CA2225126A1 - Mammalian brainiac and egghead genes - Google Patents

Description

CA 0222~126 1997-12-17 MAMMALIAN BRAINIAC AND EGGHEAD GENES

Field of the Invention This invention relates to new mammalian genes involved in cell adhesion.

Background of the Invention Analysis of the Brainiac and Egghead genes in Drosophila melanogaster has revealed that these two molecules each encode a protein with a leader sequence and therefore destined for entry into the secretory pathway. Both Brainiac and Egghead proteins are required for several Notch dependent adhesion events, relating to adhesion between epithelial cells or sheets of cells (1,2).
In addition, the Egghead protein contains at lest one transmembrane domain, suggesting that it is anchored to the cell which synthesizes it.
A model has been proposed by Goode et. al. whereby Egghead on the surface of one cell is linked, via secreted Brainiac in the extracellular space, to a Notch receptor anchored in the membrane of a neighboring cell.
This trimolecular complex is therefore expected to link two cells together. This cell-cell adhesion is required for many developmental processes such as correct follicle cell fate, growth regulation and neurogenesis, and cell contact or adhesion is required for transduction of signals between two cells, for example for tyrosine kinase-mediated signaling.
The Drosophila Brainiac and Egghead genes regulate adhesion between epithelial cells and require the presence of the Notch protein for this activity.
Mutational disruption of Brainiac, Egghead or Notch results in the loss of follicle epithelial cell adhesion, thereby reducing the efficiency of signalling through CA 0222~126 1997-12-17 other epithelial cell receptors such as the epidermal growth factor receptor.
Similarities have been noted between the amino acid sequences of the Drosophila Fringe proteins and Drosophila Brainiac protein, with conservation of particular amino acid motifs (3). The Brainiac proteins may therefore belong to the Fringe protein family although they appear to have functions which are distinct from those of the Fringe proteins.
Description of the Invention The present inventors have identified and sequenced a new mammalian gene which shows some homology with the Drosophila Brainiac gene. Table 1 shows the cDNA
sequence (Sequence ID No. 1) and Table 2 the deduced amino acid sequence (Sequence ID No. 2) of mouse Brainiac. Table 3 shows a comparison of the amino acid sequence of mouse (Brainiac 1) and Drosophila (D-Brainiac) Brainiac proteins.
The inventors have also obtained the cDNA sequence (Table 4: Sequence ID No:3) and amino acid sequence (Table 5: Sequence ID No:4) of human Brainiac. Table 6 shows a comparison of the mouse (Brainiac 1 pr) and human (Human Braini) amino acid sequences. Both Brainiac proteins contain a leader sequence for secretion from the cell.
The inventors have found that Brainiac mRNA is widely expressed in mouse tissues. Northern blot analysis has shown that a single band of approximately 3kb is expressed in adult mouse heart, brain, spleen, lung, liver, muscle, kidney and testes. A strong signal was noted after 4.5 hours exposure, using 2 ~g of poly A

CA 0222~126 1997-12-17 mRNA on the blot, indicating high expression in all tissues analysed.
As the Drosophila Brainiac and Egghead proteins are involved in adhesion between epithelial cells, and as this adhesion is required for cell viability, cell growth regulation and cell fate specification, it is envisioned that wild type or mutant forms of mammalian Brainiac and/or Egghead can be used to alter epithelial cell adhesion. This should be useful in treating many diseases which present problems of cell viability, cell growth regulation and cell fate specification. For example, these proteins, or active fragments or analogues of these proteins and these genes can be used to treat diseases such as cancer, psoriasis and other skin lesions, and nervous system defects or diseases.
Many cells require specific cell-cell contacts with their neighbors for survival and/or growth.
Overexpression of mutant Brainiac protein can be envisioned to disrupt the cell-cell contact between tumour cells or between tumour and normal cells. This may result in reduced cell survival or reduced tumour growth.
Alternatively, some tumour cells have sustained mutations in genes which encode adhesion molecules like the cadherins. The adhesion mediated by Brainiac may regulate the efficiency by which growth-suppressive or differentiation-inducing signals are transmitted to tumour cells. Indeed, mutations in Brainiac or Egghead in flies result in tumourous growth of follicle tissue.
It can be expected therefore that some tumours may have suffered mutations which alter Brainiac-dependent cell adhesion. Therapeutic application of Brainiac protein or derivatives thereof may restore control of cell growth.

CA 0222~126 1997-12-17 As skin lesions can involve altered development, differentiation, adhesion or growth of epithelial cells which are normally present in specific, adhering epithelial layers, it is envisioned that Brainiac proteins, nucleic acids or derivatives could be used to treat lesions such as psoriasis to restore normal differentiation, growth and cell adhesion.
Mutations of the Drosophila Brainiac gene in fly eggs cause tumour formation in the neighboring follicle cell layers.
Mutations in the mammalian Brainiac gene are therefore likely to lead to congenital developmental defects, including cancer susceptibility syndromes.
The mammalian Brainiac genes may therefore be used to detect somatic or germline DNA-lesions which are responsible for developmental syndromes or diseases including cancer. Once mutations are detected using standard DNA analysis techniques, then Brainiac protein, derivatives of Brainiac protein or gene therapy vectors containing the Brainiac cDNA could be used to target expression of Brainiac protein function to the affected tissue. In this way, the Brainiac-dependent adhesive system could be restored.
As the Brainiac protein appears to be a secreted protein, antibodies, drugs or mutants could be used to block its activity or it could be administered therapeutically.
The inventors have noted a significant degree of homology between the Drosophila Egghead gene and Drosophila Brainiac and Fringe genes (Table 7), suggesting that Egghead also belongs to the Fringe family of proteins.

CA 0222~126 1997-12-17 Isolated Nucleic Acids In accordance with one series of embodiments, this invention provides isolated nucleic acids corresponding to or related to the nucleic acid sequences disclosed herein which encode murine and human Brainiac proteins.
One of ordinary skill in the art is now enabled to identify and to isolate mammalian Brainiac genes or cDNAs which are allelic variants of the disclosed mammalian Brainiac sequences or are homologues thereof, in other species, using standard hybridization screening or PCR
techniques.
In one embodiment, the invention provides cDNA
sequences encoding murine and human Brainiac proteins (Sequence ID NOS: 2 and 4 respectively) comprising the nucleotide sequences of Sequence ID NOS: 1 and 3 respectively.
Also provided are portions of the Brainiac gene sequences useful as probes in PCR primers or for encoding fragments, functional domains or antigenic determinants of Brainiac proteins.
The invention also provides portions of the disclosed nucleic acid sequences comprising about 10 consecutive nucleotides (eg. for use as PCR primers) to nearly the complete disclosed nucleic acid sequences.
The invention provides isolated nucleic acid sequences comprising sequences corresponding to at least 10, preferably 15 and more preferably at least 20 consecutive nucleotides of the Brainiac genes as disclosed or enabled herein or their complements.
The invention also provides recombinant vectors and host cells comprising the nucleotide sequences of the invention.

CA 0222~126 1997-12-17 Substantially Pure Proteins In accordance with a further series of embodiments, this invention provides substantially pure mammalian Brainiac proteins, fragments of these proteins and fusion proteins including these proteins and fragments.
The proteins, fragments and fusion proteins have utility, as described herein, for the preparation of polyclonal and monoclonal antibodies to mammalian Brainiac proteins, for the identification of binding partners of the mammalian Brainiac proteins and for diagnostic and therapeutic methods, as described herein.
For these uses, the present invention provides substantially pure proteins, polypeptides or derivatives of polypeptides which comprise portions of the mammalian Brainaic amino acid sequences disclosed or enabled herein and which may vary from about 4 to 5 amino acids (e.g.
for use as immunogens) to the complete amino acid sequence of the proteins. The invention provides substantially pure proteins or polypeptides comprising sequences corresponding to at least 5, preferably at least 10 and more preferably 50 or 100 consecutive amino acids of the mammalian Brainiac proteins disclosed or enabled herein.
The proteins of the invention may be isolated and purified by any conventional method suitable in relation to the properties revealed by the amino acid sequences of these proteins.
Alternatively, cell lines may be produced which overexpress the Brainiac gene products, allowing purification of the proteins for biochemical characterization, large-scale production, antibody production and patient therapy.
For protein expression, eukaryotic and prokaryotic expression systems may be generated in which a Brainiac CA 0222~126 1997-12-17 gene sequence is introduced into a plasmid or other vector which is then introduced into living cells.
Constructs in which the Brainiac cDNA sequence containing the entire open reading frame is inserted in the correct orientation into an expression plasmid may be used for protein expression. Alternatively, portions of the sequence may be inserted. Prokaryotic and eukaryotic expression systems allow various important functional domains of the protein to be recovered as fusion proteins and used for binding, structural and functional studies and also for the generation of appropriate antibodies.
Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA
corresponding to the gene. They may also include sequences allowing for their autonomous replication within the host organism, sequences that encode genetic traits that allow cells containing the vectors to be selected, and sequences that increase the efficiency with which the mRNA is translated. Stable long-term vectors may be maintained as freely replicating entities by using regulatory elements of viruses. Cell lines may also be produced which have integrated the vector into the genomic DNA and in this manner the gene product is produced on a continuous basis.
Expression of foreign sequences in bacteria such as . coli require the insertion of the sequence into an expression vector, usually a plasmid which contains several elements such as sequences encoding a selectable marker that assures maintenance of the vector in the cell, a controllable transcriptional promoter which upon induction can produce large ~mounts of mRNA from the cloned gene, translational control sequences and a polylinker to simplify insertion of the gene in the correct orientation within the vector. A relatively CA 0222~126 1997-12-17 simple E. coli expression system utilizes the lac promoter and a neighboring lacZ gene which is cut out of the expression vector with restriction enzymes and replaced by the Brainiac gene sequence. In vitro expression of proteins encoded by cloned DNA is also possible using the T7 late-promoter expression system.
Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages such as T3, T5 and SP6 may also be used for in vitro production of proteins from cloned DNA. E. coli can also be used for expression by infection with M13 Phage mGPI-

2. E. coli vectors can also be used with phage Lambda regulatory sequences, by fusion protein vectors, by maltose-binding protein fusions, and by glutathione-S-transferase fusion proteins.
Eukaryotic expression systems permit appropriatepost-translational modifications to expressed proteins.
This allows for studies of the Brainiac genes and gene products including determination of proper expression and post-translational modifications for biological activity, identifying regulatory elements in the 5' region of the gene and their role in tissue regulation of protein expression. It also permits the production of large amounts of normal proteins for isolation and purification, to test the effectiveness of pharmacological agents or as a component of a signal transduction system to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring polymorphisms and artificially produced mutated proteins.
The Brainiac DNA sequences can be altered using procedures such as restriction enzyme digestion, DNA
polymerase fill-in, exonuclease deletion, terminal CA 0222~126 1997-12-17 deoxynucleotide transferase extension, ligation of synthetic or cloned DNA sequences and site-directed in vitro mutagenesis, including site-directed sequence alteration using specific oligonucleotides together with PCR.
Once the appropriate expression vector containing the selected gene is constructed, it is introduced into an appropriate host cell by transformation techniques including calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion and liposome-mediated transfection.
The host cell which may be transfected with the vectors of this invention may be selected from the group consisting of E. Coli, Pseudomonas, Bacillus subtilis, or other bacilli, other bacteria, yeast, fungi, insect (using baculoviral vectors for expression), mouse or other animal or human tissue cells. Mammalian cells can also be used to express the Brainiac proteins using a vaccinia virus expression system.
Methods for producing appropriate vectors, for transforming cells with those vectors and for identifying transformants are described in the scientific literature, for example in Sambrook et al. (1989), Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. or latest edition thereof.
The cellular distribution of Brainiac proteins in tissues can be analyzed by reverse transcriptase PCR
- analysis. Antibodies can also be generated for several applications including both immunocytochemistry and immunofluorescence techniques to visualize the proteins directly in cells and tissues in order to establish the cellular location of the proteins.
The present invention includes effective fragments or analogues of the Brainiac proteins described herein.

CA 0222~126 1997-12-17 "Effective" fragments or analogues retain the activity of the described Brainiac proteins to mediate adhesion between cells.
The term "analogue" extends to any functional and/or chemical equivalent of a mammalian Brainiac protein and includes proteins having one or more conservative amino acid substitutions, proteins incorporating unnatural amino acids and proteins having modified side chains.
Examples of side chain modifications contemplated by the present invention include modification of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidatei acetylation with acetic anhydride; carbamylation of amino groups with cyanate;
trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH4.
The guanidino 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 -acylisourea formation followed by subsequent derivatisation, for example, to a corresponding amide.
Sulfhydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide;
performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic CA 0222~126 1997-12-17 acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercurials; carbamylation with cyanate at alkaline pH.
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 sulphonyl halides. Tyrosine residues 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 iodacetic acid derivatives of 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-methylheptanoic acid, 2-thienyl alanine and/or D-isomers or amino acids.
Examples of conservative amino acid substitutions are substitutions within the following five groups of amino acids (amino acids are identified by the conventional single letter code): Group 1: F Y W; Group 2: V L I; Group 3: H K R; Group 4: M S T P A G; Group 5:
D E.
Fragments or analogues of the mammalian Brainiac proteins of the invention may be conveniently screened for their effectiveness by a variety of methods.
For example, a Drosophila-based assay can be employed to test for the ability of mammalian Brainiac protein fragments or analogues to mediate adhesion between oocyte and follicle cells or between epithelial cells during neurogenesis in Drosophila. In addition, CA 0222~126 1997-12-17 transgenic mice may be generated with Brainiac or mutant Brainiac cDNAs targeted to an accessible tissue compartment such as skin. These transgenics could then be used to screen Brainiac fragments or analogues for activity in altering the phenotype of the transgenic anlmal .
These screening systems may also be used to screen compounds for effectiveness as antagonists of Brainiac protein activity.
Antibodies In order to prepare polyclonal antlbodies, fusion proteins containing defined portions or all of the Brainiac proteins can be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle. Fusion proteins are commonly used as a source of antigen for producing antibodies. Two widely used expression systems for E. coli are glutathione-S-tranferase or maltose binding protein fusions using the pGex vectors. The protein can then be purified, coupled to a carrier protein if desired, and mixed with Freund's adjuvant (to help stimulate the antigenic response of the animal) and injected into rabbits or other appropriate laboratory animals. Alternatively, the protein can be isolated from Brainiac protein-expressing cultured cells.
Following booster injections at weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or purified prior to use by various methods including affinity chromatography employing Protein A-Sepharose, antigen Sepharose or Anti-mouse-Ig-Sepharose. The sera can then be used to probe protein extracts from cells and tissues run on a polyacrylamide gel to identify the Brainiac protein.

CA 0222~126 1997-12-17 Alternatively, synthetic peptides can be made to the antigenic portions of the proteins and used to inoculate the animals. The most common practice is to choose a 10 to 15 residue peptide corresponding to the carboxyl or amino terminal sequence of a protein antigen and to chemically cross-link it to a carrier molecule such as keyhole limpet haemocyanin or BSA. However, if an internal sequence peptide is desired, selection of the peptide is based on the use of algorithms that predict potential antigenic sites. These predictive methods are, in turn, based on predictions of hydrophilicity (Kyte and Doolittle (29), Hopp and Woods (30) or secondary structure (Chou and Fasman (31)). The objective is to choose a region of the protein that is either surface exposed such a hydrophilic region or a region conformationally flexible relative to the rest of the structure, such as a loop region or a region predicted to form a ~-turn. The selection process is also limited by constraints imposed by the chemistry of the coupling procedures used to attach peptide to carrier protein. A
carboxyl-terminal peptide is chosen because they are often more mobile than the rest of the molecule and the peptide can be coupled to a carrier in a straightforward manner using glutaraldehyde. The amino-terminal peptide has the disadvantage that it may be modified post-translationally by acetylation or by the removal of a leader sequence. A comparison of the protein amino acid sequence between species can yield important information.
Those regions with sequence differences are likely to be immunogenic. Synthetic peptides can also be synthesized as immunogens as long as they mimic the native antigen as closely as possible.

CA 0222~l26 l997-l2-l7 It is understood by those skilled in the art that monoclonal anti-Brainiac antibodies may also be produced using Brainiac protein obtained from cells actively expressing the protein or by isolation from tissues. The cell extracts, or recombinant protein extracts, containing the Brainiac protein, are injected in Freund's adjuvant into mice. After being injected 9 times over a three week period, the mice spleens are removed and resuspended in phosphate buffered saline (PBS). The spleen cells serve as a source of lymphocytes, some of which are producing antibody of the appropriate specificity. These are then fused with a permanently growing myeloma partner cell, and the products of the fusion are plated into a number of tissue culture wells in the presence of a selective agent such as HAT. The wells are then screened by ELISA to identify those containing cells making binding antibody. These are then plated and after a period of growth, these wells are again screened to identify artibody-producing cells.
Several cloning procedures are carried out until over 90~
of the wells contain single clones which are positive for antibody production. From this procedure a stable line of clones which produce the antibody is established. The monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose, ion-exchange chromatography, as well as variants and combinations of these techniques.
Truncated versions of monoclonal antibodies may also be produced by recombinant techniques in which plasmids are generated which express the desired monoclonal antibody fragment(s) in a suitable host. Antibodies specific for mutagenic epitopes can also be generated.
The mammalian Brainiac proteins and fragments or analogues thereof are also useful as antigens in CA 0222~126 1997-12-17 immunoassays including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of the protein.

Pharmaceutical Compositions In a further embodiment, this invention provides pharmaceutical compositions for the treatment of mammalian disorders which involve Brainiac-dependent adhesion defects, comprising a therapeutic amount of a Brainiac protein, or an active fragment or analogue thereof in association with a pharmaceutical carrier.
Administration of a therapeutically active amount of a pharmaceutical composition of the present invention means an amount effective, at dosages and for periods of time necessary to achieve the desired result. This may also vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the mammalian Brainiac protein to elicit a desired response in the subject. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
By pharmaceutically acceptable carrier as used herein is meant one or more compatible solid or liquid delivery systems. Some examples of pharmaceutically acceptable carriers are sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, collagen, talc, stearic acids, magnesium stearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids, pyrogen-free water, isotonic saline, phosphate buffer, and other suitable non-toxic substances used in pharmaceutical formulations. Other excipients such as CA 0222~l26 l997-l2-l7 wetting agents and lubricants, tableting agents, stabilizers, anti-oxidants and preservatives are also contemplated.
The compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers and formulations adapted for particular modes of administration are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA
1985). On this basis the compositions of the invention include solutions of the substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The pharmaceutical compositions of the invention may be administered therapeutically by various routes such as by injection or by oral, nasal, buccal, rectal, vaginal, transdermal or ocular routes in a variety of formulations, as is known to those skilled in the art.
Binding partners The mammalian Brainiac proteins, expressed as fusion proteins, can be utilized to identify small peptides that bind to these proteins. In one approach, termed phage display, random peptides (up to 20 amino acids long) are expressed with coat proteins (geneIII or geneVIII) of filamentous phage such that they are expressed on the surface of the phage thus generating a library of phage that express random sequences. A library of these random CA 0222~126 1997-12-17 sequences is then selected by incubating the library with the mammalian Brainiac protein or fragments thereof and phage that bind to the protein are then eluted either by cleavage of Brainiac from the support matrix or by elution using an excess concentration of soluble Brainiac protein or fragments. The eluted phage are then repropagated and the selection repeated many times to enrich for higher affinity interactions. The random peptides can either be completely random or constrained at certain positions through the introduction of specific residues. After several rounds of selection, the final positive phage are sequenced to determine the sequence of the peptide.
An alternate but related approach uses affinity purification techniques. Brainiac proteins are immobilised on a suitable solid support. Preparations such as cell extracts which may contain Brainiac protein binding partners are passed over the affinity matrix and any bound material is eluted and microsequenced.
Suitable methods are available in the scientific literature, for example in Bartley et al., Nature (1994), 368, 558-560.
Expression cloning, for example through expression of cDNA libraries in Cos or other cells followed by binding of labelled Brainiac protein to the transfected cells, may also be used to screen for Brainiac protein binding partners, for example as described in Matthews et al., Cell (1991) 65, 973-982.
The identification of proteins or peptides that interact with Brainiac proteins can provide the basis for the design of peptide antagonists or agonists of Brainiac protein function. Further, the structure of these peptides determined by standard techniques such as CA 0222~l26 l997-l2-l7 protein NMR or X-ray crystallography can provide the structural basis for the design of small molecule drugs.

Animal Models The present invention also provides for the production of transgenic non-human animal models for the study of mammalian Brainiac gene function, for the screening of candidate pharmaceutical compounds, for the creation of explanted mammalian cell cultures which express the Brainiac proteins or in which a Brainiac gene has been inactivated by knock-out deletion, and for the evaluation of potential therapeutic interventions.
The invention enables a transgenic animal, including a transgenic insect, wherein a genome of the animal or of an ancestor of the animal has been modified by introduction of a transgene comprising a mammalian Brainiac gene under the transcriptional control of tissue restricted regulatory elements including the mouse mammary-tumour virus long term repeat sequences.
Transgenic fruit flies which express mammalian Brainiac genes may be made by methods previously described in relation to transgenic flies having mammalian Fringe genes (4). Such transgenic flies may be used to screen for compounds which can repair developmental defects observed in these transgenic flies.
Transgenic animals may also be made and used similarly. Further, transgenic animals with inappropriate expression of Brainiac proteins may be examined for phenotypic changes, for example tumour development, and may be used to screen for compounds with potential as pharmaceuticals. Compounds which provide reversal of the phenotypic changes are candidates for development as pharmaceuticals.

CA 0222~126 1997-12-17 Transgenic animal models in accordance with the invention can be created by introducing a DNA sequence encoding a selected mammalian Brainiac protein either into embryonic stem (ES) cells of a suitable animal, for example a mouse, by transfection or microinjection, or into a germ line or stem cell by a standard technique of oocyte microinjection.
The ES cells are inserted into a young embryo and this embryo or an injected oocyte are implanted into a pseudo-pregnant foster mother to grow to term.
The techniques for generating transgenic animals are now widely known and are described in detail, for example, in Hogan et al., (1986), and M. Capecchi (1989).

Methods of Treatment In accordance with one embodiment, the present invention enables a method for preventing or treating a disorder in a mammal characterised by an abnormality in a signal transduction pathway which requires Brainiac-dependent cell adhesion.

Screening Methods In a further embodiment, the invention enables amethod for identifying compounds which can modulate the expression of a mammalian Brainiac gene comprising contacting a cell with a candidate compound wherein the cell includes a regulator of a Brainiac gene operably joined to a coding region; and detecting a change in expression of the coding region.
In a further embodiment, the invention enables a method for identifying compounds which can selectively bind to a mammalian Brainiac protein comprising providing a preparation including at least one mammalian Brainiac protein;
contacting the preparation with a candidate compound; and determining binding of the Brainiac protein to the compound.
Suitable methods for such screening include affinity chromatography, co-immunoprecipitation, biomolecular interaction assay.
In a further embodiment, the invention enables a method for diagnosing in a subject a disorder characterised by abnormal expression of a Brainiac protein comprising obtaining a tissue sample from the subject;
determining Brainiac protein expression in the tissue sample.
Tissue samples could be used for isolation of RNA
which would then be subjected to RT-PCR analysis using specific primers for Brainiac genes in order to amplify 20 the cDNA for sequencing. Control tissues could be used for comparison of sequence.
With the identification of the mammalian Brainiac gene sequences and gene products, nucleotide probes and antibodies raised to the gene products can be used in a 25 variety of hybridisation and immunological assays to screen for and detect the presence of either a normal or mutated gene or gene product.
Patient therapy through removal or blocking of a mutant gene product, as well as supplementation with a normal gene product by amplification, by genetic and recombinant techniques or by immunotherapy can now be achieved.
Correction or modification of the defective gene product by protein treatment immunotherapy (using CA 0222~126 1997-12-17 antibodies to the defective protein) or knock-out of the mutated gene together with wild-type supplementation is now also possible. Suitable methods are described or referenced for example, in Crystal, R.G. (1995), Science, 270, 404-410.

Examples Example 1: Cloning and sequencing of murine Brainiac cDNA
The public database of expressed sequence tags was screened for DNA sequences which could be translated to produce a reading frame with significant amino acid sequence similarity to Drosophila Brainiac. Two such sequences were identified (clones AA119132 and AA204363).
The oligonucleotide 5' AGGTATGAGAGATGAGTGTGG and 5' CTCACTGGGATGTAGTACTTC, based on sense and antisense sequences from the predicted Brainiac-related open reading frame from these two EST sequences respectively, were synthesised. These two oligonucleotides were used in PCR reactions from several tissues and in several cases yielded product of 895 nucleotides (based on the predicted sequence contig generated from the two EST
sequences discussed above). This product was cloned and sequenced from either end to confirm its similarity or identity with the EST sequences. Finally, the cloned 895 bp fragment was excised and used as probe to screen filters from a mouse mammary gland cDNA library.
Positive plaques were isolated and clones were sequenced.
One such clone, the nucleotide sequence of which is shown in Table 1, encoded full length mouse Brainiac protein.
Mouse Brainiac cDNA was epitope tagged with a C-terminal myc-epitope tag. This chimeric cDNA was CA 0222~126 1997-12-17 transfected into COS cells and expressed, yielding a protein of approximately 55 kDa.

Example 2: The sequence of human Brainiac cDNA
The human Brainiac partial sequence has been derived using the sequence of the mouse gene in searches and analysis of the human EST sequence databases.
The complete mouse cDNA sequence was used to identify human EST gene sequences which were part of the human Brainiac cDNA. These included EST sequences which could not be unambiguously identified as being from Brainiac based solely on their homology to the Drosophila gene or its predicted protein. This group of human EST
sequences, determined to encode part of human Brainiac, were then connected and translated. Ambiguities were then resolved through comparison with the sequence of the mouse Brainiac cDNA and its encoded protein. In this way, the human Brainiac cDNA sequence was constructed from the mouse sequence and the EST database.

References 1. Goode, S., Morgan, M., Liang, Y.-P. & Mohawald, A.P., (1996), Dev. Biol. 178, 35-50.

2. Goode, S., Melnick, M., Chou, T.-B. & Perrimon, N., (1996), Development 122, 3863-3870.

3. Yuan, Y.P. et al., (1997), Cell, 88, 9-11.

4. Cohen, B. et al., (1997), Nature Genetics, 16, 283-288.

CA 0222~126 1997-12-17 Table 1 - mouse Brainiac cDNA
GAATTCGGCACGAGGCGGCAACAAGTGCTGGAGCTGAGGCGAGCCGGAGCCGCCCAG
ACCCCGCCGGGCCGCCCGTCCGCGCATTGCGCATGGAGCGAGAGCGCGGCGGTCGCG
GGGCTGAGCCGCAAGACCGGCTGGGACGTGGATGCGGCCGCGGTCTTCCGCCCCGCC
CCGCCGAGCTGGAGGTGTCCCTAGACAAGGTATGAGAGATGAGTGTGGGGCGTCGAA
GAGTCAAGTTGCTGGGCATCCTGATGATGGCA~ATGTCTTCATTTATTTGATTGTGG
AAGTCTCCA~AAACAGTAGCCAAGACA~AAATGGA~AGGGAGGAGTAATAATCCCGA
AAGAGAAGTTCTGGAAGCCACCCAGCACTCCCCGGGCATACTGGAACAGGGAACAGG
AGAAGCTGAACAGGTGGTACA~TCCCATCTTGAACAGGGTGGCCAATCAGACAGGGG
AGCTAGCCACATCTCCA~ACACAAGTCACCTGAGCTATTGTGAACCAGACTCGACGG
TCATGACAGCTGTGACAGATTTTA~TAATCTGCCGGACAGATTTA~AGACTTTCTCT
TGTATTTGAGATGCCGGAATTACTCGCTGCTTATAGATCAACCGAAGA~ATGTGCAA
AGAAGCCCTTCTTACTATTGGCGATA~AGTCCCTCATTCCACATTTTGCCAGAAGGC
AAGCAATTCGGGAGTCTTGGGGCCGAGA~ACCAACGTAGGGAACCAGACAGTAGTGA
GGGTCTTCCTGTTGGGCAAGACACCCCCAGAGGACAACCACCCTGACCTTTCGGACA
TGCTTAAGTTTGAGAGTGACAAGCACCAGGACATCCTCATGTGGAACTATAGAGACA
CATTCTTCAACCTGTCCCTGAAGGAAGTGCTGTTTCTTAGGTGGGTGAGCACTTCCT
GTCCAGACGCAGAGTTTGTCTTCA~GGGCGATGATGACGTGTTTGTGAACACCCATC
ACATCCTTAATTACTTGAATAGCTTATCCAAGAGCA~AGCCA~AGACTTGTTCATAG
2 0 GTGACGTGATCCACAATGCTGGGCCTCACCGGGATAAGA~ACTGAAGTACTACATCC
CAGAAGTCTTCTACACCGGCGTCTACCCACCGTATGCCGGGGGTGGTGGATTCCTGT
ACTCCGGCCCCCTTGCCTTGAGGCTGTACAGTGCGACTAGCCGGGTCCATCTCTACC
CTATTGATGATGTTTATACGGGAATGTGCCTTCAGA~ACTGGGCCTTGTTCCAGAGA
AGCACAAAGGCTTCAGGACATTTGATATTGAAGAGA~AAATAAGA~AAATATTTGTT
2 5 CCTATATAGACCTAATGTTAGTACATAGCAGA~ACCTCAAGAGATGATTGATATCT
GGTCTCAGTTGCA~AGTCCTAATTTA~AATGCTGA~ATAGACATGAGCTGCATTTCA
CAGA~AGGCCTAGCCTGACTAGTTCCCATGGTGTGCTCTCACAATAGGTGAGTTCTG
TGTGAGGCTATTAGCCTTCATGAGCAGGTAGCCCCTGGGCTCCCAAGCCCTCAGTCC
TTCCCTTGCCTTGTGAAGAGGGA~GGCTGAAGACAGCTCAGCATGGCAGGGTGAGTG
GTTATGACCCTTCCTCTGGCTGCCGGTCCTCAGTTTCTAATTTGTTTTCTTTCTCCT
CCACAATTATGTATGTATGTGTGTATATATGTGTGTGTGTACATACATACATATATA
TATATGTAGGACACAACCTGGTGGCTTTGTGA~ATGGAATTCCTATGTATTTTCATA
AGATBTTGA~AGTTGTCTAGA~AGTAGACTGATGTCAATCTCCCGTCACCCAGCAGT
ATTGTCCTTGTTACTAGA~ACCGTTACTTCCTTTATGCAAGGA~AGCCACGCAGGCG

CA 0222~l26 l997-l2-l7 TGTAGTTCATCTTGTCAGGGCTTATGGCCATGAGGACAGAGGGGATTTTCTTTTTAC
TTGTGTTTGGTTTCCTGGGTGGCATCATGGTAGTTAACCTATTTTTAGTATTTGAAG
ATCATGAGTGTGATTCCCTAATGGCCAACTGGAGACTGAGTAGCCCGACAGCCATGG
GTCTGTGAGTGTTCAGAGACTGGGAAGCATTCGCCACTTCTGAGCTTTGGACGTGAT
TAGTCAGTTAA~ACCCCAAGATTCTATTCTTGCCATATTATCACGTATTCCTTAGAT
A~AATTCTGGGTAGTGACACTTCCCTGTCTCAGTGTAGAAGTGCCTGTGCTTTTATT
TATTGTTCAGATCA~ACACCA~AACATTTTCTTAAA~AATATTTTGTGTAATATTTT
ATTTGTATACAGTGTTTGTGA~ATATTTAACTAGAGCATGATATTTTATTTTTTCTC
ATTTTTAATTCTTTGAGAATTTCATACAATGTGTTTTGATTGTATTCACCTCCCCAT
CTCCTCCCAGATCCAGCATGATGTTTTA~ATGTTAAGCTGTAACATGTTAGATA~AG
TTAACTCTTATTTTTGAATTTTAA~ATTTGGATGGGGGGGTATGAACTGCTAGAGAA
AATA~AGTTCTGCCAAAATATTGCATATACTAGTATCTTGTAACATGCTTTCTTGAA
ATATTTTTGTGCTTTAGAGGGGTCTCACCTGTGCTACAGGGGACTGGGA~AAGTGGA
ATA~AGTGATTGTATTTTTTAATC
start codon underlined Table 2 - mouse Brainiac amino acid sequence MSVGRRRVKLLGILMMANVFIYLIVEVSKNSSQDKNGKGGVIIPKEKFWKPPSTPRA
YWNREQEKLNRWYNPILNRVANQTGELATSPNTSHLSYCEPDSTVMTAVTDFNNLPD
RFKDFLLYLRCRNYSLLIDQPKKCAKKPFLLLAIKSLIPHFARRQAIRESWGRETNV
GNQTW RVFLLGKTPPEDNHPDLSDMLKFESDKHQDILMWNYRDTFFNLSLKEVLFL
RWVSTSCPDAEFVFKGDDDVFVNTHHILNYLNSLSKSKAKDLFIGDVIHNAGPHRDK
KLKYYIPEVFYTGVYPPYAGGGGFLYSGPLALRLYSATSRVHLYPIDDVYTGMCLQK
LGLVPEKHKGFRTFDIEEKNKKNICSYIDLMLVHSRKPQEMIDIWSQLQSPNLKC

D-Brainiac 1 ~ SKH ~ LL~ RCL ~ LPLI- -- T,~ YC-- ---------~ LLTHLHEL--50 Br~;n;~ VGR~VK~ LGI33~M~NVF IY3r~VSKN SSQDKNGKG~ Vll~K~K~K 50 D-Brainiac 51 ---------- ----NFE ~ F HYP ~ ---- -DDTGSGSAS ~ ~ ------ 100 Br~;n;~cl 51 PPSTPRAYWN REQEKLN~Y NPI~RVANQ TGELATSPNT ~HeSYCEPDS 100 D-Brainiac 101 ---------- ---~ ~ --- - ~ PSFTA EVPV ~ R- ----- ~ L~ 150 Br~;n;~c1 101 TVMTAVTDFN NLE~ L~D~RNys- -T.T.T~bkKC AKKPF13LLAa 150 160 170 ~ 180 190 200 D-Brainiac 151 ~ VGNSR ~ ~ 1' ~ 3 GRFSDVHLR~ ~ l'A--3 ~ EK ~A--- 200 BrA;n;Ac1 151 ~ IPHFA~ Q ~ S~R~ TNVGNQTV~ ~~J l~hl~p~ ~NHP~SDML 200 210 220. 230 240 250 D-Brainiac 201 -~ ADFT ~Y3 ~ T ~ ~hM ~ ~ QFNRS ~ y 250 BrA;n;A~l 201 K~29UK~ ~ NYR ~ ~ ~S ~ !FL l~ ~rSCPDA ~ ~KC ~ 250 D-Brainiac 251 Y ~ AKN~n3~F ~ RGRQ~HQP E ~ FAGHVFQ TS ~ ~ ~ ~ SL .YPF 300 BrA;n;Act 251 F~ ln~ Y ~SLSK ~AK D ~ IGDVIHN AG ~ ~ ~ Y~ P-l~ YT 300 D-Brainiac 301 DRW ~ TA~ A~ L ~ KALR Q ~ P ~-K~ '~ IVAL ~ ~ SL 350 BrA;n;~cl 301 GVY ~ GG~ G ~Y ~PLAL ~ KVH ~L~ MCLQ ~ ~VP350 D-Brainiac 351 QHCDD~L-- FHRPAY ~ PD ~ SVIAS~ FGD ~ ~ n~ ~ ECR ~ A 400 BrA; n; A~l 351 ~K~K~ ~ ~ 1 ~ ~C ~FTnT.MT.V~ - K~ r.l)l ~ QLQ ~ ~ K 400 D-Brainiac 401 -.......... .......... .................... .......... 450 BrA;n;Arl 401 C.......... .......... .................... .......... 450 Table 3 CA 0222~126 1997-12-17 Table 4 - human Brainiac nucleotide sequence NNNNNNNNNNNNNNNNNNNNNNNNNNNTTGCTGGGCATCCTGATGATGGCAAATGTC
TTCATTTATTTGATTATGGAAGTCTCCAAAAACAGTAGCCAAGAAAAAAATGGAAAA
GGNNNNGTAATAATACCCA~AGAGAAGTTCTGGAAGATATCTACCCCTCCCNNGGCA
TACTNGAACAGAGAACAAGAGAAGCTGAACAGGCAGTACAACCCCATCCTGAACAGG
CTGACCAACCAGACAGGGGAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTTACGGGTTTTAACAACTTGCCGGAC
AGATTTA~AGACTTTCTGCTGTATTTGAGATGCCGCAATTATTCACTGCTTATAGAT
CAGCCGGATAAGTGTGCA~AGA~ACCTTTCTTGTTGCTGGCGATTAAGTCCCTCACT
CCACATTTTGCCAGAAGGCAAGCAATCCGGGAATCCTGGGGCCAAGAAAGCAACGCA
GGGAACCA~ACGGTGGTGCGAGTCTTCCTGCTGGGCCAGACACCCCCAGAGGACAAC
CACCCCGACCTTTCAGATATGCTGA~ATTTGAGAGTGAGAAGCACCAAGACATTCTT
ATGTGGAACTACAGAGACACTTTCTTCAACTTGTCTCTGAAGGAAGTGCTGTTTCTC
AGGTGGGTAAGTACTTCCTGCCCAGACACTGAGTTTGTTTTCAAGGGCGATGACGAT
GTTTTTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGACCA~A
GCCA~AGATCTCTTCATAGGTGATGTGATCCACAATGCTGGACCTCATCGGGATAAG
AAGCTGAAGTACTACATCCCAGAAGTTGTTTACTCTGGCCTCTACCCACCCTATGCA
GGGGGAGGGGGGTTCCTCTACTCCGGCCACCTGGCCCTGAGGCTGTACCATATCACT
2 0 GACCAGGTCCATCTCTACCCCATTGATGACGTTTATACTGGAATGTGCCTTCAGA~A
CTCGGCCTCGTTCCAGAGA~ACACA~AGGCTTCAGGACATTTGATATCGAGGAGAAA
AACAAAAATAACATCTGCTCCTATGTAGATCTGATGTTAGTACATAGTAGAAAACCT
CAAGAGATGATTGATATTTGGTCTCAGTTGCAGAGTGCTCATTTAAAATGCTAA

Table 5 - human Brainiac amino acid sequence XXXXXXXXXLLGILMMANVFIYLIMEVSKNSSQEKNGKGXVIIPKEKFWKISTPPXA
YXNREQEKLNRQYNPILNRLTNQTGEXXXXXXXXXXXXXXXXXXXXXXVTGFNNLPD
RFKDFLLYLRCRNYSLLIDQPDKCAKKPFLLLAIKSLTPHFARRQAIRESWGQESNA
GNQTVVRVFLLGQTPPEDNHPDLSDMLKFESEKHQDILMWNYRDTFFNLSLKEVLFL
RWVSTSCPDTEFVFKGDDDVFVXXXXXXXXXXXXXKTKAKDLFIGDVIHNAGPHRDK
KLKYYIPE W YSGLYPPYAGGGGFLYSGHLALRLYHITDQVHLYPIDDVYTGMCLQK
LGLVPEKHKGFRTFDIEEKNKNNICSYVDLMLVHSRKPQEMIDIWSQLQSAHLKC

CA 02225l26 l997-l2-l7 _ 28 _ 30 40 50 Human Braini~ 1 xxxxxxxxx~ r ~ ~ h L ~ r ~ 50 Brainiacl pr~ 1 MSVGRRRVK~ dr~ 17~ 50 C~O~>

Human Braini51 ISTF~ ~ ~ r~:3~ y~T ~ F--~xxxxxx xxxxxxxxxx 100 Br~;n;~cl pr51 PPST ~ ~ d~ 3 ~ ATSPNT SHLSYCEPDS 100 Human Braini101 xxxxx~ n-3 n~ 1 r~k ~ 150 Br~;n;~cl pr 101 TVMTA ~ ~ -S~ 3~.~d~ 150 Human Braini 151 ~ n:Y~ e~ F~ 200 Br~;n;A~l pr 151 ~ :r:~@~3Rar~ 200 Human Braini 201 ;;~1rr:l731~n~ n~ ~n ~ Z: ~ 250 Br~;n;~c1 pr 201 ~ 'X ~~ 250 Human Braini 251 xxxxxxxxxx xxx~ ::L'~ ~ ~ r r ~ ~
BrA;n;Acl pr 251 NTHHILNYLN SL~ t~L~ 1~ 300 Human Braini 301 ~ ~lrA~ ~ y~s 1 ~ 5 l~ ~j :: 350 BrA;n;Ac1 pr 301 : ~ ~ ~ S~ 5.~ 350 Human Braini 351 ~ ?:~ n~ t:?~ ~H~. . . 400 BrA;n;~c1 pr 351 ::~ P~I ~ t ~ ~~ ~ ......... 400 ~' 1 ' ~._ ~ -Table 6 LDDlFI sv~ N yHul~r~I IKlwFQLAkuu ~ r ~ 5 . . KcsQGHF . . l 7 . . ~jAA~ L ~t~ v~ .l ~vpRLviu~LD . . 3 l . . KKITFwFATGGAGFcLsRA- - -LTLKML- pI . . l l . . AIRFpDDv rMGFI IE
PRDVFIAVA~ Hl~ARLDLLFETWI~it~HK~ ~rltlL~;. .14. .NCSSAHS. .17. .SGKK~ HvuwNYvNLE~ALLRLLA. .30. .RPVHFWFATCGAGFCISRG---LALRMG-PW. .11. .RIRL~VL~ll~iYlVE
LGDlFrAvKT~FHRcRT~nT~rT~rr~nl~Q~lrlt~lv~..15..NC.SAEHS..17..sGLR~vt~HvuuvrJyvNp~ rQT~T~K~.30..KLVRFWFATGGAGFCINRQ---LAkKMV-PW..11..LIRLPDDCTVGYIIE
PDDVFIAVKlTR~HGPPLRLLLRTWISR~4 rlt.L~u. .15. .NCSAVRT. .17. .SGRK~tC:Hvvw~.~\rNPKSLLHLLs. .31. .NTVKFWFATGGAGFCLSRG---LALKMS-PW. .11. .RVRLPDDCTvGYIVE
pARr~rMT~TT~ GNsRRRE-AIRRTl~iGyE~GR---FsDvHLR..28..DFTDAyF..l7..NRsEFyLtvuDvy-y~AKNvLK-FLG..36..~tul ~vTAGAFILSQKA--LRQLYAA....4...PLFRFwvYrJGIvAL
PF-LLIAIKSLIPHFARRQ-AIRE~ GN~rvvR..24..NyROTFF..l7..pDA:-~vrA~uuvv~vN~HHTTNyTN..35..~ vlt~yAG~GFLysGpL--ALRLy~sA....g...H-LypIDrN~McLQ
RGDFPDLv~rNVLRN~N---TCLr;TGLEN-----FLIEvvT..19..EYKTRTG..19..NDsrJwIvi~r~r~r~rTT ~T ~ tN~V}~llN..19..EtrJvNwLTrLAD~txv~uv..~ALRLQFH-ApL..15..h~V~tvN~ilu~ ~E
P.D.. VAT.V.N.R.R.. ILRTW.. R.. F....... N.. A.F N.S.W.. hVDDD.YV.. KSVLK.L. .. VR.W.. T.A.. F.LS.. LRL.. P. R.. FDDV.. G.VAE
FRINGE
LUNATIC FRINGE D
r~ANIC FRINGE o RADICAL FRINGE
BRAINIAC

EGGHEAD ~
AMINO ACIDS CO~ON TO TWO GENE GROUPS (NOT NECESSARILY IDENTICAL) I_ T~G)n t i f'A
SirnilE~riti~ (I:L:V:A) (R:K) ~ (Y:F:W) (T-S: S-N) ~E:D) Takle 7

Claims