h-TRAAK polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing h-TRAAK polypeptides and polynucleotides in diagnostic assays.

This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in diagnosis and in identifying compounds that may be agonists, antagonists that are potentially useful in therapy, and to production of such polypeptides and polynucleotides.
The drug discovery process is currently undergoing a fundamental revolution as it embraces xe2x80x9cfunctional genomicsxe2x80x9d, that is, high throughput genome- or gene-based biology. This approach as a means to identify genes and gene products as therapeutic targets is rapidly superseding earlier approaches based on xe2x80x9cpositional cloningxe2x80x9d. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on high-throughput DNA sequencing technologies and the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery.
The present invention relates to h-TRAAK, in particular h-TRAAK polypeptides and h-TRAAK polynucleotides, recombinant materials and methods for their production. Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to, pain, psychiatric disorders including depression and schizophrenia, neurodegenerative disease including Alzheimer""s, stroke and head trauma and neurological disorders including migraine, epilepsy, hereinafter referred to as xe2x80x9cdiseases of the inventionxe2x80x9d. In a further aspect, the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with h-TRAAK imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate h-TRAAK activity or levels.
In a first aspect, the present invention relates to h-TRAAK polypeptides. Such polypeptides include:
(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO:1;
(b) an isolated polypeptide comprising a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO:2;
(d) an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(e) the polypeptide sequence of SEQ ID NO:2; and
(f) an isolated polypeptide having or comprising a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2;
(g) fragments and variants of such polypeptides in (a) to (f).
Polypeptides of the present invention are believed to be members of the potassium channel family of polypeptides. They are therefore of interest because potassium channels are a ubiquitous group of ion channels that are important in controlling excitability and modulating secretory processes. They have a number of roles including neuronal integration, volume regulation, maintenance of the resting membrane potential and an important role in determining the frequency and duration of action potentials. The changes in cell excitability that follow modulation of potassium channels give rise to a broad number of potential therapeutic uses of such modulators. One example is the potassium channel blocker, dofetilide, which is an effective antiarrhythmic. A new structural family of potassium channels has recently been identified, the 2P domain potassium channels, which includes the mouse TRAAK (M. Fink et al. EMBO J. 17: 3297-3308, 1998, and references therein).
The biological properties of the h-TRAAK are hereinafter referred to as xe2x80x9cbiological activity of h-TRAAKxe2x80x9d or xe2x80x9ch-TRAAK activityxe2x80x9d. Preferably, a polypeptide of the present invention exhibits at least one biological activity of h-TRAAK.
Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allelic forms and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3 to 2, from 2 to 1 or 1 amino acids are inserted, substituted, or deleted, in any combination.
Preferred fragments of polypeptides of the present invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, or an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO: 2. Preferred fragments are biologically active fragments that mediate the biological activity of h-TRAAK, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or immunogenic in an animal, especially in a human.
Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention. The polypeptides of the present invention may be in the form of the xe2x80x9cmaturexe2x80x9d protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.
Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods. Means for preparing such polypeptides are well understood in the art.
In a further aspect, the present invention relates to h-TRAAK polynucleotides. Such polynucleotides include:
(a) an isolated polynucleotide comprising a polynucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide sequence of SEQ ID NO:1;
(b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO:1;
(c) an isolated polynucleotide having at least 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide of SEQ ID NO:1;
(d) the isolated polynucleotide of SEQ ID NO:1;
(e) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(f) an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2;
(g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;
(h) an isolated polynucleotide encoding the polypeptide of SEQ ID NO:2;
(i) an isolated polynucleotide having or comprising a polynucleotide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polynucleotide sequence of SEQ ID NO:1;
(j) an isolated polynucleotide having or comprising a polynucleotide sequence encoding a polypeptide sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO:2; and
polynucleotides that are fragments and variants of the above mentioned polynucleotides or that are complementary to above mentioned polynucleotides, over the entire length thereof.
Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
Preferred variants of polynucleotides of the present invention include splice variants, allelic variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
Polynucleotides of the present invention also include polynucleotides encoding polypeptide variants that comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.
In a further aspect, the present invention provides polynucleotides that are RNA transcripts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that:
(a) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
(b) is the RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
(c) comprises an RNA transcript of the DNA sequence of SEQ ID NO:1; or
(d) is the RNA transcript of the DNA sequence of SEQ ID NO:1; and RNA polynucleotides that are complementary thereto.
The polynucleotide sequence of SEQ ID NO:1 shows homology with mouse TRAAK (M. Fink et al., EMBO J. 17: 3297-3308. 1998). The polynucleotide sequence of SEQ ID NO:1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2. The polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO:1 or it may be a sequence other than SEQ ID NO:1, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2. The polypeptide of the SEQ ID NO:2 is related to other proteins of the potassium channel family, having homology and/or structural similarity with mouse TRAAK (M. Fink et al., EMBO J. 17: 3297-3308. 1998).
Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one h-TRAAK activity.
The present invention also relates to partial or other polynucleotide and polypeptide sequences which were first identified prior to the determination of the corresponding full length sequences of SEQ ID NO:1 and SEQ ID NO:2.
Accordingly, in a further aspect, the present invention provides for an isolated polynucleotide which:
(a) comprises a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3;
(b) has a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity, to SEQ ID NO:3 over the entire length of SEQ ID NO:3;
(c) comprises the polynucleotide of SEQ ID NO:3; or
(d) has a nucleotide sequence encoding a polypeptide which has at least 95% identity, even more preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4;
as well as the polynucleotide of SEQ ID NO:3.
The present invention further provides for a polypeptide which:
(a) comprises an amino acid sequence which has at least 95% identity, preferably at least 97-99% identity, to that of SEQ ID NO:4 over the entire length of SEQ ID NO:4;
(b) has an amino acid sequence which is at least 95% identity, preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:4 over the entire length of SEQ ID NO:4;
(c) comprises the amino acid of SEQ ID NO:4; and
(d) is the polypeptide of SEQ ID NO:4;
as well as polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO:3.
The nucleotide sequence of SEQ ID NO:3 and the peptide sequence encoded thereby are derived from EST (Expressed Sequence Tag) sequences. It is recognised by those skilled in the art that there will inevitably be some nucleotide sequence reading errors in EST sequences (see Adams, M. D. et al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotide sequence of SEQ ID NO:3 and the peptide sequence encoded therefrom are therefore subject to the same inherent limitations in sequence accuracy. Furthermore, the peptide sequence encoded by SEQ ID NO:3 comprises a region of identity or close homology and/or close structural similarity (for example a conservative amino acid difference) with the closest homologous or structurally similar protein.
Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA in cells of human brain, (see for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques.
When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence, or other fusion peptide portions. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5xe2x80x2 and 3xe2x80x2 sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO:1, may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than human) that have a high sequence similarity to SEQ ID NO:1, typically at least 95% identity. Preferred probes and primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.
A polynucleotide encoding a polypeptide of the present invention, including homologs from species other than human, may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42xc2x0 C. in a solution comprising: 50% formamide, 5xc3x97SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5xc3x97Denhardt""s solution, 10% dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1xc3x97SSC at about 65xc2x0 C. Thus the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO:1 or a fragment thereof, preferably of at least 15 nucleotides.
The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5xe2x80x2 terminus. This is a consequence of reverse transcriptase, an enzyme with inherently low xe2x80x9cprocessivityxe2x80x9d (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template during first strand cDNA synthesis.
There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon (trade mark) technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon (trade mark) technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an xe2x80x98adaptorxe2x80x99 sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the xe2x80x9cmissingxe2x80x9d 5xe2x80x2 end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using xe2x80x98nestedxe2x80x99 primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3xe2x80x2 in the adaptor sequence and a gene specific primer that anneals further 5xe2x80x2 in the known gene sequence). The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5xe2x80x2 primer.
Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Polynucleotides may be introduced into host cells by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ibid). Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
Representative examples of appropriate hosts include bacterial cells, such as Streptococci, Staphylococci, E. coli, Streptonyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., (ibid). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and/or purification.
Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO:1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques well known in the art.
Nucleic acids for diagnosis may be obtained from a subject""s cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled h-TRAAK nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401).
An array of oligonucleotides probes comprising h-TRAAK polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Such arrays are preferably high density arrays or grids. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M. Chee et al., Science, 274, 610-613 (1996) and other references cited therein.
Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
Thus in another aspect, the present invention relates to a diagnostic kit comprising:
(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA transcript thereof;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO:2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
The polynucleotide sequences of the present invention are valuable for chromosome localisation studies. The sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co-inheritance of physically adjacent genes). Precise human chromosomal localisations for a genomic sequence (gene fragment etc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M. Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) A method for constructing radiation hybrid maps of whole genomes, Nature Genetics 7, 22-28). A number of RH panels are available from Research Genetics (Huntsville, Ala., USA) e.g. the GeneBridge4 RH panel (Hum Mole Genet 1996 Mar;5(3):339-46 A radiation hybrid map of the human genome. Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D, Muselet D, Prud""Homme J F, Dib C, Auffray C, Morissette J, Weissenbach J, Goodfellow P N). To determine the chromosomal location of a gene using this panel, 93 PCRs are performed using primers designed from the gene of interest on RH DNAs. Each of these DNAs contains random human genomic fragments maintained in a hamster background (human/hamster hybrid cell lines). These PCRs result in 93 scores indicating the presence or absence of the PCR product of the gene of interest. These scores are compared with scores created using PCR products from genomic sequences of known location. This comparison is conducted at http://www.genome.wi.mit.edu/. The gene of the present invention maps to human chromosome 11 between the markers D11 S1357 and D 11S913.
The polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them. The techniques used are well known in the art and include in situ hydridisation techniques to clones arrayed on a grid, such as cDNA microarray hybridisation (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR. A preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropriate expression thereof in disease. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.
The polypeptides of the present invention are expressed in brain.
A further aspect of the present invention relates to antibodies. The polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention. The term xe2x80x9cimmunospecificxe2x80x9d means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et aL, Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).
Techniques for the production of single chain antibodies, such as those described in U.S. Pat. No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography. Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.
Polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not. An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention. One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid. For use a vaccine, a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition). The formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide. Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, collections of chemical compounds, and natural product mixtures. Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)) or a small molecule. Such small molecules preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g. agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a h-TRAAK activity in the mixture, and comparing the h-TRAAK activity of the mixture to a control mixture which contains no candidate compound.
Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening (HTS) formats. Such HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997).
Fusion proteins, such as those made from Fc portion and h-TRAAK polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al., J Mole Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).
The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
A polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 125I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
Examples of antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
Screening methods may also involve the use of transgenic technology and h-TRAAK gene. The art of constructing transgenic animals is well established. For example, the h-TRAAK gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts. Particularly useful transgenic animals are so-called xe2x80x9cknock-inxe2x80x9d animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-in transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target. Other useful transgenic animals are so-called xe2x80x9cknock-outxe2x80x9d animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled. The gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal. Transgenic animal technology also offers a whole animal expression-cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
Screening kits for use in the above described methods form a further aspect of the present invention. Such screening kits comprise:
(a) a polypeptide of the present invention;
(b) a recombinant cell expressing a polypeptide of the present invention;
(c) a cell membrane expressing a polypeptide of the present invention; or
(d) an antibody to a polypeptide of the present invention;
which polypeptide is preferably that of SEQ ID NO:2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
Glossary
The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.
xe2x80x9cAntibodiesxe2x80x9d as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
xe2x80x9cIsolatedxe2x80x9d means altered xe2x80x9cby the hand of manxe2x80x9d from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not xe2x80x9cisolated,xe2x80x9d but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is xe2x80x9cisolatedxe2x80x9d, as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is xe2x80x9cisolatedxe2x80x9d even if it is still present in said organism, which organism may be living or non-living.
xe2x80x9cPolynucleotidexe2x80x9d generally refers to any polyribonucleotide (RNA) or polydeoxribonucleotide (DNA), which may be unmodified or modified RNA or DNA. xe2x80x9cPolynucleotidesxe2x80x9d include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, xe2x80x9cpolynucleotidexe2x80x9d refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term xe2x80x9cpolynucleotidexe2x80x9d also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. xe2x80x9cModifiedxe2x80x9d bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, xe2x80x9cpolynucleotidexe2x80x9d embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. xe2x80x9cPolynucleotidexe2x80x9d also embraces relatively short polynucleotides, often referred to as oligonucleotides.
xe2x80x9cPolypeptidexe2x80x9d refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. xe2x80x9cPolypeptidexe2x80x9d refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. xe2x80x9cPolypeptidesxe2x80x9d include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteinsxe2x80x94Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, 1-12, in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., xe2x80x9cAnalysis for protein modifications and nonprotein cofactorsxe2x80x9d, Meth Enzymol, 182, 626-646, 1990, and Rattan et al., xe2x80x9cProtein Synthesis: Post-translational Modifications and Agingxe2x80x9d, Ann N.Y. Acad Sci, 663, 48-62, 1992).
xe2x80x9cFragmentxe2x80x9d of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide. xe2x80x9cFragmentxe2x80x9d of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO:1.
xe2x80x9cVariantxe2x80x9d refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof. A typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide. Generally, alterations are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe and Tyr. A variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C-terminal glycines.
xe2x80x9cAllelexe2x80x9d refers to one of two or more alternative forms of a gene occurring at a given locus in the genome.
xe2x80x9cPolymorphismxe2x80x9d refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population.
xe2x80x9cSingle Nucleotide Polymorphismxe2x80x9d (SNP) refers to the occurrence of nucleotide variability at a single nucleotide position in the genome, within a population. An SNP may occur within a gene or within intergenic regions of the genome. SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 primers are required. A common primer is used in reverse complement to the polymorphism being assayed. This common primer can be between 50 and 1500 bps from the polymorphic base. The other two (or more) primers are identical to each other except that the final 3xe2x80x2 base wobbles to match one of the two (or more) alleles that make up the polymorphism. Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.
xe2x80x9cSplice Variantxe2x80x9d as used herein refers to cDNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of that may encode different amino acid sequences. The term splice variant also refers to the proteins encoded by the above cDNA molecules.
xe2x80x9cIdentityxe2x80x9d reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
xe2x80x9c% Identityxe2x80x9dxe2x80x94For sequences where there is not an exact correspondence, a xe2x80x9c% identityxe2x80x9d may be determined. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting xe2x80x9cgapsxe2x80x9d in either one or both sequences, to enhance the degree of alignment. A % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defmed lengths (so-called local alignment), that is more suitable for sequences of unequal length.
xe2x80x9cSimilarityxe2x80x9d is a further, more sophisticated measure of the relationship between two polypeptide sequences. In general, xe2x80x9csimilarityxe2x80x9d means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated xe2x80x9cscorexe2x80x9d from which the xe2x80x9c% similarityxe2x80x9d of the two sequences can then be determined.
Methods for comparing the identity and similarity of two or more sequences are well known in the art. Thus for instance, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al, Nucleic Acids Res, 12, 387-395, 1984, available from Genetics Computer Group, Madison, Wis. USA), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polynucleotides and the % identity and the % similarity between two polypeptide sequences. BESTFIT uses the xe2x80x9clocal homologyxe2x80x9d algorithm of Smith and Waterman (J Mole Biol, 147,195-197, 1981, Advances in Applied Mathematics, 2, 482-489, 1981) and finds the best single region of similarity between two sequences. BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer. In comparison, GAP aligns two sequences, finding a xe2x80x9cmaximum similarityxe2x80x9d, according to the algorithm of Neddleman and Wunsch (J Mole Biol, 48, 443-453, 1970). GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length. Preferably, the parameters xe2x80x9cGap Weightxe2x80x9d and xe2x80x9cLength Weightxe2x80x9d used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Preferably, % identities and similarities are determined when the two sequences being compared are optimally aligned.
Other programs for determining identity and/or similarity between sequences are also known in the art, for instance the BLAST family of programs (Altschul S F et al, J Mole Biol, 215, 403-410, 1990, Altschul S F et al, Nucleic Acids Res., 25:389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Md., USA and accessible through the home page of the NCBI at www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, Methods in Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448,1988, available as part of the Wisconsin Sequence Analysis Package).
Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison.
Preferably, the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore described.
xe2x80x9cIdentity Indexxe2x80x9d is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence. Thus, for instance, a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion. These differences may occur at the 5xe2x80x2 or 3xe2x80x2 terminal positions of the reference polynucleotide sequence or anywhere between these terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polynucleotide sequence having an Identity Index of 0.95 compared to a reference polynucleotide sequence, an average of up to 5 in every 100 of the nucleotides of the in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.
Similarly, for a polypeptide, a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These differences may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. In other words, to obtain a polypeptide sequence having an Identity Index of 0.95 compared to a reference polypeptide sequence, an average of up to 5 in every 100 of the amino acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described. The same applies mutatis mutandis for other values of the Identity Index, for instance 0.96, 0.97, 0.98 and 0.99.
The relationship between the number of nucleotide or amino acid differences and the Identity Index may be expressed in the following equation:
naxe2x89xa6xaxe2x88x92(xaxc2x7I),
in which:
na is the number of nucleotide or amino acid differences,
xa is the total number of nucleotides or amino acids in SEQ ID NO:1 or SEQ ID NO:2, respectively,
I is the Identity Index,
xc2x7is the symbol for the multiplication operator, and in which any non-integer product of xa and I is rounded down to the nearest integer prior to subtracting it from xa.
xe2x80x9cHomologxe2x80x9d is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling within this generic term are the terms xe2x80x9corthologxe2x80x9d, and xe2x80x9cparalogxe2x80x9d. xe2x80x9cOrthologxe2x80x9d refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. xe2x80x9cParalogxe2x80x9d refers to a polynucleotide or polypeptide that within the same species which is functionally similar.
xe2x80x9cFusion proteinxe2x80x9d refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 533-A discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.
All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

Polynucleotides encoding a human potassium channel


A simple, low cost energy absorbing shear strip bender that provides constant load, tailored load, or adjustable load/displacement profiles for use in any application where a single use energy absorber is required. Examples include, but are not limited to: a spinal load attenuator for use in crash-resistant aircraft seats; crash-resistant aircraft landing gear; aircraft or automotive restraint harness attachments; cargo or high-mass item tie-downs; automotive bumper attachments; and collapsible steering columns. The present invention comprises a shear plate or other base material, a shear strip integral to the shear plate, and a shear strip tab, all of which are formed from a single sheet of ductile material. The energy absorber attaches to two objects and begins to displace when opposing forces applied to the shear plate and shear strip tab reach the design limit value such that shear strip shears and plastically deforms, i.e., bends. The load that must be applied to stroke the energy absorber is the sum of the shearing force and the bending force. The direction of motion is generally parallel to the shearing plate, but may deviate from parallel by as much as 90xc2x0.

1. Field of the Invention
The present invention relates to energy absorbing devices. More specifically, the present invention relates to an energy absorbing and load limiting device that can be used in a wide variety of applications, such as helicopter, aircraft, and space vehicle seating mechanisms, automotive or aircraft restraint harness attachments, cargo or high-mass item tie-downs, automotive bumper attachments, and collapsible steering columns.
2. Background of the Invention
Most prior art energy absorbing devices use either bending or shearing to absorb energy by converting kinetic energy into work. Also, most prior art energy absorbers use multiple components, which increases the cost and complexity of these devices.
U.S. Pat. No. 4,358,136 discloses an energy absorbing device for use with a vehicular seat belt. This energy absorber requires multiple parts, including a housing and a metal strip. The energy absorption of this device is a function of friction and plastic deformation of the metal strip, but not shearing. Friction loads will vary, as a function of the speed of operation, and may be altered by contaminants such as oil or dirt.
U.S. Pat. No. 5,639,144 discloses an energy absorbing child seat fastener. This device uses a combination of shear and plastic deformation of a plate to provide energy absorption. However, the weight efficiency of the device is limited because of the single shear plane and the limited amount of bending that occurs during operation. Because the shearing and plastic deformation follow a spiral path, the resultant load from the absorber cannot, in practice, be a constant value or follow a tailored profile. Additionally, the absorber only responds effectively to loads that are applied essentially perpendicular to the plate.
The present invention is a simple, low cost energy absorbing shear strip bender that can be configured to provide constant loads, tailored loads, or adjustable loads. As shown in FIGS. 1a and 1b, the energy absorber of the present invention is a single part comprising a shear plate, a shear strip tab integral to the shear plate, guide grooves in the shear plate, a shear strip integral to the shear plate, a means for attaching the shear strip tab to a first object, and a means for attaching the shear plate to a second object.
The invention may be fabricated from a single sheet or plate of ductile material, e.g., aluminum, steel, polyethylene, polypropylene, or composite materials. Two through-cuts are made on one side of the sheet to form a shear strip tab, preferably, but not necessarily, of a rectangular shape. This shear strip tab is bent about 180xc2x0, preferably to produce the smallest possible radius without fracture of the material. The remaining (un-bent) sheet material forms the shear plate.
As used herein in the specification and in the claims, the term plate means any base material from which the invention is formed. The plate could be flat, spherical, cylindrical, or any shape compatible with the shear strip process. Preferably, the shape of the plate is the shape most compatible with the mating parts within the system.
Starting from both sides of the shear strip tab, guide grooves are cut or otherwise formed on the surface of the shear plate, to establish the paths along which the shear strip will bend and tear. These grooves reduce the material thickness, making the structure weaker than the remaining portions of the shear plate. The material thus shears along the grooves longitudinally, forming an increasing tab length that plastically deforms. In addition, although grooves are described in this specification and the claims for illustration purposes, it should be understood that other forms of structural weakening, e.g., perforations, would suffice to establish the paths along which the shear strip bends and tears.
The present invention absorbs energy by shearing the shear strip tab from the shear plate, along the guide grooves and bending it in the direction of the applied force. To create this action, two opposing forces (one is in reaction to the other) must be independently applied to the shear plate and shear strip tab. Thus, the shear plate attaches to a first object (such as a seat bucket) while the shear strip tab attaches to a second object (such as a structural component of a vehicle), wherein there would be relative motion between the first and second objects (in the event of a crash). As the objects pull in different directions, the load on the energy absorber increases until it reaches a predetermined load limit whereupon the shear strip begins to shear and bend away from the shear plate. The shearing and bending of the shear strip results in a controlled load during displacement, separating the two objects. This displacement is also referred to as xe2x80x9cstroke.xe2x80x9d The direction of relative motion of the objects is generally parallel to the shearing plate but may vary up to 90xc2x0.
In addition to vehicle seats, the present invention could be used in many other applications. For instance, in an automobile bumper system, the shear plate could be attached to the vehicle structure while the shear strip tab attaches to the bumper itself In a seat belt application, the shear plate could be attached to the seat structure while the shear strip tab attaches to a seat belt fastener. As a final example, for an automobile steering column assembly, the shear plate could be attached to the vehicle structure while the shear strip tab attaches to the steering column. In essence, the present invention is suitable for any application in which the energy displacing two connected objects must be absorbed and the load controlled. As mentioned above, further examples of objects that can be coupled together by the energy absorber include aircraft or other vehicle seating mechanisms, energy absorbing landing gear, automotive or aircraft restraint harness attachments, cargo or high-mass item tie-downs, automotive bumper attachments, and collapsible steering columns.
Many other applications exist. These include, but are not limited to, emergency elevator braking mechanisms, shock snubbers, and earthquake protection attachments that absorb energy from relative motion between structures such as buildings or bridges. There are many applications where loads need to be limited to a predetermined value, where energy needs to be absorbed, or where relative motion between objects is needed without losing structure attachment. Shock load snubbing could be used inside electronic devices to limit the loads and protect sensitive electronic gear from experiencing excessive loads if dropped or impacted by another object.
The load limit of the energy absorber is the sum of the force required to shear the shear strip and the force required to bend the shear strip. The predetermined load of the energy absorber can be controlled by selecting parameters such as the material shear strength, the material tensile strength, the material thickness, the material modulus, the shear guide groove depth relative to the base material thickness, the shear guide groove placement, the distance between shear guide grooves, the direction of loading and the bend radius. Using combinations of these parameters and selectively changing the parameters as the system strokes, the present invention can be implemented as an energy absorber with a constant load/displacement profile, a tailored load/displacement profile, or an adjustable load/displacement profile, as desired. It is also possible to provide a tailored or adjustable load/displacement profile by selectively engaging multiple shear strips.
In a preferred embodiment of the present invention, a constant load energy absorber is formed using two substantially parallel shear guide grooves of uniform depth. With the forces applied parallel to the surface of the shear plate of a given thickness, the constant load limit is a function of the guide groove depth, the width of the shear strip (the distance between the guide grooves), and the structural properties of the energy absorber material.
In another preferred embodiment of the present invention, a tailored load energy absorber is formed by varying the material thickness of the shear plate and shear strip, the depth of the guide grooves, direction of the applied load, and/or the width of the shear strip. Changing these variables either increases or decreases the force required to shear or bend the shear strip, to facilitate the tailoring of the energy absorber for specific varying load patterns.
In another preferred embodiment of the present invention, the load/displacement profile of the energy absorber is adjustable, such that a user can select from a predetermined series of loads. The adjustable load energy absorber features multiple shear strip tabs, each providing a specific load limit. By attaching to one or more of the shear strip tabs, the user can adjust the load limit to suit the particular application for which the energy absorber is being used. The more shear strip tabs that are attached, the higher the load limit.
As an alternative to multiple shear strip tabs, an adjustable load energy absorber could use a dial or other mechanical device to set a cutting or scoring blade to produce a specific guide groove depth. Adjusting the blade to furnish a deeper cut would lower the load limit of the energy absorber.
Accordingly, an object of the present invention is to provide a simple, low cost, tailored load (including constant load) energy absorber that is free from the difficulties and disadvantages found in the prior art.
Another object of the present invention is to provide a simple energy absorber requiring only one primary component.
Another object of the present invention is to provide high efficiency load limiting through a combination of plastic deformation and shearing action.
Another object of the present invention is to provide an energy absorber that absorbs energy applied in any direction ranging from parallel to the shear plate to perpendicular to the shear plate.
These and other objects of the present invention are described in greater depth in the detailed description of the invention, the appended drawings, and the attached claims.


A display apparatus comprises a device enclosure supporting a front panel and a monitor housing supporting a first monitor screen. The monitor housing is pivotally engaged with the device enclosure, and enabled for pivotal movement between a closed position wherein the first monitor screen is approximately parallel to front panel, and an open position, placing the first monitor screen at an angle to the front panel. A sliding assembly supports a second monitor screen. The sliding assembly is enabled for sliding movement between a concealed position within the monitor housing and a viewing position lateral to the monitor housing. The sliding assembly is further enabled, when placed in the viewing position, for pivotal movement relative to the monitor assembly for improved viewing, jointly, of the first and the second monitor screens.

1. Field of the Invention
This invention relates generally to portable computers such as laptop models and more particularly to a portable computer with a primary monitor screen and a secondary monitor screen with side-by-side viewing and storage of one screen behind the other.
2. Description of Related Art
The following art defines the present state of this field:
Leveridge et al., U.S. D395,041 describes a computer monitor assembly design.
Ambroe, U.S. D397,998 describes a closable display device design.
Moscovitch, U.S. RE36,978 describes a display system including a base, a pair of electronic displays, and an arm assembly that supports the displays from the base in vertical or horizontal registration. In one implementation, the arm assembly is a single telescopic member that rotates relative to the base and locks in vertical and horizontal orientations, the displays rotate relative to the member between corresponding extreme angular positions in which the operative angular orientation of the displays relative to horizontal is maintained, and the length of the member is adjusted to minimize separation of the displays. In another implementation, the arm assembly has separate arms rotating about vertically spaced axes and linked to minimize the separation of the displays automatically when vertically or horizontally registered. In a simple implementation, the arm assembly is a rigid arm that releasably attaches to the base only in vertical and horizontal orientations, the displays mounted releasably to the arm in pre-defined angular orientations that preserve their operative angular orientation, and one display can be connected to the arm at spaced apart position to adjust separation of the displays.
Matheny et al., U.S. Pat. No. 4,884,068 describes a multiple display system having a single light pen made to operate with a plurality of synchronized monitors in an array. A determination of which monitor in the array the light pen is being used with, is made to allow simultaneous usage of the light pen on any of the monitors in the array. Other positioning or pointing input devices can be arranged to work with the array of monitors.
Buisson et al., U.S. Pat. No. 4,988,995 describes a display device including several flat screens. Two sides of a case each form a flat screen display face controlled by the same control system. Under normal operation, the case is held in slides and only the screen disposed on one side is visible and in the emergency mode the case is pulled out of the slides and is pivoted so as to cause the other screen disposed on the other side to appear.
Kuno et al., U.S. Pat. No. 5,467,102 describes a portable display device capable of presenting a document in an easy to read format, in which a simultaneous display of different parts of the document in reasonable sizes can be made, by a simple operation of the device. The display device comprises a document memory for storing documents to be displayed, at least two display screens for displaying the documents stored in the document memory means, and a display control unit for controlling displays of the documents on the display screens to be in a linked mode in which the displays on the display screens are linked together and in a separate mode in which the displays on the display screens are independent from each other.
Register, U.S. Pat. No. 5,590,021 describes an apparatus and method for a secondary display system for a computer. A secondary display system of the present invention includes a flat panel display configured such that the display is conveniently located both during and between use thereof. The system comprises a liquid crystal display module (LCDM), a corresponding display controller connected to the LCDM via an appropriate interface, and a mounting device connected to the LCDM for mounting and positioning the LCDM in close proximity to the monitor of a computer. In a preferred embodiment, the mounting device comprises a rectangular plate, which is horizontally disposed between a chassis and a monitor of the computer, and the positioning means comprises a single axis hinge, which is connected to a front corner of the plate such that the hinge rotates on a vertical axis. One edge of the LCDM is attached to the hinge so that the LCDM may be rotated on a vertical axis. During use, the LCDM may be rotated such that its display screen is coplanar with the display screen of the monitor. Between uses, the LCDM may be rotated such that its display screen is substantially orthogonal to the display screen of the monitor.
Ouchi et al, U.S. Pat. No. 5,796,577 describes a notebook computer comprising a keyboard positioned on the upper surface of a computer body, an operating ball, function keys and the like. A cover member fixed to the computer body by a hinge so that it opens and closes against the computer body is supported in an open position substantially perpendicular to the body. A first display device is formed on the inner surface of the cover member, and a second display device is formed on the outer surface thereof. The operator and the client facing each other with the notebook computer positioned between them could share the same screen information by watching each of the two display devices in front of them.
Chee, U.S. Pat. No. 5,694,141 describes a computer system including a pair of display devices, such as cathode ray tubes (CRT""s) or liquid crystal displays (LCD""s) for providing a visible display to a user of the computer system. The computer system includes a video display controller (VDC) providing for simultaneous display of different images on the pair of display devices. The VDC includes a display data processing circuit (DDPC) which is variably configurable to provide decoding of data words from a first bit-word format as received from a display first-in-first-out (FIFO) memory to a second bit-word format as required by a particular one of the pair of display devices. The DDPC is variably configurable to allow the pair of display devices to each receive driving signals providing the simultaneous differing images, and which driving signals originate with the bit-words allocated to each particular one of the pair of display devices. Accordingly, the DDPC simultaneously decodes bit-words from the first bit-word format to a pair of second bit-word formats, which second bit-word formats need not be the same, but can differ depending on the type of display device receiving the corresponding driving signals.
Haneda et al., U.S. Pat. No. 5,90,848 describes an information processing apparatus having a main body and a lid body configured so that the lid body can be switched between a closed state, a stacked state, a double screen state and an inverted state. Each of the main body and the lid body has a display section for displaying information and an input section composed of a transparent tablet and provided on the display section. As a sensor or the like detects the lid body in one of the four states, a control section of the information processing apparatus controls the display section so that a screen on the display section is appropriate to the detected state of the lid body. The control section also controls the lighting section for lighting the display section appropriately to the detected state of the lid body. This realizes appropriate display in accordance with usages of the information processing apparatus and offers an easy-to-operate and easy-to-use information processing apparatus. Besides, the lighting section is turned on/off according to needs, and therefore it is possible to prevent heat generation and to restrain power consumption.
Batio, U.S. Pat. No. 5,949,643 describes a retrofitting, folding, portable keyboard for a notebook computer consisting of two, pivotally-hinged halves. The two halves are hinged so that may be assume a perfectly flat, horizontal position, or, alternatively, a laterally raised and sloping configuration that provides an enhanced, ergonometric configuration to the user. Each half has its own set of keys and space bar. The keyboard of the invention has its own pointing device that takes over the pointing function of the pointing device of the dedicated keyboard provided with the notebook computer. The portable, folding keyboard of the invention also has a joystick-adapter by which a conventional joystick may be operatively coupled to the notebook computer by which games may be played. Four, bottom, pivotal feet support the keyboard on and/or above the existing keyboard provided with the notebook computer. A carrying case is provided in which the folded-up keyboard may be stored and transported. Also provided is a dual split screen, where each half of the split screen is pivotally mounted for universal rotation.
Reddy et al., U.S. Pat. No. 6,215,459 describes a video controller for controlling at least two video displays incorporating a video memory for storing first and second video frames of interleaved pixel data. A video memory controller connected to the video memory sequentially reads data for a first pixel from the first video frame and data for a second pixel from the second video frame. Each pixel data is in turn transferred to a look-up table connected to the video memory controller which converts the first and second pixel data to first and second display data. A selector coupled to the look-up table alternately routes the first display data to one video display and routes the second display data to the other video display.
Gouko, U.S. Pat. No. 6,222,507 describes a personal computer which displays a plurality of images and which includes a body of the personal computer, a primary display panel provided on a front side of the body, and a secondary display panel provided adjacently to the primary display panel, the secondary display panel is slid to be contained into the primary display panel or outer sides of the primary display panel by the use of rack and pinion mechanism. The secondary display panel is rotatable towards both upper or lower side and left or right side with respect to the primary display panel by a hinge mechanism.
Chee et al., WO 96/41328 describes a computer system including a dual-panel monochrome or color liquid crystal display (LCD). A dynamic random access memory (DRAM) of the computer including a defined virtual memory array representative of pixel locations of the dual-panel LCD. Pixel values are read from the virtual array of the DRAM and written to corresponding locations of the display by a display pipeline. The writing of pixel values to the display proceeds pixel-by-pixel across a row of pixels in a panel, and then to the next row of pixels until a panel is refreshed. The panels of the array are refreshed one at a time alternating between an upper panel of the display and a lower panel of the display. While one panel is being refreshed, the other panel is blanked. Consequently, the dual-panel display may be driven with a simplied structure of display pipeline, and with a reduced time requirement for access to the DRAM.
The prior art teaches the use of dual screen monitors, but does not teach a sliding withdrawal of a second screen into a position lateral to the first with hinged adjustment of both. The present invention fulfills these needs and provides further related advantages as described in the following summary.
The present invention teaches certain benefits in construction and use which give rise to the objectives described below.
A display apparatus comprises a device enclosure supporting a front panel and a monitor housing supporting a first monitor screen. The monitor housing is pivotally engaged with the device enclosure, and enabled for pivotal movement between a closed position wherein the first monitor screen is approximately parallel to front panel, and an open position, placing the first monitor screen at an angle to the front panel. A sliding assembly supports a second monitor screen. The sliding assembly is enabled for sliding movement between a concealed position within the monitor housing and a viewing position lateral to the monitor housing. The sliding assembly is further enabled, when placed in the viewing position, for pivotal movement relative to the monitor assembly for improved viewing, jointly, of the first and the second monitor screens.
A primary objective of the present invention is to provide an apparatus and method of use of such apparatus that provides advantages not taught by the prior art.
Another objective is to provide such an invention capable of displaying two monitor screens in side-by-side fashion for joint viewing.
A further objective is to provide such an invention capable of storing the two screens in a single integral assembly when not in use.
A still further objective is to provide such an invention capable of separate hinged adjustment of each of the two screens.
A still further objective is to provide such an invention wherein a second of the two screens is enclosed for mounting onto a first screen housing.
Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of the invention.

Dual display device with lateral withdrawal for side-by-side viewing