+

WO2003054187A1 - Gene de l'helicase - Google Patents

Gene de l'helicase Download PDF

Info

Publication number
WO2003054187A1
WO2003054187A1 PCT/GB2002/005653 GB0205653W WO03054187A1 WO 2003054187 A1 WO2003054187 A1 WO 2003054187A1 GB 0205653 W GB0205653 W GB 0205653W WO 03054187 A1 WO03054187 A1 WO 03054187A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
helicase
sequence
polynucleotide
activity
Prior art date
Application number
PCT/GB2002/005653
Other languages
English (en)
Inventor
Ian George Charles
Weiming Xu
Lizhi Liu
Original Assignee
University College London
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University College London filed Critical University College London
Priority to AU2002347395A priority Critical patent/AU2002347395A1/en
Publication of WO2003054187A1 publication Critical patent/WO2003054187A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a new gene which encodes a helicase.
  • the invention also relates to use of the helicase and modulators thereof in the treatment or diagnosis of a cancer and premature aging.
  • Unwinding double-stranded helical DNA (dsDNA) to form the single- stranded (ssDNA) intermediate is a fundamental biological process and is important in transcription, replication, recombination and repair.
  • a super-family of DNA helicases catalyses these overall reactions.
  • genomic integrity is vital to all organisms.
  • a number of human genetic disorders exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. These syndromes are all characterised by defects in DNA helicases.
  • a novel human gene pi 62 XLC helicase, which may play an important role in human genetic processes, such as gene transcription, replication, recombination and DNA repair.
  • This new gene encodes a novel helicase and was identified using a cDNA microarray technique using RNA derived from a nitric oxide-generating cell line as a probe (Xu et al., Nature Cell Biol. 2(6), 339-345). According to the invention there is thus provided a polypeptide comprising:
  • an expression vector incorporating a polynucleotide of the invention which expression vector is capable of expressing a polypeptide of the invention
  • a method for producing a polypeptide of the invention comprises maintaining a cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide;
  • a polypeptide, polynucleotide or a substance of the invention for use in a method of treatment of the human or animal body by therapy; - use of a polypeptide, polynucleotide or a substance of the invention in the manufacture of a medicament for treatment of a cancer or a premature ageing disorder;
  • a method for treating a human or animal suffering from a cancer or a premature ageing disorder comprises administering to the host a therapeutically effective amount of a polypeptide, polynucleotide or a substance of the invention
  • composition comprising a polypeptide, polynucleotide or a substance of the invention
  • a polynucleotide of the invention in the identification of a human or animal which has or is predisposed to a cancer or a premature ageing disorder
  • a method for identifying a human or animal which has or is predisposed to a cancer or a premature ageing disorder comprises typing a polynucleotide of the invention in the human or animal, thereby to determine whether the human or animal has or is susceptible to a cancer or a premature ageing disorder;
  • a method for treating a human or animal suffering from a cancer or a premature ageing disorder comprises: (a) identifying a substance that modulates helicase expression and/or activity by a method as set out above;
  • Figure 1 sets out the results of cDNA microarray array experiments. Plates in the left-hand column represent the results of probing with RNA derived from uninduced cells (non-NO expressing). Plates in the right-hand column represent the results of probing with RNA derived from induced cells (NO expressing).
  • the EST (G12021KA4T3) corresponding to the pi 62 XLC helicase gene is indicated by an arrow.
  • Figure 2 shows the results of using the gene predication program, GENSCAN, to predict the open reading frame and genomic structure of the pi 62 XLC helicase gene.
  • Figure 3 sets out the full length coding sequence of the pi 62 XLC helicase gene and the translated polypeptide sequence (SEQ ID NOS: 1 and 2).
  • Figure 4 sets out the results of database searching carried out with the p 162 XLC helicase gene sequence, which identified the pi 63 XLC helicase C-terminal domain as sharing homology with the E. coli UvrD-helicase.
  • Figure 5 sets out a prosite scan identifying multiple TPR repeats within the pi 62 XLC helicase gene sequence.
  • Figure 6 sets out a plasmid map of the pi 62 XLC helicase-ET33 construct.
  • Figure 7 sets out the results of T7-mediated in vitro translation of the p 162 XLC helicase gene using the EcoPro System (Novagen).
  • the left hand lane corresponds to control plasmid; the middle lane corresponds to the pi 62 XLC helicase-ET33 plasmid and the right hand lane contains size markers.
  • Figure 8 sets out the results of western blot hybridisation using a His-tag specific monoclonal antibody carried out on fractions collected in the course of purification of the pi 62 XLC helicase polypeptide purification.
  • Recombinantly expressed pi 62 XLC helicase was purified using anNi-NTA resin column.
  • Pre-column is an aliquot of the bacterial protein preparation before being added to Ni-NTA column;
  • column flowthrough is an aliquot of the liquid collected after mixing the bacterial protein preparation with the Ni-NTA resin; elution 1 and 2 are aliquots of the two elutions carried out with Ni-NTA wash buffer.
  • Figure 9 sets out the results of a pi 62 XLC helicase assay.
  • the right hand lane shows a reaction carried out with the addition of 50 ⁇ g of protein; the left hand lane shows a reaction carried out with no protein in the presence of ImM ATP; the two middle lanes show reactions carried out with lO ⁇ g or 50 ⁇ g protein in the presence of ImM ATP.
  • SEQ ID NO: 1 sets out the full length coding sequence of the pl62 XLC helicase gene.
  • SEQ ID NO: 2 sets out the polypeptide sequence of the pl62 XLC helicase gene product.
  • SEQ ID NO: 3 sets out the sequence of an oligonucleotide used in helicase activity assays.
  • SEQ ID NO: 4 sets out the sequence of an oligonucleotide used in helicase activity assays.
  • the present invention relates to a human gene, pi 62 XLC helicase, which encodes a helicase protein and to variants and fragments thereof.
  • Sequence information for the helicase-encoding gene is provided in SEQ ID NO: 1 (nucleotide) and in SEQ ID NO: 2 (amino acid).
  • Polynucleotides and polypeptides of the invention may comprise or may consist essentially of the nucleic acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 respectively, or may be variants or fragments of those sequences .
  • the polynucleotide, polypeptides, vectors, cells or antibodies of the invention may be provided in substantially isolated form.
  • isolated is intended to convey that the polynucleotide/polypeptide is not in its native state, insofar as it has been purified at least to some extent or has been synthetically produced, for example by recombinant methods.
  • polynucleotides, polypeptides, vectors, cells or antibodies of the invention may be mixed with carriers or diluents which will not interfere with the intended purpose of the polynucleotide, polypeptide, vector, cell or antibody and still be regarded as substantially isolated.
  • isolated therefore includes the possibility of the polynucleotide, polypeptide, vector, cell or antibody being in combination with other biological or non-biological material, such as cells, suspensions of cells or cell fragments, proteins, peptides, expression vectors, organic or inorganic solvents, or other materials where appropriate, but excludes the situation where the product in question in is in a state as found in nature.
  • a polynucleotide, polypeptide, vector, cell or antibody of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%>, e.g. more than 80%, 90%, 95%> or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.
  • Routine methods can be employed to purify and/or synthesise the polynucleotide proteins of the invention. Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd Edition, CSH Laboratory Press (1989), the disclosure of which is included herein in its entirety by way of reference.
  • variants in relation to polypeptides of the invention refers to polypeptides, which have the same essential character or basic biological functionality of the p 162 XLC helicase gene product.
  • the product of that gene has helicase activity and a variant of the invention will also have helicase activity. That is, a variant of the invention will have the ability to unwind a DNA duplex substrate.
  • a variant will have substantially the same amount of helicase activity (as determined by, for example, the helicase assay set out in Example 6 below) as the pi 62 XLC helicase polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2.
  • a variant may display less helicase activity than the pi 62 XLC helicase polypeptide, for example about at least 50%>, preferably about at least 60%, more preferably about at least 70%, for example about at least 80%> or most preferably about at least 90%> of the helicase activity of that polypeptide.
  • a variant may comprise multiple tetratricopeptide repeats, for example at least about 10, at least about 25 or at least about 50, up to about 100 such repeats.
  • variants may include allelic variants, species variants (sometimes referred these two types of variants are referred to as homologues) and variants derived from the modification of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains the basic biological functionality of the pi 62 XLC helicase gene product as discussed above.
  • Amino acid modifications may be made, for example 1, at least 5, at least 10, at least 20, at least 30, at least 50 up to about 70, 80, 100 or 150 modifications.
  • the polypeptide may be modified by insertion, deletion, N-terminal or C-terminal addition, or substitution of an amino acid with another amino acid. These types of modification may be combined to give a polypeptide of the invention. Deletions may be from one or both ends of the polypeptide.
  • Amino acid substitutions will preferably be conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. Again, the resulting peptide/polypeptide typically maintains the basic functionality of the pi 62 XLC helicase gene product.
  • Shorter polypeptide sequences are also within the scope of the invention.
  • a peptide of from at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100 amino acids up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or 1300 amino acids in size is considered to fall within the scope of the invention and will typically demonstrate the basic biological functionality of the p 162 XLC helicase gene product.
  • this aspect of the invention encompasses the situation when the protein is a fragment of the complete protein sequence and may represent a region which interacts with cytoskeleton or membrane skeleton components (N-terminal intracellular domain) or an anion exchange region (C-terminal transmembrane domain).
  • Such fragments can be used to construct chimeric proteins.
  • fragments of the invention include those which include an epitope. Suitable fragments will be at least 5, for example at least 10, at least 12, at least 15 or at least 20 amino acids in size. Epitope fragments may typically be up to 50, 60, 70, 80, 100, 150, or 200 amino acids in size. Epitopes may be determined by techniques known to those skilled in the art. These fragments will be useful in obtaining antibodies to polypeptides of the invention. Such fragments may comprise an epitope of the pi 62 XLC helicase gene product and may otherwise not demonstrate the enzymatic (i.e. helicase) activity, ligand binding or other properties of that polypeptide.
  • Polypeptides of the invention may be chemically modified, e.g. post- translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term "polypeptide" of the invention. Polypeptides of the invention may be modified for example by the addition of
  • Histidine residues for example His 6 , or a T7 tag to assist their identification or purification or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence.
  • the invention also includes nucleotide sequences that encode for the pi 62 XLC helicase polypeptide, variants and fragments thereof, as well as nucleotide sequences which are complementary thereto.
  • the polynucleotide may be single stranded or double stranded.
  • the nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA.
  • the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence.
  • Nucleotide sequence information is provided in SEQ ID NO: 1.
  • Such polynucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al.
  • a polynucleotide of the invention may be a coding sequence.
  • a coding sequence is a nucleotide sequence that is capable of being transcribed by the transcriptional mechanisms endogenous to a cell comprising the polynucleotide.
  • the coding sequence encodes an mRNA. More preferably, the mRNA is capable of being translated by the translation mechanisms of a cell comprising the polynucleotide.
  • a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.
  • a polynucleotide of the invention can hydridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library.
  • the signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1.
  • the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32 P.
  • Selective hybridisation may typically be achieved using conditions of medium to high stringency (for example, 2 x SSC [0.3M sodium chloride and 0.03M sodium citrate] or 1 x SSC [0.15M sodium chloride and 0.015M sodium citrate] at about 50°C to about 60°C or at about 65°C, or even at about 68°C).
  • medium to high stringency for example, 2 x SSC [0.3M sodium chloride and 0.03M sodium citrate] or 1 x SSC [0.15M sodium chloride and 0.015M sodium citrate] at about 50°C to about 60°C or at about 65°C, or even at about 68°C.
  • suitable conditions include from 0.1 to 0.2 x SSC at about 60°C to about 65°C.
  • suitable conditions include 2 x SSC at 60°C.
  • the coding sequence of SEQ ID No: 1 may be modified by nucleotide substitutions, for example from 1, 2 , 3, at least 5, at least 10 up to at least 25, at least 50, at least 100 or at least 200 substitutions.
  • the polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. For example from 1, 2 , 3, at least 5, at least 10 up to at least 25, at least 50, at least 100 or at least 200 of such modifications can be made to give a polynucleotide of the invention.
  • a combination of the above-mentioned modifications may be combined to give a polynucleotide of the invention.
  • substitutions are made so that there is no effect of the resulting polypeptide encoded. That is, preferably a substitution is a degenerate substitution. Alternatively, the substitution may result in a conservative amino acid substitution. Conservative substitutions are defined above.
  • a polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included.
  • the modified polynucleotide generally encodes a polypeptide which has helicase activity and optionally tetratricopeptide repeats.
  • a nucleotide sequence which is capable of selectively hybridizing to the coding sequence of SEQ ID NO: 1 or the complement thereof will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99%> sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100, for example at least 200 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1. Any combination of sequence identity and polynucleotide length can be used to define a polynucleotide of the invention.
  • nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo.
  • nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides complementary to those encoding a polypeptide having helicase activity, or antisense sequences, may also be used in gene therapy.
  • Polynucleotides of the invention may include within them synthetic or modified nucleotides.
  • a number of different types of modification to polynucleotides are known in the art. Such modifications may be carried out in order to enhance the in vivo activity, lifespan, nuclease resistance or ability to enter cells.
  • phosphorothioate oligonucleotides may be used.
  • Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide methylphosphonates.
  • MBOs Mixed backbone oligonucleotides
  • MBOs contain segments of phosphothioate oligodeoxynucleotides and appropriately placed segments of modified oligodeoxy- or oligoribonucleotides.
  • MBOs have segments of phosphorothioate linkages and other segments of other modified oligonucleotides, such as methylphosphonate, which is non-ionic, and very resistant to nucleases or 2'- O-alkyloligoribonucleotides .
  • Polynucleotides of the invention may be used as a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe or the polynucleotides may be cloned into vectors.
  • a primer e.g. a PCR primer, a primer for an alternative amplification reaction, a probe or the polynucleotides may be cloned into vectors.
  • Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700, 1000, 1500, 2000, 2500, 3000, 3500 or 4000 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO: 1.
  • Such polynucleotides, primers or probes may carry a revealing label, for example they may be labelled by conventional means using a radioactive label, for example 32 P or 35 S, or non-radioactive labels, for example an enzyme label, a fluorescent label or biotin.
  • a radioactive label for example 32 P or 35 S
  • non-radioactive labels for example an enzyme label, a fluorescent label or biotin.
  • Sequence identity for both polypeptides and polynucleotides, can be calculated using the UWGCG Package, which provides the BESTFIT program (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • the PILEUP and BLAST algorithms can be used to calculate sequence identity or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et ⁇ / (1990) J MolBiol 215:403-10.
  • Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.i-lm.--ili.gov/).
  • This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighbourhood word score threshold (Altschul et al, supra).
  • These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • polynucleotides of the invention Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred.
  • a polynucleotide/polypeptide which has at least 90%> sequence identity over 25, preferably over 30 nucleotides/ainino acids forms one aspect of the invention, as does a polynucleotide/polypeptide which has at least 95%> sequence identity over 40 nucleotides/amino acids.
  • the present invention also includes expression vectors that comprise nucleotide sequences, for example a polynucleotide of the invention, encoding the proteins or variants or fragments thereof of the invention.
  • Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression.
  • Other suitable vectors would be apparent to persons skilled in the art.
  • Polynucleotides according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA.
  • Antisense RNA or other antisense polynucleotides may also be produced by synthetic means.
  • Such antisense polynucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.
  • a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • control sequence such as a promoter
  • "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.
  • the control sequence will typically comprise a promoter and optionally also comprise other types of control sequence, for example an enhancer and/or terminator.
  • a control sequence may be positioned 5', 3' or internal to (for example in an intron) a coding sequence.
  • a coding sequence may be operably linked to more than one control sequence, for example two, three, four or five control sequences. Such multiple control sequences may be positioned, for example, entirely 5' to the coding sequence. However, more typically control sequences will be located both 5 1 and 3' to the coding sequence, with optional internal control sequences.
  • a promoter is a nucleotide sequence capable of initiating transcription of a coding sequence.
  • a coding sequence is positioned 3 1 (i.e. downstream) to a promoter, although promoters may be situated in introns.
  • An enhancer is any polynucleotide sequence capable of increasing the level of transcription initiating from a promoter and may act on a cis or trans basis .
  • a terminator is any polynucleotide sequence capable of promoting dissociation of an RNA polymerase from the said sequence. Control sequences may be derived from any suitable source and may be generated by recombinant techniques or synthetic means.
  • the vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector.
  • Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell.
  • the vectors may also be adapted to be used in vivo, for example in a method of gene therapy.
  • Promoters and other expression regulation signals/control sequences may be selected to be compatible with the host cell for which expression is designed.
  • bacterial promoters include the T7 and T3 promoters
  • yeast promoters include S. cerevisiae GAL4 and ADH promoters
  • S. pombe nmtl and adh promoter include the metallothionein promoter which can be induced in response to heavy metals such as cadmium.
  • Viral promoters such as the SN40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art. Mammalian promoters, such as ⁇ -actin promoters, may be used.
  • Tissue-specific promoters are especially preferred.
  • Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMN) IE promoter, adenovirus, HSN promoters (such as the HSN IE promoters), or HPN promoters, particularly the HPN upstream regulatory region (URR).
  • MMLV LTR Moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • HSN promoters such as the HSN IE promoters
  • HPN promoters particularly the HPN upstream regulatory region (URR).
  • Viral promoters are readily available in the art.
  • the vector may also include sequences encoding a selectable marker, for example a polypeptide that provides resistance to an antibiotic, such as ampicillin kanamycin (eg. the nptl or nptll genes), neomycin or chloramphenicol (eg. the CAT gene), or a polypeptide that can be colorimetrically or fluorometrically detected.
  • a selectable marker for example a polypeptide that provides resistance to an antibiotic, such as ampicillin kanamycin (eg. the nptl or nptll genes), neomycin or chloramphenicol (eg. the CAT gene), or a polypeptide that can be colorimetrically or fluorometrically detected.
  • the vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.
  • sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.
  • sequences flanking the polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences.
  • viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses, HSV and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.
  • the invention also includes cells that have been modified to express a polypeptide of the invention or a variant thereof.
  • Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells.
  • the cell will not be a human embryonic cell, for example a human embryonic stem cell.
  • cells which may be modified by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa and COS cells.
  • the cell line selected will be one which is not only stable, but also allows for mature glycosylation and cell surface expression of a polypeptide. Expression may be achieved in bacterial cells, for example inE. coli cells, using an expression vector such as the pET33b (Novagene) expression vector. Expression may be achieved in transformed oocytes.
  • a polypeptide of the invention may be expressed in a cell-free system, for example by use of a rabbit reticulate lysate or an HeLa cell nuclear extract.
  • a polypeptide of the invention may be expressed in cells of a transgenic non-human animal.
  • a transgenic non-human animal for example a rodent, such as a mouse, a rat, a guinea pig, a ferret, a gerbil or a hamster expressing a polypeptide of the invention is included within the scope of the invention.
  • a polypeptide of the invention may also be expressed in Xenopus laevis oocytes or melanophores, in particular for use in an assay of the invention.
  • proteins of the invention can be transiently expressed in a cell line or on a membrane, such as for example in a baculovirus expression system.
  • a cell line or on a membrane such as for example in a baculovirus expression system.
  • Such systems which are adapted to express the proteins according to the invention, are also included within the scope of the present invention.
  • the present invention also relates to antibodies which have been raised by standard techniques and are specific for a polypeptide of the invention.
  • Such antibodies could for example, be useful in purification, isolation or screening methods involving immunoprecipitation techniques and may be used as tools to further elucidate the function of the pi 62 XLC helicase gene, or a variant or fragment thereof, or indeed as therapeutic agents in their own right.
  • Antibodies may also be raised against specific epitopes of the proteins according to the invention.
  • An antibody, or other compounds "specifically binds" to a protein when it binds with high affinity to the protein for which it is specific but substantially does not bind, or binds with only low affinity to other proteins.
  • a variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art. Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.
  • Antibodies of the invention may be antibodies to human polypeptides or fragments thereof.
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which maintain their binding activity for a polypeptide encoded by a polynucleotide of the invention, a polypeptide of the invention or a fragment thereof. Such fragments include Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies.
  • the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.
  • Antibodies may also be used in a method for detecting polypeptides of the invention present in a biological sample, which method comprises:
  • a sample may be for example a tissue extract, blood, serum and saliva.
  • Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions, etc. Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in vivo pi 62 XLC helicase gene expression imaging.
  • Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the
  • a method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the serum.
  • the animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.
  • a method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody.
  • Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).
  • An immortalized cell producing the desired antibody may be selected by a conventional procedure.
  • the hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
  • Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
  • the experimental animal is suitably a goat, rabbit, rat or mouse.
  • the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.
  • the carrier molecule is typically a physiologically acceptable carrier.
  • the antibody obtained may be isolated and, if desired, purified. Humanized antibodies may be obtained by replacing components of a non- human antibody with human components, without substantially interfering with the ability of the antibody to bind antigen.
  • An important aspect of the present invention is the use of polynucleotides, polypeptides, vectors and cells of the invention in screening methods.
  • the screening methods may be used to identify substances that modulate activity and/or expression of a helicase, in particular substances which modulate the activity of the pi 62 XLC helicase gene and/or expression of the pi 62 XLC helicase gene product.
  • Screening methods may thus be used to identify modulators, for example inhibitors or stimulators of the pi 62 XLC helicase gene or gene product, inhibitors or stimulators of the activity of the p 162 XLC helicase gene product or modulators which up-regulate or down-regulate pi 62 XLC helicase gene expression.
  • modulators for example inhibitors or stimulators of the pi 62 XLC helicase gene or gene product, inhibitors or stimulators of the activity of the p 162 XLC helicase gene product or modulators which up-regulate or down-regulate pi 62 XLC helicase gene expression.
  • a modulator of the invention may modulate a factor which regulates or is part of the expression pathway of pi 62 XLC helicase.
  • the factor is preferably specific to that expression pathway and does not play a role in the expression of other proteins.
  • the factor may be the gene from which p 162 XLC helicase is expressed, an RNA polymerase that can express mRNA from the gene, the unspliced mRNA which is transcribed from the gene, further factors that aid splicing of the mRNA, the spliced mRNA, nuclear factors that bind to the mRNA and/or transport the mRNA from the nucleus to the cytoplasm, translation factors that contribute to translating the mRNA to protein, or the p 162 XLC helicase gene product.
  • a modulator of the invention may modulate transcription and/or translation of the pi 62 XLC helicase gene or activity of the pi 62 XLC helicase gene product.
  • the substance is a specific modulator of transcription, translation and does not activate transcription from other genes.
  • the modulator may bind to the gene 5' to the coding sequence 3' to the coding sequence.
  • a modulator may bind to the promoter, and stimulate or inhibit the initiation of transcription.
  • the agent may bind and stimulate or inhibit the action of a protein that is required for transcription from the pi 62 XLC helicase gene.
  • a modulator may bind to the untranslated or translated regions of the mRNA. This could modulate the initiation of translation.
  • the modulator may modulate expression by modulating the rate at which the pi 62 XLC helicase gene product is broken down.
  • the modulator may modulate the expression of different variants of the pi 62 XLC helicase gene (e.g. different isoforms produced by different splicing of the mRNA). Any suitable format may be used for carrying our an assay for identifying a modulator of a substance that modulates p 162 XLC helicase gene expression and/or pi 62 XLC gene product activity.
  • the assay will be carried out in a single medium and preferably it will be possible for the assay to be carried out in a single well of a plastics microtitre plate.
  • the screen is preferably adapted for high through-put screening.
  • screening methods may involve contacting a polypeptide of the invention with a test product. Activity of the polypeptide is monitored, thereby to determine whether the test product substance activity of the polypeptide.
  • Suitable polypeptides may be produced, for example recombinantly according to Examples 3 and 5 below and helicase activity may be determined according to Example 6 below.
  • modulator activity can be determined by directly contacting a polypeptide of the invention with a substance under investigation and by monitoring the effect mediated by the polypeptide.
  • the contacting step is carried out under conditions that in the absence of the test substance would permit activity of the polyppetide.
  • control experiments are carried out on a polypeptide different to that of the invention in order to establish whether the test substance specifically modulates the polypeptide of the invention or whether the effect of the test substance is non-specific.
  • the binding of a test substance to a polypeptide of the invention can be determined directly.
  • a radiolabeled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored.
  • the radiolabeled test substance can be incubated with the polypeptide until equilibrium is reached.
  • the polypeptide can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting.
  • Nonspecific binding of the test substance may also be determined by repeating the experiment in the presence of a saturating concentration of a non-radioactive ligand.
  • test products may be assayed for their ability to modulate, ie. up-regulate or down-regulate, expression of the reporter polypeptide.
  • Suitable reporter polypeptides include the lacZ gene product, luciferase and fluorescent proteins such as green fluorescent protein.
  • test products which can be tested in the above assays include combinatorial libraries, defined chemical entities, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e.g. phage display libraries) and antibody products.
  • organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons.
  • Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or stimulation tested individually. Test substances may be used at a concentration of from lnM to lOOO ⁇ M, preferably from l ⁇ M to lOO ⁇ M, more preferably from l ⁇ M to lO ⁇ M.
  • a modulator of p 162 XLC helicase gene expression and/or p 162 XLC helicase gene product activity is one which produces a measurable reduction or increase in pi 62 XLC helicase gene expression and/or activity in the assays described above.
  • modulators of pi 62 XLC helicase gene expression and/or pi 62 XLC helicase gene product activity may be inhibitors or stimulators of pi 62 XLC helicase gene expression and/or p 162 XLC helicase gene product activity.
  • Preferred inhibitors are those which inhibit pi 62 XLC helicase gene expression and/or pi 62 XLC helicase gene product activity by at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%> at a concentration of the inhibitor of lng ml- 1 , lOng nl "1 , lOOng nl "1 , l ⁇ g ml "1 , lO ⁇ g ml "1 , lOO ⁇ g ml' 1 , SOO ⁇ g ml "1 , lmg ml" 1 ' lOmg ml "1 or lOOmg ml "1 .
  • Preferred stimulators are those which stimulate pi 62 XLC helicase gene expression and/or pi 62 XLC helicase gene product activity by at least 10%, at least 25%, at least 50%, at least 100%, at least, 200%, at least 500% or at least 1000% at a concentration of the stimulator of lng ml "1 , lOng nl “1 , lOOng nl “1 , l ⁇ g ml "1 , lO ⁇ g ml “1 , lOO ⁇ g ml “1 , 500 ⁇ g ml "1 , lmg ml "1, lOmg ml "1 or lOOmg ml "1 .
  • the percentage inhibition or stimulation represents the percentage decrease or increase in expression/activity in a comparison of assays in the presence and absence of the test substance. Any combination of the above mentioned degrees of percentage inhibition or stimulation and concentration of inhibitor or stimulator may be used to define an inhibitor or stimulator of the invention, with greater inhibition or stimulation at lower concentrations being preferred.
  • a preferred modulator is one which is a specific modulator of a polypeptide of the invention.
  • a specific modulator is one which modulates a polypeptide of the invention, while having substantially no modulatory effect on any other polypeptide.
  • a specific modulator may have, for example at least 2-fold, at least 3 -fold, at least 5 -fold or at least 10-fold the activity on a polypeptide of the invention that it has for other polypeptides, in particular for other helicase polypeptides.
  • a modulator of the invention may be capable of specifically inhibiting a particular type or class of helicases.
  • a modulator may be specific for 5' ⁇ -3' helicases or specific for 3'- 5' helicases.
  • Candidate substances which show activity in assays such as those described above can be tested in in vivo systems, for example in an animal model (an -inimal disease model, for instance).
  • Another aspect of the present invention is the use of polynucleotides encoding helicase polypeptides to identify mutations, for example single nucleotide polymorphisms, deletions, or insertions in the pi 62 XLC helicase gene. Identification of such mutations may be used to assist in diagnosis of or susceptibility to a cancer or a premature ageing disorder.
  • a variety of methods may be employed for the diagnosis of a cancer or premature aging; predisposition to a cancer or a premature ageing disorder and for monitoring patients undergoing clinical evaluation for the treatment of a cancer or a premature ageing disorder. These methods may be performed utilizing pre-packaged diagnostic kits comprising at least one specific pi 62 XLC helicase gene polynucleotide or antibody specific for an pi 62 XLC helicase gene epitope. Typically, such polynucleotides or antibodies may be capable of discriminating between polymorphic versions of the pi 62 XLC helicase gene and thus may be conveniently used in clinical settings to diagnose patients exhibiting a cancer or a premature ageing disorder.
  • the present invention provides a method for detecting variation in the expressed products encoded by pi 62 XLC helicase gene. This may comprise determining the level of the p 162 XLC helicase gene product expressed in cells or determining specific alterations in the expressed product. In particular, a reduction in the expression of the pi 62 XLC helicase gene as compared to a normal individual may be indicative of the presence of or susceptibility to a cancer or a premature ageing disorder. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and conservation of intron/exon structure. This sort of assay has importance in the diagnosis, prognosis and monitoring treatments of a cancer or a premature ageing disorder. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest co-segregates with disease phenotype in a family.
  • Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed "in situ” directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate procedures are described in, for example, Nuovo, G.J., 1992, “PCR-f ⁇ Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression pattern of the pi 62 XLC helicase gene, and qualitative aspects of pi 62 XLC helicase gene expression and/or composition.
  • Alternative diagnostic methods for the detection of the pi 62 XLC helicase gene may involve the amplification of a polynucleotide of the invention, e.g., by PCR (the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y et al., 1989, Proc. Natl. Acad. Sci.
  • PCR the experimental embodiment set forth in Mullis, K.B., 1987, U.S. Patent No. 4,683,202
  • ligase chain reaction Barany, F., 1991, Proc. Natl. Acad. Sci. USA 88:189-193
  • Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et al., 1996, Nature Genetics 14:441-447 and Shoemaker et al, 1996, Nature Genetics 14:450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization.
  • Another aspect of the present invention is the use of polypeptides and polynucleotides of the invention and products identified by the screening techniques referred to above, in methods of treatment of the human or animal body by therapy.
  • polypeptides, polynucleotides and modulators may be used in the treatment or prophylaxis of a cancer or a premature ageing disorder. It is to be understood that mention of these specific disorders is by way of example only and is not intended to be limiting on the scope of the invention as described.
  • products of the invention may be used in the manufacture of a medicament for use in the treatment of one of the above mentioned conditions. The conditions of a host suffering from one of the above mentioned condition can therefore be improved by administration of a product of the invention. A therapeutically effective amount of a product of the invention may be given to a in need thereof.
  • the host may be a human or an animal.
  • cancers examples include primary and secondary cancers.
  • the cancers may be, for example, a leukemia, a lymphoma, a sarcoma, a carcinoma or an adenocarcinoma.
  • Specific types of cancers which may be treated according to the invention include breast, colon, brain, lung, ovary, pancreatic, stomach, skin, testicular and tongue cancers.
  • Examples of premature ageing disorders that may be treated according to the invention includes Bloom's, Werner's, and Rothmund-Thomson syndrome. In addition, symptoms of premature ageing syndromes may be treated according to the invention.
  • unusual patterns of loss of subcutaneous fat, skin ulceration or sarcomas may be treated (for example in the case of Werner's syndrome).
  • Immuno-deficiency, male infertility and female sub -fertility, small body size, sun- induced facial erythema of a cancer may be treated (for example in the case of Bloom's syndrome).
  • Growth deficiency, photosensitivity with poikilodermatous skin changes, early greying and hair loss, cataracts or a cancer, in particular osteogenic sarcoma may be treated (for example in the case of Rothmund-Thomson syndrome).
  • a chemotherapeutic agent may be used in combination with a polypeptide, polynucleotide or modulator, in particular a stimulator.
  • Polypeptides, polynucleotides and modulators, in particular stimulators, of the invention may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the polypeptides or modulators may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • a polypeptide or modulator may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular patient.
  • polypeptide or modulator of the invention will depend upon factors such as the nature of the exact antagonist, whether a pharmaceutical or veterinary use is intended, etc.
  • a polypeptide or modulator of the invention may be formulated for simultaneous, separate or sequential use.
  • a polypeptide or modulator of the invention is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
  • Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
  • The. syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective amount of a polypeptide or modulator of the invention is administered to a patient.
  • the dose of a modulator of a polypeptide or modulator of the invention may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g. That dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.
  • a nucleic acid encoding a polypeptide of the invention may be administered to the a human or animal.
  • Nucleic acid such as RNA or DNA, and preferably, DNA
  • a vector such as the polynucleotides described above, which may be expressed in the cells of the human or animal.
  • Nucleic acid encoding the peptide may be administered to the human or animal by any available technique.
  • the nucleic acid may be introduced by injection, preferably intradermally, subcutaneously or intramuscularly.
  • the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery.
  • the nucleic acid may be administered topically to the skin, or to the mucosal surfaces for example by intranasal, oral, intravaginal, intrarectal administration.
  • Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.
  • these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam.
  • the dosage of the nucleic acid to be administered can be altered.
  • the nucleic acid is administered in the range of lpg to lmg, preferably to lpg to lO ⁇ g nucleic acid for particle mediated gene delivery and lO ⁇ g to lmg for other routes.
  • a polynucleotide of the invention may be delivered in a viral vector.
  • a virus incorporating a polynucleotide of the invention is administered in the range of from 10 6 to 10 10 pfu, preferably in the range of from 10 7 to 10 9 pfu, more preferably about 10 8 pfu for adenoviral vectors.
  • EcR293 clone 11 cells or Tex2P3 clone 22 cells (Xu et al, Nature Cell Biol. 2(6), 339-345; WO-A-01/09307 (PCT/GB00/02926)) which are capable of regulatable high level NO expression were grown either in non-inducing or inducing conditions (i.e. in the presence or absence of lO ⁇ M muristerone A or lOng/ml tetracycline) for 24 hours.
  • 50ng polyA+ RNA prepared from control cells was labelled with the fluorescent molecule Cy3.
  • 50ng polyA+ RNA prepared from NO-generating cells was labelled with the fluorescent molecule Cy5.
  • the two fluorescently-labelled probe samples were simultaneously applied to a single microarray (Gem array V; Incyte Genomics, Palo Alto, California), which contained 8,372 unique annotated genes/EST clusters. Analysis of the expression data showed that one of the elements, G12021KA4T3 (indicated by the arrow in Fig. 1), a short EST (expression Tag) cDNA sequence, (Genebank accession number: AI478977), was down-regulated 2.6-fold in the presence of NO.
  • the cDNA sequence on the array was an EST tag containing 722bp of mRNA sequence originating from a cDNA library derived from human B -cells (chronic lymphomatic leukaemia).
  • GenBank The corresponding genomic sequence was found to be located on human chromosome 2.
  • the genomic sequence of the corresponding gene was predicated as having an open reading frame of 4.2kb, containing 31 coding exons, and spanning 81kb of genomic DNA (Fig. 2)
  • pairs of gene-specific PCR primers were designed to the predicted open reading frame sequence. These were used to amplify the open reading frame directly from mRNA samples isolated from human cells (for example, the human embryonic kidney cell line EcR293 and the human carcinoma cell line DLD-1). The identity of the resulting PCR fragments were confirmed by direct PCR sequencing.
  • a cDNA library from human B-cells was also screened to find AI478977-related cDNA clones.
  • the coding cDNA sequence is 4248 bp, and capable of encoding a protein of 1415 amino acids (Fig. 3).
  • the C-terminal domain of the novel protein was identified as sharing considerable homology with the E. coli UvrD-helicase, with a 3D structure CD score of 37.3 (E value 0.006; Fig. 4). Overall, there was a 20%> sequence identity over 175 residues in the region corresponding to the active site of the E. coli UvrD-helicase.
  • the gene was widely represented in eukarotic cells, including yeast, C.elegans, Drosophila and was also found in the mouse genome.
  • the protein was also shown to contain multiple tetratricopeptide repeats (Fig.
  • Example 3 Cloning of the o!62 XLC helicase cDNA into pET-33b
  • the 4.2 kb cDNA sequence of the p 162 XLC helicase was cloned in-frame with a 5 ' -His-tag into the E. coli expression vector pET-33b (Novagene).
  • the resulting construct was sequenced in both orientations to confirm the predicated open reading frame.
  • the construct was given the name, pl62 XLC helicase-ET33 (Fig.6), Example 4: T7-mediated in vitro translation of the pi 62 XLC helicase cDNA, sequence T7-mediated in vitro translation (Novagene) was used to determine whether or not the construct was capable of generating a protein. As shown in Fig. 7, the translation product which had a molecular weight of 160 kDa corresponded to the predicted size of the protein without major modifications.
  • the p 162 XLC helicase protein was purified.
  • the bacteria were recovered by centrifugation at 4° C for 20 min at 4000rpm, the supernatant was removed and the cell pellet retained.
  • 500 ⁇ l of lx Ni-NTA buffer Na-NTA kit - Novagen Cat. No: 70899-3
  • lO ⁇ l lysozyme 50 mg/ml was then added and the resuspended pellet was allowed to stand on ice for 30 min.
  • the bacterial cells were then sonicate on ice for 4 x 10 seconds at an amplitude of 20-24 ⁇ .
  • the cell debris was removed by centrifugation at 13,000 rpm, for 20 min.
  • Ni-NTA His.Bind Ni-NTA binding-buffer
  • Ni-NTA kit - Novagen Cat. No: 70899-3 Ni-NTA kit - Novagen Cat. No: 70899-3
  • the resin was allowed to settle by gravity and the 4 ml of binding buffer was removed with a pipette.
  • 1ml of Ni-NTA His-binding slurry was then added to the (protein-containing) supernatant and mixed with the Ni-NTA resin at 4° for approximately 1 hr with rolling.
  • the total material protein + Ni-NTA resin
  • Example 6 Determination of helicase activity of the pi 62 XLC helicase The purified extract obtained according to Example 5 was used to assess
  • DNA 5 '-3' helicase activity as follows.
  • a DNA oligonucleotide (Oligo 2 - SEQ ID NO: 4) was 5'-labeled with 32 P using T4 polynucleotide kinase.
  • Equal -molar quantities of an unlabelled DNA oligonucleotide (Oligo 1 - SEQ ID NO: 3) and the [ 32 P]-labeled Oligo 2 were annealed at 90°C for 5 minutes and cooled to room temperature.
  • Varying amounts of purified pi 62 XLC-helicase (0 ⁇ g, 10 ⁇ g, 50 ⁇ g and 50 ⁇ g) in 50 ⁇ L of assay buffer were incubated in the presence or absence of 1 mM ATP (no ATP is shown in the last lane of Fig. 9.
  • the reaction was incubated at 37 °C, for 10 min and samples were subsequently loaded on a 10% acrylamide gel.
  • the double-stranded DNA duplex could be unwound by the pl62 XLC helicase (lO ⁇ g).
  • the overall unwinding reaction begins at the single/double-strand junction, and results in the release of the [ 32 P]-labeled DNA strand (22 mer).
  • the assay was also performed in the absence of ATP.
  • the level of DNA unwinding activity was shown to be dependent upon the dosage of pi 62 XLC helicase added ( Figure 9).

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un polypeptide comprenant : (i) la séquence d'acides aminés décrite dans la SEQ ID NO: 2; (ii) une variante d'une séquence telle que définie sous (i) qui est capable d'une activité de type hélicase ; ou (iii) un fragment d'une séquence telle que définie sous (i) ou sous (ii) qui est capable d'une activité hélicase. Le polypeptide de l'invention, un polynucléotide codant ledit polypeptide ou un modulateur de l'expression et/ou de l'activité dudit polypeptide peuvent être utilisés dans le traitement ou le diagnostic d'un cancer ou d'un trouble de vieillissement prématuré.
PCT/GB2002/005653 2001-12-13 2002-12-13 Gene de l'helicase WO2003054187A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002347395A AU2002347395A1 (en) 2001-12-13 2002-12-13 Helicase gene

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0129870.2 2001-12-13
GB0129870A GB0129870D0 (en) 2001-12-13 2001-12-13 Helicase gene

Publications (1)

Publication Number Publication Date
WO2003054187A1 true WO2003054187A1 (fr) 2003-07-03

Family

ID=9927578

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2002/005653 WO2003054187A1 (fr) 2001-12-13 2002-12-13 Gene de l'helicase

Country Status (3)

Country Link
AU (1) AU2002347395A1 (fr)
GB (1) GB0129870D0 (fr)
WO (1) WO2003054187A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1160324A1 (fr) * 1999-01-19 2001-12-05 Agene Research Institute, Co., Ltd. Gene a l'origine du syndrome de rothmund-thomson et produit genique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1160324A1 (fr) * 1999-01-19 2001-12-05 Agene Research Institute, Co., Ltd. Gene a l'origine du syndrome de rothmund-thomson et produit genique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 31 October 2001 (2001-10-31), ISOGAI ET AL.: "Homo sapiens cDNA FLJ32706 fis, clone TESTI2000627", XP002241411, Database accession no. AK057268 *
MOHAGHEGH PAYAM ET AL: "DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders.", HUMAN MOLECULAR GENETICS, vol. 10, no. 7, 2001, pages 741 - 746, XP002241410 *

Also Published As

Publication number Publication date
GB0129870D0 (en) 2002-02-06
AU2002347395A1 (en) 2003-07-09

Similar Documents

Publication Publication Date Title
EP1454146A2 (fr) Compositions, kits et procedes d'identification, d'evaluation, de prevention et de traitement de l'arthrite rhumatoide
WO2005032328A2 (fr) Compositions, trousses et methodes d'identification, d'evaluation, de prevention et de traitement de la polyarthrite rhumatoide
US20050170500A1 (en) Methods for identifying risk of melanoma and treatments thereof
US7544475B2 (en) Depression gene
WO2000052173A2 (fr) Homologues de sphingosine kinase humaine clones
JP2006508642A (ja) 脂肪の蓄積を減少させる方法と、関連疾患を治療するための方法
US20080199480A1 (en) Methods for Identifying Risk of Type II Diabetes and Treatments Thereof
US20070275872A1 (en) Peptides with Anti-Obesity Activity and Other Related Uses
US5756700A (en) Nucleic acid encoding human signal transducer and activator of transcription 4
US7449561B1 (en) Alterations in the dystrophin gene associated with sporadic dilated cardiomyopathy
US20020127692A1 (en) Protein
WO2003054187A1 (fr) Gene de l'helicase
WO2000071751A1 (fr) Gene du diabete
US20030113811A1 (en) Method of identifying modulators of presenilin
US6355430B1 (en) Diagnostic and screening methods employing KIAA0101
JP2004505637A (ja) ガン関連ｓｉｍ２遺伝子
US6660483B1 (en) Diagnostic marker for neurological conditions
JPH11164692A (ja) ヒトｒｃｅ１
US6300473B1 (en) SLM-1: a novel Sam68-like mammalian protein
WO2001016596A2 (fr) Crible
US20020076758A1 (en) Polypeptide
JP2004000195A (ja) Ｙａｋ−１関連セリン／トレオニンプロテインキナーゼ−ｈｔｌａｒ３３
US20020076800A1 (en) Protein
US20040018497A1 (en) Human obesity LIPIN3 polynucleotide and polypeptide sequences and methods of use thereof
GB2365432A (en) Neurotransmitter transporter polypeptide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载