of the isolated MUC nucleic acid of Claim 1, together with a polynucleotide sequence amplification technique, to produce a MUC gene amplification product from said extract; and (iii) detecting said MUC gene amplification product.

21. The method of Claim 20, wherein the one or more primers is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID
NO: 10, SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO:15.

22. The method of Claim 21, wherein the polynucleotide sequence amplification technique is RT-PCR.

23. Use of the isolated MUC nucleic acid of Claim 1, or a portion thereof, to detect a polymorphism, mutation, deletion, truncation and/or expansion in a corresponding MUC gene or MUC gene transcript.

24. A pharmaceutical composition comprising a pharmaceutically acceptable amount of the MUC polypeptide of Claim 10 or Claim 11, together with a pharmaceutically-acceptable carrier and/or diluent.

25. A pharmaceutical composition comprising a pharmaceutically acceptable amount of an anti-MUC antibody according to any one of Claims 13-15, together with a pharmaceutically-acceptable carrier and/or diluent.

26. A method of treating of a mammal suffering from a disease condition, said method including the step of administering to said mammal a pharmaceutical composition according to Claim 24 or Claim 25 to thereby alleviate or prevent one or more symptoms of said disease condition in said mammal.

27. A method of gene therapy of a mammal suffering from a disease condition, said method including the step of administering a gene therapy construct to said mammal, said gene therapy construct comprising the isolated MUC nucleic acid of Claim 1, or a portion thereof, to thereby alleviate or prevent one or more symptoms of said disease condition in said mammal.

28. The method of Claim 27, wherein the isolated MUC nucleic acid has a nucleotide sequence selected from the group consisting of SEQ
ID NO:2 and SEQ ID NO:5.

29. The method of any one of Claims 26-28, wherein said disease condition is associated with aberrant Mucin expression, altered properties of mucus or epithelial inflammatory processes involving Mucins

30. The method of any one of Claims 26-28, wherein said disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD), breast cancer (BC), Crohn's disease, ulcerative colitis, asthma and chronic bronchitis.

31. The method of Claim 30, wherein said disease condition is selected from the group consisting of colorectal cancer (CRC), cystic fibrosis (CF), inflammatory bowel disease (IBD) and breast cancer (BC).

32. The method of any one of Claims 26-31, wherein the mammal is a human.