WO2001029213A1 - Proteines analogues aux proteines humaines associees au sit4 (sapl), genes codant pour ces proteines, et leurs utilisations - Google Patents

Proteines analogues aux proteines humaines associees au sit4 (sapl), genes codant pour ces proteines, et leurs utilisations Download PDF

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Publication number
WO2001029213A1
WO2001029213A1 PCT/GB2000/004027 GB0004027W WO0129213A1 WO 2001029213 A1 WO2001029213 A1 WO 2001029213A1 GB 0004027 W GB0004027 W GB 0004027W WO 0129213 A1 WO0129213 A1 WO 0129213A1
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nucleic acid
polypeptide
sequence
sapl
amino acid
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PCT/GB2000/004027
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English (en)
Inventor
John Andrew Todd
Rebecca Christina Joan Twells
John Wilfred Hess
Patricia Hey
Charles Thomas Caskey
Holly Hammond
Michael Lee Metzker
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The Wellcome Trust Limited As Trustee To The Wellcome Trust
Merck & Co., Inc.
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Priority to CA002387041A priority Critical patent/CA2387041A1/fr
Priority to EP00969687A priority patent/EP1222264A1/fr
Priority to JP2001532196A priority patent/JP2003512055A/ja
Priority to AU79343/00A priority patent/AU7934300A/en
Publication of WO2001029213A1 publication Critical patent/WO2001029213A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to nucleic acids, polypeptides, oligonucleotide probes and primers, methods of diagnosis or prognosis, and other methods relating to and based on the cloning and characterisation of a gene which the present inventors have termed SAPL, found in at least two isoforms terms SAPLa and SAPLb .
  • Mammals and yeast use a similar mechanism that relies upon cyclins and cyclin-dependent kinases (CDKs) to regulate the cell cycle.
  • CDKs cyclin-dependent kinases
  • the components of this mechanism include the cyclins, CLN1 and CLN2, and the cyclin-dependent kinases CDC28 (cell division control) in yeast (Dynlacht, 1997. Nature 389, 149-152) .
  • the activity of the serine/t recnine kinase CDC28, also known as CDKl is essential for the completion of G ⁇ START, the controlling event in the yeast cell cycle.
  • CDC28 activity is modulated by the level of the cyclins, CLNJ and CLN2. The level of expression of CDC28 remains relatively constant throughout the cell cycle.
  • the SIT4 Ppase protein phosphatase
  • the SIT4 Ppase is a type 2A phosphatase which is encoded by the si t4 (sporulation-induced transcript 4)gene (Sutton et al . 1991. Mol. Cell. Biol . 11, 2133-2148).
  • the SIT4 protein is 55% identical to the catalytic subunit of mammalian type 2A phosphatase and 40% identical to mammalian type 1 phosphatase.
  • SAP proteins act as positive modulators of SIT4 Ppase.
  • the mechanism by which the SAP proteins modulate SIT4 activity is unknown.
  • One possibility that has been suggested is that the SAP proteins increase the substrate specificity of SIT4 Ppase, in a fashion analogous to that found for the glycogen- targeting subunit of type 1 phosphatases (Luke et al . , 1996. Mol. Cell. Biol. 16, 2744-2755).
  • SIT4 would be a SAP-dependent phosphatase in a manner similar to CDC28 being a cyclin-dependent kinase (Luke et al., 1996. Mol. Cell. Biol. 16, 2744-2755) .
  • CDC28 being a cyclin-dependent kinase
  • the present inventors now disclose for the first time two isoforms of a novel gene, arising from alternative splicing and encoding highly related proteins, from the IDDM4 locus on human chromosome llql3.
  • the isoforms have been termed by the inventors " SAPLa” (SAP like) and " SAPLb” .
  • Figure 1(a) shows the nucleotide sequence of SAPLa cDNA. Nucleotide numbering herein is by reference to this sequence.
  • Figure 1 (c) shows the amino acid sequence translation of the open reading frame in the SAPLa cDNA producing SAPL isoform a. Amino acid residue numbering herein is by reference to this sequence .
  • Figure 1 (d) shows the amino acid sequence translation of an alternative open reading frame in the SAPLa cDNA which starts with a sequence that conforms with the Kozak consensus sequence for efficient initiation of translation.
  • Figure 2 (b) shows the longest open reading frame of the SAPLb cDNA.
  • Figure 5 shows a multipoint linkage curve of the IDDM4 region.
  • SAPLa is most likely localized to the cytoplasmic portion of the cell. A nonoptimal initiation can lead to multiple start sites (Kozak, (1996) Mamm. Genome 7, 563-574) .
  • the first ATG codon that conforms to the Kozak consensus sequence is at nucleotide 482, GACATGG, this resulting in a protein of 725 amino acids ( Figure 1(d)).
  • the SAPLa cDNA contains consensus signals for polyadenylation at nucleotides 3592-3597 and 4115 to 4120.
  • the second cDNA ( Figure 2(a)), SAPLb, of 3228 nucleotides contains an open reading frame ( Figure 2 (b) ) that encodes a protein ( Figure 2(c)), SAPLB, of 791 amino acids.
  • the two proteins, SAPLa and SAPLb are 100% identical for the first 776 amino acids.
  • SAPLb has a predicted molecular weight of 89 kdal with an isoelectric point of 4.30, like SAPLa, it is predicted to be expressed in the cytoplasm.
  • Both SAPLa and SAPLb contain a tandem repeat of the amino acid sequence Ser- Thr-Asp-Ser-Glu-Glu (STDSEE) from amino acids 562-573.
  • the amino acid sequence identity between amino acid 94-724 of SAPL and SAP190 is 19%. Over a similar region SAPL is 18% identical to SAP185.
  • SAPL protein A number of potential protein phosphorylation sites are found in the SAPL protein (Table 4) . These include sites for the cAMP dependent protein kinase, protein kinase C, and casein kinase 2. Protein phosphorylation is a reversible modification of proteins and an important mechanism for modulating protein function. Furthermore in yeast the deletion of the si t4 gene results in hyperphosphorylation of the SAP proteins (Luke et al . , 1996. Mol. Cell. Biol. 16, 2744-2755) , therefore there is direct evidence that this family of proteins is subject to protein phosphorylation. Thus it is likely that SAPL is phosphorylated at least at some of the sites listed in Table 4.
  • SAPL function is modulated by protein phosphorylation.
  • Protein phosphorylation of SAPL may be used in assays for compounds that modulate the level of SAPL phosphorylation. These compounds may inhibit either kinases or phosphatases that act on SAPL. Compounds isolated in such a fashion may have therapeutic utility in modifying the function of SAPL.
  • the cloning of the SAPL cDNA permits overexpression of the SAPL protein, and various isoforms, and testing of ability to complement SAP mutants in yeast. Similarly, expression of the human SAPL in yeast allows for the testing of a physical association between SAPL and SIT4. The cloning of the SAPL cDNA also allows the testing of the ability of SAPL to interact and modulate the activity of human protein phosphatase 6 and related phosphatases. Usefulness of SAPL in screening for molecules of pharmaceutical potential is discussed further below.
  • SAPL protein may act in a manner analogous to the SAP proteins in yeast either to activate the phosphatase or modify its specificity. This too inicates usefulness of SAPL in assays for compounds that modulate the activity of the phosphatase, e.g. inhibit it.
  • Compounds that modify the activity of SAPL may be tested in assays of T-cell proliferation or apoptosis.
  • Compounds able to modify SAPL activity may be identified by ability to stimulate or inhibit SAPL complementation in mutant yeast deleted for all four yeast SAP genes (Luke et al . (1996) Mol . Cel l . Biol . 16: 2744-2755).
  • polymorphisms within the SAPL gene and the location of this gene within the IDDM4 locus allow for use of certain of the polymorphisms as diagnostic markers. These polymorphisms may be used to assay for the presence of a chromosomal region that confers susceptibility to type 1 diabetes. This susceptibility may be due to functional polymorphisms within the SAPL gene itself or may be due to a functional polymorphism within a neighboring gene that is in linkage disequilibrium with a SAPL polymorphism.
  • a nucleic acid molecule encoding a SAPL polypeptide, which may be any of the SAPLa polypeptide isoforms of which the amino acid sequences are shown in Figure 1 (c) and Figure 1 (d) and the SAPLb polypeptide isoform of which the amino acid sequence is shown in Figure 2 (c) .
  • nucleic acid encoding a polypeptide including the amino acid sequence shown in Figure 1(c), Figure 1(d) or Figure 2(c).
  • an additional aspect of the present invention provides nucleic acid encoding a polypeptide which includes the first 776 amino acids shown in Figure 1(c) and Figure 2(c) which are identical for the respective SAPL isoforms a and b.
  • a coding sequence of the present invention may be that shown included in Figure 1 (a) , Figure 1 (b) , Figure 2 (a) or Figure 2 (b) , or it may be a mutant, variant, derivative or allele of one of the sequences shown.
  • the sequence may differ from that shown in a said figure by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in a figure herein yet encode a polypeptide with the same amino acid sequence .
  • the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in Figure 1 (c) , Figure 1 (d) or Figure 2 (c) .
  • Nucleic acid encoding a polypeptide which is an amino acid sequence mutant, variant, derivative or allele of the sequence shown in one of these figures is further provided by the present invention. Such polypeptides are discussed below.
  • Nucleic acid encoding such a polypeptide may show at the nucleotide sequence and/or encoded amino acid level greater than about 60% homology with the coding sequence shown in the relevant figure and/or the amino acid sequence shown in the relevant figure, greater than about 70% homology, greater than about 80% homology, greater than about 90% homology or greater than about 95% homology.
  • amino acid "homology” this may be understood to be similarity (according to the established principles of amino acid similarity, e.g. as determined using the algorithm GAP (Genetics Computer Group, Madison, WI) or identity.
  • GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps.
  • GAP GAP
  • FASTA Altschul et al . (1990) J. Mol . Biol . 215: 405-410
  • FASTA which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448
  • Smith-Waterman algorithm Smith and Waterman (1981) J. Mol Biol . 147: 195-197
  • suitable conditions include hybridization overnight at 42°C in 0.25M Na 2 HPOregister, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C in 0. IX SSC, 0.1% SDS.
  • suitable conditions include hybridization overnight at 65°C in 100.25M Na 2 HP0 4 , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60°C in 0. IX SSC, 0.1% SDS.
  • the coding sequence may be included within a nucleic acid molecule which has the sequence shown in Figure 1 (a) or Figure
  • Alterations in a sequence according to the present invention which are associated with IDDM or other disease may be 25 preferred in accordance with embodiments of the present invention. Implications for screening, e.g. for diagnostic or prognostic purposes, are discussed below.
  • Particular nucleotide sequence alleles according to the present invention have sequences with a variation indicated in Table 2. One or more of these may be associated with susceptibility to IDDM or other disease.
  • nucleic acid according to the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence (s) for expression.
  • Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA.
  • the coding sequence shown herein is a DNA sequence. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as encompassing reference to the RNA equivalent, with U substituted for T.
  • Nucleic acid may be provided as part of a replicable vector, and also provided by the present invention are a vector including nucleic acid as set out above, particularly any expression vector from which the encoded polypeptide can be expressed under appropriate conditions, and a host cell containing any such vector or nucleic acid.
  • An expression vector in this context is a nucleic acid molecule including nucleic acid encoding a polypeptide of interest and appropriate regulatory sequences for expression of the polypeptide, in an in vi tro expression system, e.g. reticulocyte lysate, or in vivo, e.g. in eukaryotic cells such as COS or CHO cells or in prokaryotic cells such as E. coli . This is discussed further below.
  • the nucleic acid sequence provided in accordance with the present invention is useful for identifying nucleic acid of interest (and which may be according to the present invention) in a test sample.
  • the present invention provides a method of obtaining nucleic acid of interest, the method including hybridisation of a probe having a sequence shown herein, or a complementary sequence, to target nucleic acid. Hybridisation is generally followed by identification of successful hybridisation and isolation of nucleic acid which has hybridised to the probe, which may involve one or more steps of PCR. It will not usually be necessary to use a probe with the complete sequence shown in any of these figures. Shorter fragments, particularly fragments with a sequence encoding the conserved motifs may be used.
  • Nucleic acid according to the present invention is obtainable using one or more oligonucleotide probes or primers designed to hybridise with one or more fragments of the nucleic acid sequence shown in any of the figures, particularly fragments of relatively rare sequence, based on codon usage or statistical analysis.
  • a primer designed to hybridise with a fragment of the nucleic acid sequence shown in any of the figures may be used in conjunction with one or more oligonucleotides designed to hybridise to a sequence in a cloning vector within which target nucleic acid has been cloned, or in so-called "RACE" (rapid ampli ication of cDNA ends) in which cDNA's in a library are ligated to an oligonucleotide linker and PCR is performed using a primer which hybridises with a sequence shown and a primer which hybridises to the oligonucleotide linker.
  • RACE rapid ampli ication of cDNA ends
  • oligonucleotide probes or primers as well as the full- length sequence (and mutants, alleles, variants and derivatives) are also useful in screening a test sample containing nucleic acid for the presence of alleles, mutants and variants, with diagnostic and/or prognostic implications as discussed in more detail below.
  • Nucleic acid isolated and/or purified from one or more cells may be probed under conditions for selective hybridisation and/or subjected to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR) (reviewed for instance in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant.
  • PCR polymerase chain reaction
  • PCR comprises steps of denaturation of template nucleic acid (if double-stranded) , annealing of primer to target, and polymerisation.
  • the nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA.
  • Other specific nucleic acid amplification techniques include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction and ligation activated transcription.
  • PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.
  • cloning it may be necessary for one or more gene fragments to be ligated to generate a full-length coding sequence. Also, where a full-length encoding nucleic acid molecule has not been obtained, a smaller molecule representing part of the full molecule, may be used to obtain full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries. The full-length clones isolated may be subcloned into expression vectors and activity assayed by transfection into suitable host cells, e.g. with a reporter plasmid.
  • promoter is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3' direction on the sense strand of double-stranded DNA) .
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • the present invention extends to a promoter which has a nucleotide sequence which is allele, mutant, variant or derivative, by way of nucleotide addition, insertion, substitution or deletion of a promoter sequence as provided herein.
  • Preferred levels of sequence homology with a provided sequence may be analogous to those set out above for encoding nucleic acid and polypeptides according to the present invention.
  • Systematic or random mutagenesis of nucleic acid to make an alteration to the nucleotide sequence may be performed using any technique known to those skilled in the art.
  • One or more alterations to a promoter sequence according to the present invention may increase or decrease promoter activity, or increase or decrease the magnitude of the effect of a substance able to modulate the promoter activity.
  • Promoter activity is used to refer to ability to initiate transcription.
  • the level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter.
  • the amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction.
  • Use of a reporter gene facilitates determination of promoter activity by reference to protein production .
  • nucleic acid construct comprising a SAPL promoter region or a fragment, mutant, allele, derivative or variant thereof able to promoter transcription, operably linked to a heterologous gene, e.g. a coding sequence.
  • a "heterologous” or “exogenous” gene is generally not a modified form of SAPL .
  • the gene may be transcribed into mRNA which may be translated into a peptide or polypeptide product which may be detected and preferably quantitated following expression.
  • a gene whose encoded product may be assayed following expression is termed a "reporter gene", i.e. a gene which "reports" on promoter activity.
  • the reporter gene preferably encodes an enzyme which catalyses a reaction which produces a detectable signal, preferably a visually detectable signal, such as a coloured product.
  • a detectable signal preferably a visually detectable signal, such as a coloured product.
  • Many examples are known, including ⁇ -galactosidase and luciferase.
  • ⁇ -galactosidase activity may be assayed by production of blue colour on substrate, the assay being by eye or by use of a spectro-photometer to measure absorbance. Fluorescence, for example that produced as a result of luciferase activity, may be quantitated using a spectrophotometer .
  • Radioactive assays may be used, for instance using chloramphenicol acetyltransferase, which may also be used in non-radioactive assays.
  • the presence and/or amount of gene product resulting from expression from the reporter gene may be determined using a molecule able to bind the product, such as an antibody or fragment thereof.
  • the binding molecule may be labelled directly or indirectly using any standard technique.
  • Nucleic acid constructs comprising a promoter (as disclosed herein) and a heterologous gene (reporter) may be employed in screening for a substance able to modulate activity of the promoter.
  • a substance able to up-regulate expression of the promoter may be sought.
  • a method of screening for ability of a substance to modulate activity of a promoter may comprise contacting an expression system, such as a host cell, containing a nucleic acid construct as herein disclosed with a test or candidate substance and determining expression of the heterologous gene.
  • the level of expression in the presence of the test substance may be compared with the level of expression in the absence of the test substance.
  • a difference in expression in the presence of the test substance indicates ability of the substance to modulate gene expression.
  • An increase in expression of the heterologous gene compared with expression of another gene not linked to a promoter as disclosed herein indicates specificity of the substance for modulation of the promoter.
  • a promoter construct may be introduced into a cell line using any technique previously described to produce a stable cell line containing the reporter construct integrated into the genome.
  • the cells may be grown and incubated with test compounds for varying times.
  • the cells may be grown in 96 well plates to facilitate the analysis of large numbers of compounds.
  • the cells may then be washed and the reporter gene expression analysed. For some reporters, such as luciferase the cells will be lysed then analysed.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a substance identified using a nucleic acid molecule as a modulator of promoter activity, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for increasing SAPL expression for instance in treatment (which may include preventative treatment) of IDDM or other disease, use of such a substance in manufacture of a composition for administration, e.g. for increasing SAPL expression for instance in treatment of IDDM or other disease, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a further aspect of the present invention provides a polypeptide which has the amino acid sequence shown in Figure 1(c), Figure 1(d) or Figure 2(c), or includes the first 776 amino acids of Figure 1(c) and Figure 2(c), which are identical between SAPLa and SAPLb, which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated, such as other polypeptides or such as human polypeptides other than that for which the amino acid sequence is shown in a said figure, or (for example if produced by expression in a prokaryotic cell) lacking in native glycosylation, e.g. unglycosylated.
  • Preferred such polypeptides have SAPL function, that is to say have one or more of the following properties: immunological cross-reactivity with an antibody reactive the polypeptide for which the sequence is given in a figure herein; sharing an epitope with the polypeptide for which the amino acid sequence is shown in a figure herein (as determined for example by immunological cross-reactivity between the two polypeptides) ; a biological activity which is inhibited by an antibody raised against the polypeptide whose sequence is shown in a figure herein; ability to complement the yeast mutation; containing one or more of the conserved sequences identified in Figure 3; containing the STDSEE repeat. Alteration of sequence may change the nature and/or level of activity and/or stability of the SAPL protein.
  • Preferred fragments of SAPL include those with any of the following amino acid sequences: HPSQEEDRHSNASQ
  • PSSSPEQRTGQPSAPGDTS which may be used for instance in raising or isolating antibodies .
  • Variant and derivative peptides, peptides which have an amino acid sequence which differs from one of these sequences by way of addition, insertion, deletion or substitution of one or more amino acids are also provided by the present invention, generally with the proviso that the variant or derivative peptide is bound by an antibody or other specific binding member which binds one of the peptides whose sequence is shown.
  • a peptide which is a variant or derivative of one of the shown peptides may compete with the shown peptide for binding to a specific binding member, such as an antibody or antigen-binding fragment thereof.
  • a "derivative" of a polypeptide or a fragment thereof may include a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve one or more of insertion, addition, deletion or substitution of one or more amino acids, which may be without fundamentally altering the qualitative nature of biological activity of the wild type polypeptide. Also encompassed within the scope of the present invention are functional mimetics of active fragments of the SAPL polypeptides provided (including alleles, mutants, derivatives and variants) .
  • the term "functional mimetic” means a substance which may not contain an active portion of the relevant amino acid sequence, and probably is not a peptide at all, but which retains in qualitative terms biological activity of natural SAPL polypeptide. The design and screening of candidate mimetics is described in detail below.
  • a polypeptide according to the present invention may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below) .
  • a polypeptide may be provided free or substantially free from contaminants with which it is naturally associated (if it is a naturally-occurring polypeptide) .
  • a polypeptide may be provided free or substantially free of other polypeptides.
  • Polypeptides according to the present invention may be generated wholly or partly by chemical synthesis.
  • the isolated and/or purified polypeptide may be used in formulation of a composition, which may include at least one additional component, for example a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier.
  • a composition including a polypeptide according to the invention may be used in prophylactic and/or therapeutic treatment as discussed below.
  • a method according to one aspect of the invention includes providing a polypeptide or peptide of the invention and bringing it into contact with a substance, which contact may result in binding between the polypeptide or peptide and the substance. Binding may be determined by any of a number of techniques available in the art, both qualitative and quantitative.
  • One aspect of the present invention provides an assay which includes :
  • Fragments may be generated and used in any suitable way known to those of skill in the art. Suitable ways of generating fragments include, but are not limited to, recombinant expression of a fragment from encoding DNA. Such fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Small fragments (e.g. up to about 20 or 30 amino acids) may also be generated using peptide synthesis methods which are well known in the art .
  • the interaction between the polypeptides may be studied in vi tro by labelling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support.
  • Suitable detectable labels include 35 S-methionine which may be incorporated into recombinantly produced peptides and polypeptides.
  • Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.
  • the protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se .
  • a preferred in vi tro interaction may utilise a fusion protein including glutathione-S-transferase (GST) . This may be immobilized on glutathione agarose beads.
  • GST glutathione-S-transferase
  • a test compound can be assayed by determining its ability to diminish the amount of labelled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS- polyacrylamide gel electrophoresis .
  • the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.
  • a method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium, testing the activity of the treated polypeptide and comparing that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances.
  • an assay method which includes:
  • the kinase may be, for example, cAMP dependent protein kinase, protein kinase C, or casein kinase 2
  • Phosphorylation may be determined for example by immobilising a polypeptide of the invention, or fragment, mutant, variant or derivative thereof, e.g. on a bead or plate, and detecting phosphorylation using an antibody or other binding molecule which binds the relevant site of phosphorylation with a different affinity when the site is phosphorylated from when the site is not phosphorylated.
  • Such antibodies may be obtained by means of any standard technique as discussed elsewhere herein, e.g. using a phosphorylated peptide (such as a fragment of a SAPL polypeptide) .
  • Binding of a binding molecule which discriminates between the phosphorylated and non-phosphorylated form of the polypeptide or relevant fragment, mutant, variant or derivative thereof may be assessed using any technique available to those skilled in the art, which may involve determination of the presence of a suitable label, such as fluorescence.
  • Phosphorylation may be determined by immobilisation of the polypeptide or a fragment, mutant, variant or derivative thereof, on a "suitable substrate such as a bead or plate, wherein the substrate is impregnated with scintillant, such as in a standard scintillation proximetry assay, with phosphorylation being determined via measurement of the incorporation of radioactive phosphate.
  • Phosphate incorporation into a polypeptide or a fragment, mutant, variant or derivative thereof may be determined by precipitation with acid, such as trichloroacetic acid, and collection of the precipitate on a suitable material such as nitrocellulose filter paper, followed by measurement of incorporation of radiolabeled phosphate. SDS-PAGE separation of substrate may be employed followed by detection of radiolabel .
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.
  • test substances Prior to or as well as being screened for modulation of activity, test substances may be screened for ability to interact with the polypeptide, e.g. in a yeast two-hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid) . This may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide.
  • test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM. Greater concentrations may be used when a peptide is the test substance.
  • Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.
  • a further class of putative inhibitor compounds can be derived from the SAPL polypeptide and/or a ligand which binds. Peptide fragments of from 5 to 40 amino acids, for example from 6 to 10 amino acids from the region of the relevant polypeptide responsible for interaction, may be tested for their ability to disrupt such interaction.
  • candidate inhibitor compounds may be based on modelling the 3-dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals .
  • a substance identified using as a modulator of polypeptide or promoter function may be peptide or non-peptide in nature.
  • Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses.
  • a mimetic or mimick of the substance may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
  • a range of sources e.g. spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modelling process.
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
  • a polypeptide, peptide or substance able to modulate activity of a polypeptide according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment. Such a kit may include instructions for use .
  • a convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it, by use of the nucleic acid in an expression system.
  • the present invention also encompasses a method of making a polypeptide (as disclosed) , the method including expression from nucleic acid encoding the polypeptide (generally nucleic acid according to the invention) .
  • This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide.
  • Polypeptides may also be expressed in in vi tro systems, such as reticulocyte lysate.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E . coli .
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).
  • Animal models may also be useful for any of the various diseases discussed elsewhere herein.
  • host cells may be used as a nucleic acid factory to replicate the nucleic acid of interest in order to generate large amounts of it. Multiple copies of nucleic acid of interest may be made within a cell when coupled to an amplifiable gene such as dihyrofolate reductase (DHFR) , as is well known.
  • DHFR dihyrofolate reductase
  • Host cells transformed with nucleic acid of interest, or which are descended from host cells into which nucleic acid was introduced may be cultured under suitable conditions, e.g. in a fermentor, taken from the culture and subjected to processing to purifiy the nucleic acid. Following purification, the nucleic acid or one or more fragments thereof may be used as desired, for instance in a diagnostic or prognostic assay as discussed elsewhere herein .
  • a further aspect of the present invention provides an antibody able to bind specifically to the polypeptide whose sequence is given in a figure herein.
  • Such an antibody may be specific in the sense of being able to distinguish between the polypeptide it is able to bind and other human polypeptides for which it has no or substantially no binding affinity (e.g. a binding affinity of about lOOOx less) .
  • Specific antibodies bind an epitope on the molecule which is either not present or is not accessible on other molecules.
  • Antibodies according to the present invention may be specific for the wild-type polypeptide.
  • Suitable peptides for use in immunising an animal and/or isolating anti-SAPL antibody include any of the following amino acid sequences: HPSQEEDRHSNASQ RIQQFDDGGSDEEDI PESQRRSSSGSTDSE PSSSPEQRTGQPSAPGDTS
  • antibodies according to the present invention include antibodies able to bind and/or which bind specifically, e.g. with an affinity of at least 10 "7 M, to one of the following peptides: HPSQEEDRHSNASQ RIQQFDDGGSDEEDI PESQRRSSSGSTDSE PSSSPEQRTGQPSAPGDTS
  • Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and/or isolating a polypeptide according to the present invention, for instance following production of the polypeptide by expression from encoding nucleic acid therefor. Antibodies may modulate the activity of the polypeptide to which they bind and so, if that polypeptide has a deleterious effect in an individual, may be useful in a therapeutic context (which may include prophylaxis) .
  • An antibody may be provided in a kit, which may include instructions for use of the antibody, e.g. in determining the presence of a particular substance in a test sample.
  • One or more other reagents may be included, such as labelling molecules, buffer solutions, elutants and so on. Reagents may be provided within containers which protect them from the external environment, such as a sealed vial.
  • a variant form of the gene may contain one or more insertions, deletions, substitutions and/or additions of one or more nucleotides compared with the wild-type sequence (such as shown in Table 2) which may or may not disrupt the gene function. Differences at the nucleic acid level are not necessarily reflected by a difference in the amino acid sequence of the encoded polypeptide. However, a mutation or other difference in a gene may result in a frame-shift or stop codon, which could seriously affect the nature of the polypeptide produced (if any) , or a point mutation or gross mutational change to the encoded polypeptide, including insertion, deletion, substitution and/or addition of one or more amino acids or regions in the polypeptide.
  • a mutation in a promoter sequence or other regulatory region may prevent or reduce expression from the gene or affect the processing or stability of the mRNA transcript.
  • a sequence alteration may affect alternative splicing of mRNA.
  • various SAPL isoforms resulting from alternative splicing are provided by the present invention.
  • Nucleic acid may be screened using a variant- or allele-specific probe.
  • a probe corresponds in sequence to a region of the SAPL gene, or its complement, containing a sequence alteration known to be associated with IDDM or other disease susceptibility.
  • specific hybridisation of such a probe to test nucleic acid is indicative of the presence of the sequence alteration in the test nucleic acid.
  • more than one probe may be used on the same test sample.
  • Allele- or variant-specific oligonucleotides may similarly be used in PCR to specifically amplify particular sequences if present in a test sample.
  • Assessment of whether a PCR band contains a gene variant may be carried out in a number of ways familiar to those skilled in the art.
  • the PCR product may for instance be treated in a way that enables one to display the polymorphism on a denaturing polyacrylamide DNA sequencing gel, with specific bands that are linked to the gene variants being selected.
  • SSCP heteroduplex analysis may be used for screening DNA fragments for sequence variants/mutations. It generally involves amplifying radiolabelled 100-300 bp fragments of the gene, diluting these products and denaturing at 95°C. The fragments are quick-cooled on ice so that the DNA remains in single stranded form. These single stranded fragments are run through acrylamide based gels. Differences in the sequence composition will cause the single stranded molecules to adopt difference conformations in this gel matrix making their mobility different from wild type fragments, thus allowing detecting of mutations in the fragments being analysed relative to a control fragment upon exposure of the gel to X- ray film. Fragments with altered mobility/conformations may be directly excised from the gel and directly sequenced for mutation .
  • Sequencing of a PCR product may involve precipitation with isopropanol, resuspension and sequencing using a TaqFS+ Dye terminator sequencing kit. Extension products may be electrophoresed on an ABI 377 DNA sequencer and data analysed using Sequence Navigator software.
  • a further possible screening approach employs a PTT assay in which fragments are amplified with primers that contain the consensus Kozak initiation sequences and a T7 RNA polymerase promoter. These extra sequences are incorporated into the 5' primer such that they are in frame with the native coding sequence of the fragment being analysed. These PCR products are introduced into a coupled transcription/translation system. This reaction allows the production of RNA from the fragment and translation of this RNA into a protein fragment. PCR products from controls make a protein product of a wild type size relative to the size of the fragment being analysed. If the PCR product analysed has a frame-shift or nonsense mutation, the assay will yield a truncated protein product relative to controls. The size of the truncated product is related to the position of the mutation, and the relative region of the gene from this patient may be sequenced to identify the truncating mutation.
  • Allele- or variant-specific oligonucleotide probes or primers according to embodiments of the present invention may be selected from those shown in Table 1 and modified versions thereof. Approaches which rely on hybridisation between a probe and test nucleic acid and subsequent detection of a mismatch may be employed. Under appropriate conditions (temperature, pH etc.), an oligonucleotide probe will hybridise with a sequence which is not entirely complementary. The degree of base- pairing between the two molecules will be sufficient for them to anneal despite a mis-match. Various approaches are well known in the art for detecting the presence of a mis-match between two annealing nucleic acid molecules.
  • RN'ase A cleaves at the site of a mis-match. Cleavage can be detected by electrophoresing test nucleic acid to which the relevant probe or probe has annealed and looking for smaller molecules (i.e. molecules with higher electrophoretic mobility) than the full length probe/test hybrid.
  • an oligonucleotide probe that has the sequence of a region of the gene including a mutation associated with IDDM or other disease susceptibility may be annealed to test nucleic acid and the presence or absence of a mis-match determined.
  • the presence of a mis- match may indicate that the nucleic acid in the test sample has the normal sequence (the absence of a mis-match indicating that the test nucleic acid has the mutation) .
  • a battery of probes to different regions of the gene may be employed.
  • the presence of differences in sequence of nucleic acid molecules may be detected by means of restriction enzyme digestion, such as in a method of DNA fingerprinting where the restriction pattern produced when one or more restriction enzymes are used to cut a sample of nucleic acid is compared with the pattern obtained when a sample containing the normal gene shown in a figure herein or a variant or allele, e.g. as containing an alteration shown in Table 2, is digested with the same enzyme or enzymes.
  • a test sample of nucleic acid may be provided for example by extracting nucleic acid from cells or biological tissues or fluids, urine, saliva, faeces, a buccal swab, biopsy or preferably blood, or for pre-natal testing from the amnion, placenta or foetus itself.
  • Screening for the presence of one or more amino acid sequence variants in a test sample has a diagnostic and/or prognostic use, for instance in determining IDDM or other disease susceptibility.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies), specific for one or more particular variants of the polypeptide shown in a figure herein.
  • a sample may be tested for the presence of a binding partner for a specific binding member such as an antibody (or mixture of antibodies) , specific for the polypeptide shown in a figure herein.
  • the sample may be tested by being contacted with a specific binding member such as an antibody under appropriate conditions for specific binding, before binding is determined, for instance using a reporter system as discussed. Where a panel of antibodies is used, different reporting labels may be employed for each antibody so that binding of each can be determined.
  • a specific binding member such as an antibody may be used to isolate and/or purify its binding partner polypeptide from a test sample, to allow for sequence and/or biochemical analysis of the polypeptide to determine whether it has the 'sequence and/or properties of the polypeptide whose sequence is disclosed herein, or if it is a mutant or variant form.
  • Amino acid sequence is routine in the art using automated sequencing machines .
  • test sample containing one or more polypeptides may be provided for example as a crude or partially purified cell or cell lysate preparation, e.g. using tissues or cells, such as from saliva, faeces, or preferably blood, or for pre-natal testing from the amnion, placenta or foetus itself.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practioners and other medical doctors .
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells. Instead of administering an agent directly, it may be be produced in target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector (see below). The vector may be targeted to the specific cells to be treated, or it may contain regulatory elements which are switched on more or less selectively by the target cells.
  • An agent may be administered in a precursor form, for conversion to an active form by an activating agent produced in, or targeted to, the cells to be treated.
  • This type of approach is sometimes known as ADEPT or VDEPT; the former involving targeting the activating agent to the cells by conjugation to a cell-specific antibody, while the latter involves producing the activating agent, e.g. an enzyme, in .a vector by expression from encoding DNA in a viral vector (see for example, EP-A-415731 and WO 90/07936) .
  • Nucleic acid according to the present invention may be used in a method of gene therapy, to treat a patient who is unable to synthesize the active polypeptide or unable to synthesize it at the normal level, thereby providing the effect provided by the wild-type with the aim of treating and/or preventing one or more symptoms of IDDM and/or one or more other diseases.
  • Vectors such as viral vectors have been used to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
  • the transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
  • viruses have been used as gene transfer vectors, including adenovirus, papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses, including gibbon ape leukaemia virus, Rous Sarcoma Virus, Mandarin equine enchephalitis virus, Moloney murine leukaemia virus and murine mammary tumourvirus.
  • adenovirus papovaviruses
  • vaccinia virus such as SV40
  • herpesviruses including HSV and EBV
  • retroviruses including gibbon ape leukaemia virus, Rous Sarcoma Virus, Kunststoffualian equine enchephalitis virus, Moloney murine leukaemia virus and murine mammary tumourvirus.
  • retroviruses including gibbon ape leukaemia virus, Rous Sarcoma Virus, Mandarin equine enchephalitis virus, Moloney murine leukaemia virus
  • Disabled virus vectors are produced in helper cell lines in which genes required for production of infectious viral particles are expressed.
  • Helper cell lines are generally missing a sequence which is recognised by the mechanism which packages the viral genome and produce virions which contain no nucleic acid.
  • a viral vector which contains an intact packaging signal along with the gene or other sequence to be delivered e.g. encoding the SAPL polypeptide or a fragment thereof is packaged in the helper cells into infectious virion particles, which may then be used for the gene delivery.
  • the DNA fragments containing BstX I adapters were ligated into the BstX I sites of pSHOT II which was constructed by subcloning the BstX I sites from pcDNA II (Invitrogen, Carlsbad, CA) into the BssH II sites of pBlueScript (Stratagene, La Jolla, CA) .
  • pSHOT II was prepared by digestion with BstX I restriction endonuclease and purified by agarose gel electrophoresis. The gel purified vector DNA was extracted from the agarose by following the Prep-A-Gene (BioRad, Richmond, CA) protocol.
  • the digested vector was treated with calf intestinal phosphatase (GIBCO/BRL, Gaithersburg, MD.
  • Ligation reactions of the DNA fragments with the cloning vector were transformed into ultra-competent XL-2 Blue cells (Stratagene, La Jolla, CA) , and plated on LB agar plates supplemented with 100 ug/ml ampicillin. Individual colonies were picked into a 96 well plate containing 100 ul/well of LB broth supplemented with ampicillin and grown overnight at 37 °C. Approximately 25 ul of 80% sterile glycerol was added to each well and the cultures stored at -80 °C.
  • Phred/Phrap was used for DNA sequences assembly. This program was developed by Dr. Phil Green and licensed from the University of Washington (Seattle, WA) . Phred/Phrap consists of the following programs: Phred for base-calling, Phrap for sequence assembly, Crossmatch for sequence comparisons, Consed and Phrapview for visualization of data, Repeatmasker for screening repetitive sequences. Vector and E . col i. DNA sequences were identified by Crossmatch and removed from the DNA sequence assembly process. DNA sequence assembly was on a SUN Enterprise 4000 server running Solaris 2.51 operating system (Sun Microsystems Inc., Mountain View, CA) . The sequence assemblies were further analyzed using Consed and Phrapview.
  • the parser provided the following information from the database for each DNA sequence having a similarity with a P value greater than 10 "6 ; the annotated name of the sequence, the database from which it was derived, the length and percent identity of the region of similarity, and the location of the similarity in both the query and the subject.
  • the full length cDNA of one aspect of the present invention was generated by a method of cDNA screening called Reduced Complexity cDNA Analysis (RCCA) .
  • RCCA Reduced Complexity cDNA Analysis
  • the first method is effective but laborious and slow while the latter method is fast but limited in efficiency.
  • This RACE protocol is hindered by limited length of extension due to the use of the entire cellular mRNA population in a single reaction. Since smaller fragments are amplified much more efficiently than larger fragments by PCR in the same reaction, PCR products obtained using the second method are often quite small.
  • the RCCA method improves upon known methods of cDNA library screening by initially constructing and subdividing cDNA libraries followed by isolating 5'- and 3'- flanking fragments by PCR. Since each pool is unlikely to contain more than one clone for a given gene which is low to moderately expressed, competition between large and small PCR products in one pool does not exist, making it possible to isolate fragments of various sizes.
  • One definite advantage of the method as described herein is the efficiency, throughput, and its potential to isolate alternatively spliced cDNA forms.
  • the RCCA process provides for rapid extension of a partial cDNA sequence based on subdividing a primary cDNA library and DNA amplification by polymerase chain reaction (PCR) .
  • a cDNA library is constructed with cDNA primed by random, oligo-dT or a combination of both random and oligo-dT primers and then subdivided into pools at approximately 10,000 -20,000 clones per pool that are stored in a 96-well plate. Each pool (well) is amplified separately and therefore represents an independent portion of the cDNA molecules from the original mRNA source.
  • the fundamental principle of the RCCA process is to subdivide a complex library into superpools of about 10,000 to about 20,000 clones.
  • a library of two million primary clones, a number large enough to cover most mRNA transcripts expressed in the tissue, can be subdivided into 188 pools and stored in two 96-well plates. Since the number of transcripts for most genes is fewer than one copy per ⁇ 10,000 transcripts in total cellular mRNA, each pool is unlikely to contain more than one clone for a given cDNA sequence. Such reduced complexity makes it possible to use PCR to isolate flanking fragments of partial cDNA sequences larger than those obtained by known methods .
  • multiple primer combinations from an EST or other partial cDNA sequence, in combination with flanking vector primer oligonucleotides can be used to "walk" in both directions away from the internal, gene specific, sequence, and respective primers, such that a contig representing a full length cDNA can be constructed.
  • the 5' end of the cDNA was represented by an EST with GenBank accession number aal94169. Therefore the RCCA procedure was only employed to obtain sequences 3' of EST aal94169. This procedure relies on the ability to screen multiple pools which comprise a representative portion of the total cDNA library.
  • the primers 4dest4 3f and 4dest4 6f were used in combination with the vector primers, 543R and 873F, in a primary reaction using Taqara LA (Panvera, Madison, WI) .
  • the amplification conditions were 20 cycles of denaturation at 94 °C for 30 sec, primer annealing at 60 °C for 30 sec and extension for 4 min at 68 °C in a 10 ul reaction volume.
  • the primary reaction was diluted by adding 9 parts water and an aliquot was removed for a second PCR reaction containing primer 4dest 7f (4dest4 6f primary reactions) and 4dest4f (for 4dest4 3f primary reactions) .
  • RCCA Identifica tion of polymorphisms in SAPL
  • the process of RCCA generates clones that may differ in origin, i.e. the mRNA used to synthesize the cDNA, e.g. testis, mRNA may be derived from an individual heterozygous for the SAPL locus or may be from a pool of different individuals. Therefore polymorphisms between different RCCA clones may represent true differences, alternatively these differences may arise from PCR mistakes or from errors that are made by the DNA polymerase during the propagation of vectors containing SAPL inserts in E. coli . To discriminate against these types of errors polymorphisms were only noted when detected in more than one clone and where the sequence quality was excellent. All the candidate polymorphism's that were detected lie in the putative 3' untranslated portion of the cDNA and thus have no effect on the encoded protein.
  • the approximately 4.1 kb band may correspond to an as yet unidentified alternatively spliced form, in which case SAPLb would be a rare alternatively spliced transcript.
  • SAPLb would be a rare alternatively spliced transcript.
  • the highest level of SAPL expression is seen in skeletal muscle, placenta, heart, pancreas and testis. Detectable expression is also observed in brain, lung, liver, kidney, spleen, thymus, prostate, small intestine, colon, and leukocytes. No detectable expression is seen in ovary.
  • mouse EST AA684416 which is 93% identical to the SAPL cDNA sequence from 590 to 1080. This is likely the mouse ortholog of human SAPL . It and other mouse ESTs such as aa435418, which is 86% identical from 2888 to 3348, are used in the isolation of the mouse SAPL cDNA either by a PCR based or nucleic acid hybridization based strategy.
  • Type I diabetes is a multifactorial disorder, with the genetic component being oligo- or polygenic.
  • Two loci have been identified as conferring susceptibility to typ 1 diabetes by candidate gene approaches.
  • the main locus is encoded by the major histocompatibility complex (MHC) on chromosome 6p ( IDDM1 ) (Morton, N., et al. (1983) AM J HUM GENET 35, 201-213; Todd, J and Farrall, M. (1996) Hum Mol Genet ⁇ , 1443-1448) with the second locus, IDDM2, the insulin minisatellite or variable number of tandem repeats (VNTR) on chromosome lip (Bennett, S., et al (1995) Na ture Genet 9, 284-292).
  • MHC major histocompatibility complex
  • IDDM1 chromosome 6p
  • VNTR variable number of tandem repeats
  • H0570POLYA in 2042 families with type 1 diabetes confirmed the association observed with this marker and type 1 diabetes (2X2 test of heterogeneity for affected versus unaffected siblings, p COrrected ⁇ 4 • 8 x 10 ⁇ 5 ) (Nakagawa, Y., et al (1997) Hum . Mol . Genet . Submi t ted) .
  • SAPL gene is within 200kb of H0570POLYA, in a region showing strong association with IDDM, hence single nucleotide polymorphisms within this gene and its regulatory regions are candidates for the aetiological mutation IDDM4.

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Abstract

La présente invention concerne des acides nucléiques, des polypeptides, des sondes et amorces oligonucléotidiques, des procédés de diagnostic et de pronostic, et d'autres procédés (5) liés au et se basant sur le clonage et la caractérisation d'un gène dénommé SAPL (SIT4-(sporulation-induced transcript 4)-like) par ses inventeurs, existant sous au moins deux isoformes dénommées SAPLa et SAPLb.
PCT/GB2000/004027 1999-10-19 2000-10-19 Proteines analogues aux proteines humaines associees au sit4 (sapl), genes codant pour ces proteines, et leurs utilisations WO2001029213A1 (fr)

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CA002387041A CA2387041A1 (fr) 1999-10-19 2000-10-19 Proteines analogues aux proteines humaines associees au sit4 (sapl), genes codant pour ces proteines, et leurs utilisations
EP00969687A EP1222264A1 (fr) 1999-10-19 2000-10-19 PROTEINES ANALOGUES AUX PROTEINES HUMAINES ASSOCIEES AU SIT4 ( i SAPL /i ), GENES CODANT POUR CES PROTEINES, ET LEURS UTILISATIONS
JP2001532196A JP2003512055A (ja) 1999-10-19 2000-10-19 ヒトｓｉｔ４会合タンパク質様（ｓａｐｌ）タンパク質及びコード遺伝子；それらの使用
AU79343/00A AU7934300A (en) 1999-10-19 2000-10-19 Human sit4 associated proteins like (sapl) proteins and encoding genes; uses thereof

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EP1033401A2 (fr) * 1999-02-26 2000-09-06 Genset Marqueurs de séquence exprimées et protéines humaines codées
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WO1999035253A1 (fr) * 1998-01-07 1999-07-15 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
EP1033401A2 (fr) * 1999-02-26 2000-09-06 Genset Marqueurs de séquence exprimées et protéines humaines codées
WO2000058473A2 (fr) * 1999-03-31 2000-10-05 Curagen Corporation Acides nucleiques comprenant des phases de lecture ouverte codant des polypeptides; «orfx»

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DATABASE EMBL 1 November 1996 (1996-11-01), TURNER B.S., BHASKAR K.R., HADZOPOULOU-CLADARAS M., SPECIAN R.D., LAMONT J.T.: "Gastric mucin (fragment)", XP002160778 *
DATABASE EMBL 29 January 1999 (1999-01-29), NCI-CGAP: "National Cancer Institute, Cancer Genome Anatomy Project (CGAP), Tumor Gene Index", XP002160777 *
LUKE MAY M ET AL: "The SAPs, a new family of proteins, associate and function positively with the SIT4 phosphatase.", MOLECULAR AND CELLULAR BIOLOGY, vol. 16, no. 6, 1996, pages 2744 - 2755, XP000982253, ISSN: 0270-7306 *

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JP2003512055A (ja) 2003-04-02

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