WO2000005382A2 - A nucleic acid encoding a geranyl-geranyl pyrophosphate synthetase (ggpps) and polymorphic markers associated with said nucleic acid - Google Patents

A nucleic acid encoding a geranyl-geranyl pyrophosphate synthetase (ggpps) and polymorphic markers associated with said nucleic acid Download PDF

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Publication number
WO2000005382A2
WO2000005382A2 PCT/IB1999/001353 IB9901353W WO0005382A2 WO 2000005382 A2 WO2000005382 A2 WO 2000005382A2 IB 9901353 W IB9901353 W IB 9901353W WO 0005382 A2 WO0005382 A2 WO 0005382A2
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polynucleotide
seq
sequence
hggpps
marker
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PCT/IB1999/001353
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French (fr)
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WO2000005382A3 (en
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Lydie Bougueleret
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Genset
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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • a nucleic acid encoding a geranyl-geranyl pyrophosphate synthetase (GGPPS) and polymorphic markers associated with said nucleic acid.
  • GGPPS geranyl-geranyl pyrophosphate synthetase
  • the present invention relates to a purified or isolated polynucleotide encoding human geranylgeranyl pyrophosphate synthetase, the regulatory nucleic acids contained therein, a polymorphic marker thereof and the resulting encoded protein, as well as to methods and kits for detecting this polynucleotide and this protein.
  • the present invention also pertains to a polynucleotide carrying the natural regulatory regions of the hGGPS gene which is useful, for example, to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region.
  • the invention also consists in genetic markers, namely bialle c markers, which may be useful for the diagnosis of diseases related to an alteration in the regulatory or coding regions of hGGPS, such as pathologies related to a defect in the mevalomc biosynthetic pathway
  • Prenylation is the least common known lipid modification. Other lipid modifications include palmitylation, my ⁇ stylation and glycophosphohpidation. However, prenylation is a surprisingly common form of post-translational protein modification with an occurrence of 0.5 % of all cellular proteins.
  • Prenylation is a covalent modification which involves the attachment of either a C15 farnesyl or a C20 geranylgeranyl isoprenoid, both being products of the mevalomc acid biosynthetic pathway, to one or more cysteme residues at the carboxyl terminus of the protein via a thioether bond
  • the C20 geranylgeranyl modification predominates over the C15 farnesyl modification in terms of frequency of occurrence
  • the structural environment of the cysteme residue determines the specific type and number of isoprenoid groups that attach to each cysteme.
  • the covalent modification resulting from prenylation renders proteins more hydrophobic and, together with a subsequent modification cascade, facilitates their association with membranes.
  • Protein prenylation also mediates protein-protein interactions. Prenylated proteins can be involved in signal transduction, intracellular vesicular transport, cytoskeletal organization, cell growth control and polarity, viral replication and protein folding/assembly. In mammals, prenylated proteins are more frequently modified by one or more geranylgeranyl groups. Farnesylation has only been found to occur in the retinal heterot ⁇ me ⁇ c G protein transducin, in retinal rhodopsm kinase, m ras proteins, in nuclear lamins, and in yeast mating factors. Geranylgeranylation is found m all of the remaining heterot ⁇ me ⁇ c G proteins and small G proteins.
  • Heterot ⁇ me ⁇ c G-protems which are required for intracellular signal transduction between receptors and effector enzymes present one or two prenylated subumts This modification is often required for association of the functional complex with the membrane Among small G proteins, Ras proteins, which comprise oncogemc forms, regulate signal transduction pathways controlling cell proliferation and differentiation.
  • ras proteins are prenylated and this modification is critical for their transport to the inner surface of the plasma membrane and their biological functions
  • Other prenylated proteins belonging to the ras protein superfamily are involved in the regulation of intracellular vesicular transport (Rab/YPTl), in the cytoskeletal organization of polymerized actin to produce stress fibers (Rho) or membrane ruffling (Rac), in the oxydative burst of phagocytic cells (Rac), m the control of the cell cycle and pola ⁇ ty (cdc24Hs/G25K), and in negative growth control (Rap/Krev-1).
  • Prenylation is important to these activities.
  • Rab/YPT prenylation is c ⁇ tical for the association of these proteins with specific intracellular compartments and in their regulation of intracellular transport processes.
  • prenylation of nuclear lamins which are involved in the mitotic control of membrane assembly, is necessary for the proper assembly of these proteins into the nuclear lamina. Indeed, prenylation is necessary to the maturation by cleavage of prelamm A m lamin A and to obtain functional lamm B.
  • Geranylgeranyl pyrophosphate synthetase is involved in the mevalomc acid biosynthetic pathway and is located in the cytosol. It catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce GGPP. This biosynthesis of GGPPS is regulated according to requirements for protein prenylation. GGPS has been found to be expressed in human fetal heart, as desc ⁇ bed in the PCT Application No WO 96/21736.
  • the present invention pertains to nucleic acid molecules comp ⁇ smg the genomic sequence of a novel human gene which encodes a hGGPPS protein.
  • the hGGPPS genomic sequence comprises regulatory sequence located upstream (5 '-end) and downstream (3 '-end) of the transcribed portion of said gene, these regulatory sequences being also part of the invention
  • the invention also deals with the complete sequence of two cDNAs encoding the hGGPPS protein, as well as with the corresponding translation product
  • Oligonucleotide probes or p ⁇ mers hybridizing specifically with a hGGPPS genomic or cDNA sequences are also part of the present invention, as well as DNA amplification and detection methods using said p ⁇ mers and probes.
  • a further object of the invention consists of recombinant vectors comp ⁇ smg any of the nucleic acid sequences desc ⁇ bed above, and in particular of recombinant vectors comp ⁇ smg a h GGPPS regulatory sequence or a sequence encoding a hGGPPS protein, as well as of cell hosts and transgenic non human animals comp ⁇ smg said nucleic acid sequences or recombinant vectors
  • the invention also concerns a ⁇ GG S-related biallehc marker
  • the invention is directed to methods for the screening of substances or molecules that modify or inhibit the expression of hGGPPS
  • Figure 1 Map of the genomic, cDNA and coding (CDS) sequences of hGGPS : (1) upper line, genomic sequence; (2) cDNA sequence of SEQ ID No 2; (3) coding sequence (CDS).
  • FIG. 2 Map of the genomic, cDNA and coding (CDS) sequences of hGGPS : (1) upper line, genomic sequence; (2) cDNA sequence of SEQ LD No 3; (3) coding sequence (CDS).
  • SEQ ID No 1 contains a genomic sequence of hGGPPS comprising the 5 ' regulatory region (upstream untransc ⁇ bed region), the exons and introns, and the 3' regulatory region (downstream untransc ⁇ bed region).
  • SEQ LD No 2 contains a cDNA sequence of hGGPPS comp ⁇ smg the exons 1, 2, 3, and 4.
  • SEQ ID No 3 contains a cDNA sequence of hGGPPS comp ⁇ smg the exons Ibis, 2, 3, and 4.
  • SEQ ID No 4 contains the ammo acid sequence encoded by the cDNA of SEQ ID No 2 or 3.
  • SEQ LD Nos 5 and 6 contain the fragments containing a polymorphic base of the biallehc marker 5-187-77.
  • SEQ LD No 7 contains the microsequencing p ⁇ mer of the biallehc marker 5-187-77.
  • SEQ LD Nos 8 and 9 contain the amplification p ⁇ mers of the biallehc marker 5-187-77.
  • SEQ ID No 10 contains a p ⁇ mer containing the additional PU 5' sequence described further in Example 3.
  • SEQ ID No 1 1 contains a primer containing the additional RP 5' sequence described further in Example 3.
  • the hGGPS gene of the invention is located on chromosome 1, and more precisely on the Iq42-lq43 locus of this chromosome. This chromosome 1 locus has been shown to carry a predisposing gene for prostate cancer (Berthon et al., 1998).
  • the hGGPS gene of the invention is located in the vicinity of a retinoblastoma binding protein gene. Indeed, the coding sequence of this latter gene is on a strand which is opposite to the strand carrying the hGGPS Open Reading Frame.
  • the aim of the present invention is to provide polynucleotides de ⁇ ved from the hGGPS gene, particularly those useful to design suitable means for detecting the presence of this gene in a test sample or alternatively to disc ⁇ minate between the hGGPS mRNA molecules that are present in a test sample
  • Other polynucleotides of the invention are useful to design suitable means to express a desired polynucleotide of interest
  • the invention also relates to the hGGPS polypeptide having the ammo acid sequence of SEQ ID No 4.
  • hGGPPS gene when used herein, encompasses mRNA and cDNA sequences encoding the hGGPPS protein. In the case of a genomic sequence, the hGGPPS gene also includes native regulatory regions which control the expression of the coding sequence of the hGGPPS gene.
  • the term "functionally active fragment" of the hGGPPS protein is intended to designate a polypeptide carrying at least one of the structural features of the hGGPPS protein involved in at least one of the biological functions and/or activity of the hGGPPS protein
  • a “heterologous” or “exogenous” polynucleotide designates a purified or isolated nucleic acid that has been placed, by genetic enginee ⁇ ng techniques, in the environment of unrelated nucleotide sequences, such as the final polynucleotide construct does not occur naturally.
  • An illustrative, but not limitative, embodiment of such a polynucleotide construct may be represented by a polynucleotide comprising ( 1 ) a regulatory polynucleotide de ⁇ ved from the hGGPPS gene sequence and (2) a polynucleotide encoding a cytokme, for example GM-CSF.
  • the polypeptide encoded by the heterologous polynucleotide will be termed an heterologous polypeptide for the purpose of the present invention.
  • a “biologically active fragment or vanant" of a regulatory polynucleotide according to the present invention is intended a polynucleotide comp ⁇ sing or alternatively consisting m a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host.
  • a nucleic acid or polynucleotide is "functional" as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transc ⁇ ptional and translational regulatory information, and such sequences are "operatively linked" to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide.
  • An operable linkage is a linkage in which the regulatory nucleic acid and the DNA sequence sought to be expressed are linked in such a way as to permit gene expression.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • two DNA molecules are said to be "operably linked” if the nature of the linkage between the two polynucleotides does not (1) result m the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide
  • the promoter polynucleotide would be operably linked to a polynucleotide encoding a desired polypeptide or a desired polynucleotide if the promoter is capable of effecting transcription of the polynucleotide of interest
  • sample or "matenal sample” are used herein to designate a solid or a liquid material suspected to contain a polynucleotide or a polypeptide of the invention
  • a solid matenal may be, for example, a tissue slice or biopsy withm which is searched the presence of a polynucleotide encoding a hGGPPS protein, either a DNA or RNA molecule or withm which is searched the presence of a native or a mutated hGGPPS protein, or alternatively the presence of a desired protein of interest the expression of which has been placed under the control of a hGGPPS regulatory polynucleotide.
  • a liquid matenal may be, for example, any body fluid like serum, urine etc , or a liquid solution resulting from the extraction of nucleic acid or protein matenal of interest from a cell suspension or from cells in a tissue slice or biopsy.
  • biological sample is also used and is more precisely defined withm the Section dealing with DNA extraction
  • the term "purified" does not require absolute purity; rather, it is intended as a relative definition.
  • Pu ⁇ fication if starting material or natural matenal to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, punfication from 0.1% concentration to 10% concentration is two orders of magnitude.
  • isolated requires that the material be removed from its original environment (e.g. the natural environment if it is naturally occurring)
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting mate ⁇ als in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition and still be isolated m that the vector or composition is not part of its natural environment
  • polypeptide refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, ohgopeptides, and proteins are included withm the definition of polypeptide This term also does not specify or exclude post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an ammo acid (including, for example, non-naturally occurnng ammo acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurnng and non-naturally occurnng.
  • an ammo acid including, for example, non-naturally occurnng ammo acids, amino acids which only occur naturally in an unrelated biological
  • polypeptide refers to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.
  • purified is used herein to describe a polypeptide of the invention which has been separated from other compounds including, but not limited to nucleic acids, lipids, carbohydrates and other proteins.
  • a polypeptide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polypeptide sequence.
  • a substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight weight of a protein sample, more usually about
  • Polypeptide punty or homogeneity is indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.
  • non-human animal refers to any non-human vertebrate, birds and
  • mammals 15 more usually mammals, preferably primates, farm animals such as swme, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice.
  • animal is used to refer to any vertebrate, preferable a mammal. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • antibody refers to a polypeptide or group of polypeptides which
  • an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distnbution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.
  • Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab,
  • an “antigenic determinant” is the portion of an antigen molecule, in this case a hGGPPS polypeptide, that determines the specificity of the antigen-antibody reaction.
  • An “epitope” refers to an antigenic determinant of a polypeptide.
  • An epitope can comprise as few as 3 ammo acids in a spatial conformation which is unique to the epitope. Generally an epitope consists
  • ammo acids 30 of at least 6 such amino acids, and more usually at least 8-10 such ammo acids.
  • Methods for determining the ammo acids which make up an epitope include x-ray crystallography, 2-d ⁇ mens ⁇ onal nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method descnbed by Geysen et al. 1984; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506.
  • nucleotide sequence may be any sequence known in the art.
  • nucleotide sequence encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically charactenzes a specific DNA or RNA molecule.
  • oligonucleotides and “polynucleotides” include RNA, DNA. or RNA/DNA hybrid sequences of more than one nucleotide m either single chain or duplex form.
  • nucleotide as used herein as an adjective to descnbe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form.
  • nucleotide is also used herein as a noun to refer to individual nucleotides or va ⁇ eties of nucleotides, meaning a molecule, or individual unit a larger nucleic acid molecule, comprising a punne or py ⁇ midine, a nbose or deoxynbose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides withm an oligonucleotide or polynucleotide.
  • nucleotide is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of punne, (c) an analogous form of py ⁇ midme, or (d) an analogous sugar, for examples of analogous linking groups, punne, py ⁇ midines, and sugars see for example PCT publication No WO 95/04064.
  • the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxynbose nucleotides, and most preferably greater than 90% conventional deoxynbose nucleotides.
  • the polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any punfication methods known m the art.
  • the term "heterozygosity rate" is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallehc system, the heterozygosity rate is on average equal to 2P a (l-P a ), where P a is the frequency of the least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.
  • genotype refers the identity of the alleles present in an individual or a sample.
  • a genotype preferably refers to the desc ⁇ ption of the biallehc marker alleles present m an individual or a sample.
  • genotypmg a sample or an individual for a biallehc marker consists of determining the specific allele or the specific nucleotide earned by an individual at a biallehc marker.
  • polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals.
  • Polymo ⁇ hic refers to the condition in which two or more variants of a specific genomic sequence can be found in a population.
  • a "polymorphic site” is the locus at which the variation occurs.
  • a single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms.
  • single nucleotide polymorphism preferably refers to a single nucleotide substitution Typically, between different genomes or between different individuals, the polymo ⁇ hic site may be occupied by two different nucleotides
  • biasehc polymo ⁇ hism and “biallehc marker” are used interchangeably herein to refer to a single nucleotide polymo ⁇ hism having two alleles at a fairly high frequency in the population.
  • a "biallelic marker allele” refers to the nucleotide variants present at a biallehc marker site Typically, the frequency of the less common allele of the bialle c markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (I e. heterozygosity rate of at least 0.42)
  • a biallehc marker wherein the frequency of the less common allele is 30% or more is termed a "high quality biallelic marker"
  • nucleotides in a polynucleotide with respect to the center of the polynucleotide are desc ⁇ bed herein in the following manner.
  • the nucleotide at an equal distance from the 3' and 5' ends of the polynucleotide is considered to be "at the center" of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be "within 1 nucleotide of the center.”
  • any of the five nucleotides positions in the middle of the polynucleotide would be considered to be withm 2 nucleotides of the center, and so on.
  • defining a biallehc marker means that a sequence includes a polymo ⁇ hic base from a biallehc marker.
  • the sequences defining a biallelic marker may be of any length consistent with their intended use, provided that they contain a polymo ⁇ hic base from a bialle c marker.
  • the sequence has between 1 and 500 nucleotides in length, preferably between 5, 10 , 15, 20, 25, or 40 and 200 nucleotides and more preferably between 30 and 50 nucleotides in length.
  • Each biallehc marker therefore corresponds to two forms of a polynucleotide sequence included in a gene, which, when compared with one another, present a nucleotide modification at one position.
  • the sequences defining a bialle c marker include a polymo ⁇ hic base of the biallehc marker 5-187-77.
  • the sequences defining a bialle c marker comprise one of the sequences selected from the group consisting of SEQ ID Nos 5 and 6.
  • the term "marker” or “biallehc marker” requires that the sequence is of sufficient length to practically (although not necessa ⁇ ly unambiguously) identify the polymo ⁇ hic allele, which usually implies a length of at least 4, 5, 6, 10, 15, 20, 25, or 40 nucleotides.
  • base paired and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymme or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanme residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4 th edition, 1995)
  • complementary or “complement thereof are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & C ⁇ ck base painng with another specified polynucleotide throughout the entirety of the complementary region.
  • a first polynucleotide is deemed to be complementary to a second polynucleotide when each base m the first polynucleotide is paired with its complementary base.
  • Complementary bases are, generally, A and T (or A and U), or C and G.
  • “Complement” is used herein as a synonym from “complementary polynucleotide”, “complementary nucleic acid” and “complementary nucleotide sequence”. These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
  • the invention also relates to vanants and fragments of the polynucleotides desc ⁇ bed herein, particularly of a hGGPPS gene containing one or more biallehc markers according to the invention.
  • Vanants of polynucleotides are polynucleotides that differ from a reference polynucleotide.
  • a variant of a polynucleotide may be a naturally occurnng variant such as a naturally occurring allehc variant, or it may be a variant that is not known to occur naturally.
  • Such non-naturally occurnng variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the vanant are closely similar overall and, in many regions, identical.
  • Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences that are at least 95% identical to any of SEQ LD Nos 1-3 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ LD Nos 1-3 or the sequences complementary thereto, and preferably at least 98% identical, more particularly at least 99.5% identical, and most preferably at least 99.9% identical to any of SEQ ID Nos 1 -3 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ LD Nos 1 -3 or the sequences complementary thereto.
  • nucleotide of a vanant may be silent, which means that they do not alter the ammo acids encoded by the polynucleotide.
  • nucleotide changes may also result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence
  • substitutions, deletions or additions may involve one or more nucleotides
  • the vanants may be altered m coding or non-coding regions or both Alterations m the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions
  • particularly preferred embodiments are those m which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature hGGPPS protein
  • a polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a hGGPPS gene, and variants thereof.
  • the fragment can be a portion of an exon or of an intron of a hGGPPS gene. It can also be a portion of the regulatory sequences of the hGGPPS gene.
  • such fragments comprise the polymo ⁇ hic base of the biallelic marker 5-187-77 of SEQ LD Nos 5-6.
  • fragments may be "free-standing", I e not part of or fused to other polynucleotides, or they may be compnsed within a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide
  • polynucleotide fragments of the invention there may be mentioned those which have from about 4, 6, 8, 15, 20, 25, 40, 10 to 20, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200 nucleotides m length.
  • Preferred are those fragments having about 49 nucleotides in length, such as those of SEQ ID Nos 5-6 or the sequences complementary thereto and containing at least one of the biallehc markers of a hGGPPS gene which are descnbed herein.
  • the invention also relates to vanants, fragments, analogs and denvatives of the polypeptides described herein, including mutated hGGPPS proteins
  • the vanant may be 1) one in which one or more of the ammo acid residues are substituted with a conserved or non-conserved ammo acid residue (preferably a conserved ammo acid residue) and such substituted ammo acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the ammo acid residues includes a substituent group, or 3) one in which the mutated hGGPPS is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the mutated hGGPPS, such as a leader or secretory sequence or a sequence which is employed for pu ⁇ fication of the mutated hGGPPS or a preprotem sequence.
  • Such vanants
  • a variant hGGPPS polypeptide compnses ammo acid changes ranging from 1, 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of one ammoacid, preferably from 1 to 10, more preferably from 1 to 5 and most preferably from 1 to 3 substitutions, additions or deletions of one ammo acid.
  • the preferred ammo acid changes are those which have little or no influence on the biological activity or the capacity of the vanant hGGPPS polypeptide to be recognized by antibodies raised against a native hGGPPS protein
  • homologous peptide is meant a polypeptide containing one or several ammoacid additions, deletions and/or substitutions m the amino acid sequence of a 5 hGGPPS polypeptide.
  • an ammoacid substitution one or several -consecutive or non- consecutive- aminoacids are replaced by « equivalent » ammoacids
  • amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to 10 be substantially unchanged
  • groups of amino acids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, lie, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, T ⁇ , His.
  • ammoacid By an equivalent ammoacid according to the present invention is also meant the replacement of a residue the L-form by a residue in the D form or the replacement of a Glutamic acid (E) 15 residue by a Pyro-glutamic acid compound.
  • the synthesis of peptides containing at least one residue m the D-form is, for example, descnbed by Koch (1977).
  • polypeptide accoding to the invention could have post-translational modifications.
  • it can present the following modifications: acylation, disulfide bond formation, 25 prenylation, carboxymethylation and phosphorylation.
  • a polypeptide fragment is a polypeptide having a sequence that entirely is the same as part but not all of a given polypeptide sequence, preferably a polypeptide encoded by a hGGPPS gene and vanants thereof.
  • Preferred fragments include those regions possessing antigenic properties and which can be used to raise antibodies against the hGGPPS protein. 30
  • Such fragments may be "free-standing", i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger polypeptide of which they form a part or region. However, several fragments may be compnsed withm a single larger polypeptide.
  • polypeptide fragments of the invention there may be mentioned those which comprise at least about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 35 55 ammo acids of the hGGPPS
  • the fragments contain at least one ammo acid mutation in the hGGPPS protein Identity Between Nucleic Acids Or Polypeptides
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the va ⁇ ety of sequence comparison algo ⁇ thms and programs known in the art.
  • Such algonthms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996, Altschul et al., 1993).
  • protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see, e.g., Karhn and Altschul, 1990; Altschul et al., 1990, 1993, 1997).
  • BLAST Basic Local Alignment Search Tool
  • five specific BLAST programs are used to perform the following task:
  • BLASTP and BLAST3 compare an ammo acid query sequence against a protein sequence database
  • BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands)
  • TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a sco ⁇ ng matrix, many of which are known in the art.
  • the sco ⁇ ng matrix used is the BLOSUM62 matnx (Gonnet et al., 1992; Hemkoff and Hemkoff, 1993).
  • the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978).
  • the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology.
  • a user-specified threshold of significance such as a user- specified percent homology.
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karhn (see, e.g., Karhn and Altschul, 1990)
  • filter washes can be done at 37°C for 1 h in a solution containing 2 x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1 X SSC at 50°C for 45 mm.
  • filter washes can be performed in a solution containing 2 x SSC and 0.1% SDS, or 0 5 x SSC and 0.1% SDS, or 0.1 x SSC and 0.1% SDS at 68°C for 15 minute intervals.
  • the hyb ⁇ dized probes are detectable by autoradiography.
  • Other conditions of high stnngency which may be used are well known in the art and as cited m Sambrook et al., 1989; and Ausubel et al., 1989, are mco ⁇ orated herein in their entirety.
  • These hybndization conditions are suitable for a nucleic acid molecule of about 20 nucleotides m length.
  • the hybridization conditions desc ⁇ bed above are to be adapted according to the 0 length of the desired nucleic acid, following techniques well known to the one skilled m the art.
  • the suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgms (1985) or in Sambrook et al.(1989)
  • hGGPS gene polynucleotide cDNAs and associated regulatory regions.
  • the invention concerns a purified or isolated nucleic acid encoding the hGGPS polypeptide, wherein said nucleic acid comp ⁇ ses the nucleotide sequence of SEQ LD No 1.
  • the present invention concerns a punfied or isolated nucleic acid comprising a nucleotide sequence of SEQ LD No 1, or a nucleotide sequence complementary thereto or a fragment or a vanant thereof.
  • nucleic acids of the invention include isolated, punfied, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1-485, 547-632, 827-7291, 7385-13759, 13831-14062, 14671-15054, and
  • the invention also encompasses a punfied or isolated nucleic acid having at least 95% nucleotide identity with the nucleotide sequence of SEQ ID No 1 or a complementary sequence thereto.
  • a further object of the invention consists in a purified or isolated nucleic acid of at least 12 nucleotides in length, wherein said nucleic acid hybridizes under stnngent hybndization conditions with a polynucleotide sequence of SEQ LD No 1 or a complementary sequence thereto.
  • the hGGPS genomic nucleic acid sequence comprises five exons. These five exons are desc ⁇ bed m Table A.
  • hGGPS mtrons defined hereinafter for the pu ⁇ ose of the present invention are not exactly what is generally understood as “introns” by the one skilled in the art and will consequently be defined below.
  • an mtron is defined as a nucleotide sequence that is present both in the genomic DNA and in the unsphced mRNA molecule, and which is absent from the mRNA molecule which has undergone the splicing events.
  • the inventors have found that at least two different spliced mRNA molecules are produced when this gene is transcribed, as it will be described m detail m a further section of the specification
  • the first spliced mRNA molecule comprises Exons 1, 2, 3 and 4, as shown in Figure 1.
  • the genomic nucleotide sequence comprised between Exon 1 and Exon 2 is an lntronic sequence as regards to this first mRNA molecule, despite the fact that this lntronic sequence contains Exon Ibis.
  • Exon Ibis is of course an exomc nucleotide sequence as regards to the second hGGPS mRNA molecule shown in Figure 2.
  • the polynucleotides contained both in the nucleotide sequence of SEQ LD No 1 and in any of the nucleotide sequences of SEQ LD Nos 2 or 3 are considered as exomc sequences.
  • the polynucleotides contained in the nucleotide sequence of SEQ ID No 1 and located between Exon 1 and Exon 4, but which are absent both from the nucleotide sequence of SEQ ID No 2 and from the nucleotide sequence of SEQ ID No 3 are considered as lntronic sequences.
  • the invention embodies punfied, isolated, or recombinant polynucleotides compnsing a nucleotide sequence selected from the group consisting of the exons of the hGGPPS gene, or a sequence complementary thereto.
  • the invention also deals with punfied, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the hGGPPS gene, wherein the polynucleotides are arranged within the nucleic acid, from the 5 " -end to the 3 " -end of said nucleic acid, in the same order as in SEQ ID No 1
  • nucleic acids defining the hGGPS introns descnbed above, as well as their fragments 5 and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the hGGPS m a test sample, or alternatively in order to amplify a target nucleotide sequence within the hGGPS lntronic sequences hGGPS cD As
  • the inventors have discovered that the expression of the hGGPS gene leads to the
  • the first transcnption product comprises Exons 1, 2, 3 and 4.
  • This cDNA of SEQ ID No 2 includes a 5 " -UTR region, spanning the whole Exon 1 and part of Exon 2 This 5'-UTR region starts from the nucleotide at position 1 and ends at the nucleotide in position 84 of SEQ LD No 2.
  • 15 cDNA of SEQ ID No 2 includes a 3'-UTR region starting from the nucleotide at position 988 and ending at the nucleotide at position 1414 of SEQ ID No 2.
  • the 3'UTR carries a potential polyadenylation signal located between the nucleotide m position 1289 and the nucleotide in position 1294 of the nucleic acid of SEQ LD No 2.
  • the ORF encoding hGGPS is comprised between the nucleotide in position 85 and the nucleotide in position 987 of SEQ LD No 2. 0
  • the second transcnption product comprises Exons ibis, 2, 3 and 4. This cDNA of SEQ LD
  • No 3 includes a 5'-UTR region starting from the nucleotide at position 1 and ending at the nucleotide in position 217 of SEQ ID No 3.
  • the cDNA of SEQ ID No 3 includes a 3'-UTR region starting from the nucleotide at position 1121 and ending at the nucleotide at position 1547 of SEQ ID No 3.
  • the 3'UTR carries a potential polyadenylation signal located between the nucleotide in 5 position 1422 and the nucleotide in position 1427 of the nucleic acid of SEQ LD No 3.
  • the ORF encoding hGGPS is comp ⁇ sed between the nucleotide in position 218 and the nucleotide in position 1120 of the nucleotide sequence of SEQ LD No 3.
  • Another object of the invention consists of a punfied or isolated nucleic acid selected from the group consisting of the nucleotide sequences of SEQ ID Nos 2 and 3 or a complementary
  • nucleic acids of the invention include isolated, punfied, or recombinant polynucleotides comp ⁇ smg a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70. 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions
  • Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comp ⁇ smg a contiguous span of at least 12, 15, 18, 20, 25, 30, 35. 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3. 5, or 10 of the nucleotide positions 967- 1351 of SEQ LD No 3
  • the invention also pertains to a purified or isolated nucleic acid having at least 95% of nucleotide identity with any of the nucleotide sequences of SEQ ID Nos 2 and 3 or a complementary sequence thereto
  • a further object of the invention consists m a punfied or isolated nucleic acid of at least 12 nucleotides in length, wherein said nucleic acid hybridizes under stnngent hybndization conditions with a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ LD Nos 2 and 3, or a sequence complementary thereto.
  • Another object of the invention consists in a punfied or isolated nucleic acid comp ⁇ smg a nucleic acid fragment of a nucleotide sequence selected from the group consisting of SEQ LD Nos 2 and 3, wherein this nucleic acid fragment encodes a polypeptide having an ammo acid sequence beginning at the ammo acid in position 200 and ending at the amino acid m position 300 of the hGGPS polypeptide of SEQ ID No 4, or a nucleic acid encoding a peptide fragment thereof.
  • Regulatory sequences a nucleic acid fragment of a nucleotide sequence selected from the group consisting of SEQ LD Nos 2 and 3, wherein this nucleic acid fragment encodes a polypeptide having an ammo acid sequence beginning at the ammo acid in position 200 and ending at the amino acid m position 300 of the hGGPS polypeptide of SEQ ID No 4, or a nucleic acid encoding a peptide fragment thereof.
  • the polynucleotide of SEQ LD No 1 contains regulatory sequences both in the non-coding 5 " -flanking region and in the non-coding 3 '-flanking region that border the hGGPS coding region.
  • the longest 5 '-regulatory sequence of the hGGPS gene is localized between the nucleotide in position 1 and the nucleotide in position 632 of SEQ ID Nol .
  • a shorter 5 '-regulatory sequence of the hGGPS gene is localized between the nucleotide m position 1 and the nucleotide in position 485 of SEQ LD Nol .
  • the hGGPS 3 '-regulatory region as shown in Figure 1, comprises a nucleotide sequence starting from the nucleotide in position 15252 of SEQ LD No 1 and ending at the nucleotide in position 17131 of SEQ ID No 1
  • Polynucleotides denved from the hGGPS regulatory regions described above are useful m order to detect the presence of at least a copy of the nucleotide sequence of SEQ ID No 1 in a test sample.
  • the promoter activity of the regulatory regions contained m the h GGPS nucleotide sequence of SEQ LD No 1 can be assessed as descnbed below.
  • Genomic sequences located upstream of the hGGPS gene are cloned into a suitable promoter reporter vector, such as the pSEAP -Basic, pSEAP-Enhancer, p ⁇ gal-Basic, p ⁇ gal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech.
  • a suitable promoter reporter vector such as the pSEAP -Basic, pSEAP-Enhancer, p ⁇ gal-Basic, p ⁇ gal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech.
  • each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, beta galactosidase, or green fluorescent protein.
  • the sequences upstream the hGGPS coding region are inserted into the cloning sites upstream of the reporter gene m both onentations and introduced into an appropnate host cell
  • the level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site.
  • the presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert
  • the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcnption levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present m the inserted upstream sequence.
  • Promoter sequences within the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as Exonuclease III digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundanes of the promoters may be defined If desired, potential individual regulatory sites withm the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcnption factor binding sites within the promoter individually or m combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors
  • polynucleotides carrying the regulatory elements located both at the 5' end and at the 3' end of the hGGPS coding region may be advantageously used to control the transcnptional and translational activity of an heterologous polynucleotide of interest.
  • the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof
  • 5' regulatory region refers to the nucleotide sequence located between positions 1 and 632 of SEQ ID No 1.
  • 3' regulatory region refers to the nucleotide sequence located between positions 15252 and 17131 of SEQ LD No l
  • the present invention is also directed to a polynucleotide comp ⁇ smg a functional portion of a regulatory region contained m the contemplated hGGPS gene and to its use in a recombinant expression vector carrying a polynucleotide encoding a polypeptide or a nucleic acid of interest.
  • Preferred fragments of the 5' regulatory region have a length of about 400 nucleotides, more particularly about 300 nucleotides, more preferably 200 nucleotides and most preferably about 100 nucleotides
  • Preferred fragments of the 3' regulatory region have a length of about 600 nucleotides, more particularly about 300 nucleotides, more preferably 200 nucleotides and most preferably about 100 nucleotides In order, to identify the relevant biologically active polynucleotide derivatives of the 5' and
  • the regulatory polynucleotides of the invention may be prepared from a polynucleotide of the nucleotide sequence SEQ ID No 1 by cleavage using suitable restriction enzymes, as desc ⁇ bed for example m the book of Sambrook et al. (1989).
  • the regulatory polynucleotides may also be prepared by digestion of a polynucleotide of the nucleotide sequence SEQ LD No 1 by an exonuclease enzyme, such as for example Bal31 (Wabiko et al., 1986).
  • These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification.
  • the regulatory polynucleotides according to the invention may be advantageously part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism.
  • the recombinant expression vectors according to the invention are described elsewhere in the specification.
  • a preferred 5'-regulatory polynucleotide of the invention includes the 5 '-untranslated region (5'-UTR) located between the nucleotide at position 1 and the nucleotide at position 84 of SEQ ID No 2, or a biologically active fragment or vanant thereof.
  • Another preferred 5'-regulatory polynucleotide of the invention includes the 5 '-untranslated region (5'-UTR) located between the nucleotide at position 1 and the nucleotide at position 217 of SEQ D No 3, or a biologically active fragment or vanant thereof.
  • a preferred 3 '-regulatory polynucleotide of the invention includes the 3 '-untranslated region
  • (3'-UTR) consisting in the nucleotide sequence starting from the nucleotide in position 988 and ending a the nucleotide in position 1414 of the nucleic acid of SEQ LD No 2.
  • a further object of the invention consists of a purified or isolated nucleic acid comprising : a) a nucleic acid comprising the 5' regulatory region or a biologically active fragment or vanant thereof; b) a polynucleotide encoding a desired polypeptide or nucleic acid operably linked to the 5' regulatory region or its biologically active fragment or vanant thereof; c) optionally, a nucleic acid comprising the 3' regulatory region or a biologically active fragment or vanant thereof.
  • the desired polypeptide encoded by the above descnbed nucleic acid may be of vanous nature or ongm, encompassing proteins of prokaryotic or eukaryotic ongin.
  • polypeptides expressed under the control of a hGGPS regulatory region there may be cited bactenal, fungal or viral antigens.
  • eukaryotic proteins such as intracellular proteins, like "house keeping” proteins, membrane-bound proteins, like receptors, and secreted proteins like the numerous endogenous mediators such as cytokines.
  • the desired nucleic acids encoded by the above described polynucleotide usually a RNA molecule, may be complementary to a desired coding polynucleotide, for example to the hGGPS coding sequence, and thus useful as an antisense polynucleotide
  • Such a polynucleotide may be included a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism.
  • Suitable recombinant vectors that contain a polynucleotide such as desc ⁇ bed hereinbefore are disclosed elsewhere in the specification.
  • the hGGPS open reading frame is contained in the corresponding mRNAs of SEQ ID Nos 2 and 3.
  • the effective hGGPS coding sequence (CDS) is comp ⁇ sed between the nucleotide at position 85 (first nucleotide of the ATG codon) and the nucleotide at position 987 (end nucleotide of the TAA codon) of SEQ LD No 2.
  • a purified or isolated polynucleotide comprising the hGGPS coding region defined above is another object of the invention.
  • the above disclosed polynucleotide that contains the coding sequence of the hGGPS gene of the invention may be expressed m a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals.
  • the expression signals may be either the expression signals contained m the regulatory regions in the hGGPS gene of the invention or in contrast be exogenous regulatory nucleic sequences.
  • Such a polynucleotide, when placed under the suitable expression signals, may also be inserted m a vector for its expression
  • the inventors have discovered nucleotide polymo ⁇ hisms located withm the genomic DNA containing the hGGPS gene, and among them SNP that are also termed biallehc markers.
  • the biallehc markers of the invention can be used for example for the generation of genetic map, the linkage analysis, the association studies.
  • biallehc markers of the present invention there are two preferred methods through which the biallehc markers of the present invention can be generated.
  • a first method DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymo ⁇ hisms.
  • the genomic DNA samples from which the biallehc markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background
  • the number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually preferred to collect DNA samples from at least about 100 individuals order to have sufficient polymo ⁇ hic diversity in a given population to identify as many markers as possible and to generate statistically significant results.
  • any test sample can be foreseen without any particular limitation.
  • test samples include biological samples which can be tested by the methods of the present invention descnbed herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, unne, lymph fluids, and various external secretions of the respiratory, intestinal and genitou ⁇ nary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens.
  • the preferred source of genomic DNA used in the context of the present invention is from pe ⁇ pheral venous blood of each donor.
  • DNA samples can be pooled or unpooled for the amplification step.
  • DNA amplification techniques are well known to those skilled in the art.
  • Amplification techniques that can be used in the context of the present invention include, but are not limited to, the hgase chain reaction (LCR) described in EP-A- 320 308, WO 9320227 and
  • EP-A-439 182 the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) descnbed in Guatelli J.C., et al.(1990) and m Compton J.(l 991 ), Q-beta amplification as descnbed in European Patent Application No 4544610, strand displacement amplification as descnbed in Walker et al.(1996) and EP A 684 315 and, target mediated amplification as desc ⁇ bed m PCT Publication WO 9322461.
  • NASBA nucleic acid sequence based amplification
  • LCR and Gap LCR are exponential amplification techniques, both depend on DNA hgase to join adjacent primers annealed to a DNA molecule
  • probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target
  • the first probe hybridizes to a first segment of the target strand and the second probe hybndizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate- 3 'hydroxyl relationship, and so that a ligase can covalently fuse or hgate the two probes into a fused product.
  • a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybndize to a portion of the second probe m a similar abutting fashion.
  • the secondary probes also will hybridize to the target complement in the first instance Once the hgated strand of pnmary probes is separated from the target strand, it will hybndize with the third and fourth probes, which can be hgated to form a complementary, secondary hgated product. It is important to realize that the hgated products are functionally equivalent to either the target or its complement.
  • Gap LCR is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.
  • RT-PCR polymerase chain reaction
  • AGLCR is a modification of GLCR that allows the amplification of RNA.
  • PCR technology is the preferred amplification technique used m the present invention
  • a vanety of PCR techniques are familiar to those skilled in the art.
  • PCR pnmers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.
  • the nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample.
  • telomeres The hybndized p ⁇ mers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the pnmer sites.
  • PCR has further been descnbed in several patents including US Patents 4,683,195, 4,683,202; and 4,965,188 The PCR technology is the preferred amplification technique used to identify new biallehc markers.
  • Example 3 A typical example of a PCR reaction suitable for the pu ⁇ oses of the present invention is provided m Example 3
  • One of the aspects of the present invention is a method for the amplification of the human hGGPPS gene, particularly of a fragment of the genomic sequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID No 2 or 3, or a fragment or a variant thereof in a test sample, preferably using the PCR technology
  • This method comprises the steps of a) contacting a test sample with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side of the polynucleotide region to be amplified, and b) optionally, detecting the amplification products.
  • the invention also concerns a kit for the amplification of a hGGPPS gene sequence, particularly of a portion of the genomic sequence of SEQ LD No 1 or of the cDNA sequence of SEQ ID No 2 or 3, or a variant thereof in a test sample, wherein said kit compnses: a) a pair of oligonucleotide pnmers located on either side of the hGGPPS region to be amplified; b) optionally, the reagents necessary for performing the amplification reaction.
  • the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region.
  • pnmers comprise a sequence which is selected from the group consisting of SEQ ID Nos 7-9.
  • bialle c markers are identified using genomic sequence information generated by the inventors. Sequenced genomic DNA fragments are used to design pnmers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallehc markers. Pnmers may be designed using the OSP software (Hilher L. and Green P., 1991). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing pnmer. Those skilled in the art are familiar with pnmer extensions, which can be used for these pu ⁇ oses.
  • Preferred primers useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes.
  • a biallehc marker presents a higher probability to be an eventual causal mutation if it is located in these functional regions of the gene.
  • Preferred amplification pnmers of the invention include the nucleotide sequences of SEQ LD Nos 8 and 9
  • pnmers allow the amplification of va ⁇ ous fragments of the punfied or isolated nucleic acid of SEQ LD No 1. These pnmers are presented below as couples of forward and reverse pnmers that may be used together to amplify a desired nucleotide sequence.
  • the pnmers descnbed above are individually useful as oligonucleotide probes in order to detect the corresponding hGGPS nucleotide sequence in a sample, and more preferably to detect the presence of a hGGPS DNA molecule in a sample suspected to contain it 3 Sequencing of amplified genomic DNA and identification of polymo ⁇ hisms
  • the amplification products generated as desc ⁇ bed above, are then sequenced using any method known and available to the skilled technician.
  • Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Sambrook et al.(1989).
  • Alternative approaches include hybridization to high-density DNA probe arrays as desc ⁇ bed in Chee et al.(1996).
  • the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-pnmer cycle sequencing protocol.
  • the products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis.
  • the polymo ⁇ hism search is based on the presence of supenmposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallehc site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise.
  • the two DNA strands are sequenced and a compa ⁇ son between the peaks is earned out.
  • the polymo ⁇ hism has to be detected on both strands.
  • the above procedure permits those amplification products, which contain biallelic markers to be identified.
  • the detection limit for the frequency of biallehc polymo ⁇ hisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known alle c frequencies.
  • more than 90% of the biallehc polymo ⁇ hisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallehc markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele.
  • At least 0.2 for the minor allele and less than 0.8 for the major allele Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.
  • biallehc markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallehc marker may be less than 0.1.
  • the test samples are a pool of 100 individuals and 50 individual samples. This is the methodology used in the preferred embodiment of the present invention, in which 1 biallehc marker has been identified in a genomic region containing the hGGPS gene This bialle c marker is called 5-187-77 and is located in intron 3 of hGGPPS gene The biallehc marker consists in an insertion of a nucleotide T
  • the polymo ⁇ hisms identified above can be further confirmed and their respective frequencies can be determined through various methods using the previously descnbed primers and probes as desc ⁇ bed herein. These methods can also be useful for genotypmg either new populations in association studies or linkage analysis or individuals in the context of detection of alleles of biallehc markers which are known to be associated with a given trait. The genotypmg of the biallehc markers is also important for the mapping. It will be appreciated that the methods described below can be equally performed on individual or pooled DNA samples. b) Genotyping Of Biallelic Markers
  • biallehc markers desc ⁇ bed previously allows the design of approp ⁇ ate oligonucleotides, which can be used as probes and p ⁇ mers, to amplify a hGGPS gene containing the polymo ⁇ hic site of interest and for the detection of such polymo ⁇ hisms.
  • the invention encompasses methods of genotyping comprising determining the identity of a nucleotide at a ⁇ GGP S-related biallelic marker or the complement thereof in a biological sample; optionally, wherein said hGGPPS -related biallehc marker is the biallehc marker 5-187-77, and the complement thereof; optionally, wherein said biological sample is derived from a single subject; optionally, wherein the identity of the nucleotides at said bialle c marker is determined for both copies of said biallehc marker present in said individual's genome, optionally, wherein said biological sample is de ⁇ ved from multiple subjects;
  • the genotyping methods of the invention encompass methods with any further limitation desc ⁇ bed in this disclosure, or those following, specified alone or in any combination;
  • said method is performed in vitro; optionally, further comprising amplifying a portion of said sequence comprising the biallehc marker pnor to said determining step;
  • said amplifying is performed by PCR
  • Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallehc marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallehc markers may be used in va ⁇ ous methods and for vanous pu ⁇ oses and is not rest ⁇ cted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention. Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above in the section entitled, "DNA amplification.”
  • Some of these amplification methods are particularly suited for the detection of single nucleotide polymo ⁇ hisms and allow the simultaneous amplification of a target sequence and the identification of the polymo ⁇ hic nucleotide as it is further described below.
  • the identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention.
  • Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention.
  • the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention.
  • Preferred amplification primers are listed in Example 3. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention are also of use.
  • the spacing of the primers determines the length of the segment to be amplified.
  • amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred.
  • amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers.
  • Amplification primers may be labeled or immobilized on a solid support as described in "Oligonucleotide probes and primers".
  • the nucleotide present at a polymo ⁇ hic site can be determined by sequencing methods.
  • DNA samples are subjected to PCR amplification before sequencing as described above.
  • DNA sequencing methods are described in "Sequencing Of Amplified Genomic DNA And Identification Of Polymo ⁇ hisms".
  • the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site. 3) Microsequencing
  • the nucleotide at a polymo ⁇ hic site in a target DNA is detected by a single nucleotide primer extension reaction
  • This method involves appropnate microsequencing primers which, hybndize just upstream of the polymo ⁇ hic base of interest m the target nucleic acid.
  • a polymerase is used to specifically extend the 3 ' end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymo ⁇ hic site.
  • the identity of the inco ⁇ orated nucleotide is determined m any suitable way.
  • microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the inco ⁇ orated nucleotide as descnbed in EP 412 883, the disclosure of which is inco ⁇ orated herein by reference m its entirety.
  • capillary electrophoresis can be used in order to process a higher number of assays simultaneously.
  • An example of a typical microsequencing procedure that can be used m the context of the present invention is provided in Example 5. Different approaches can be used for the labeling and detection of ddNTPs.
  • a homogeneous phase detection method based on fluorescence resonance energy transfer has been desc ⁇ bed by Chen and Kwok (1997) and Chen et al.(1997).
  • the extended p ⁇ mer may be analyzed by MALDI-TOF Mass Spectrometry.
  • the base at the polymo ⁇ hic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, 1997).
  • Microsequencing may be achieved by the established microsequencing method or by developments or de ⁇ vatives thereof.
  • Alternative methods include several solid-phase microsequencing techniques.
  • the basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the p ⁇ mer or the target molecule is immobilized or captured onto a solid support.
  • immobilization can be earned out via an interaction between biotmylated DNA and streptavidm-coated microtitration wells or avidm-coated polystyrene particles.
  • oligonucleotides or templates may be attached to a solid support in a high-density format.
  • inco ⁇ orated ddNTPs can be radiolabeled (Syvanen, 1994) or linked to fluorescem (Livak and Hainer, 1994).
  • the detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques.
  • the detection of fluorescem-hnked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromoge c substrate (such as ⁇ -nitrophenyl phosphate).
  • reporter- detection pairs include: ddNTP linked to dmitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotmylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712).
  • DNP dmitrophenyl
  • biotmylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate
  • ELIDA enzymatic luminomet ⁇ c inorganic pyrophosphate detection assay
  • the present invention provides polynucleotides and methods to genotype one or more biallehc markers of the present invention by performing a microsequencing assay.
  • Preferred microsequencing p ⁇ mers include the nucleotide sequence of SEQ ID No 7. It will be appreciated that the microsequencing primer of SEQ ID No 7 is merely exemplary and that, any p ⁇ mer having a 3' end immediately adjacent to the polymo ⁇ hic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallehc marker or any combination of biallehc markers of the present invention.
  • One aspect of the present invention is a solid support which includes one or more microsequencing p ⁇ mers for determining the identity of a nucleotide at a biallehc marker site.
  • the present invention provides polynucleotides and methods to determine the allele of one or more biallehc markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polyme ⁇ zation reactions places particularly stnngent requirements on correct base pairing of the 3' end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the hgation site, especially at the 3' end. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in "DNA amplification".
  • Discnmination between the two alleles of a biallehc marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele.
  • allele specific amplification at least one member of the pair of p ⁇ mers is sufficiently complementary with a region of a hGGPPS gene comprising the polymo ⁇ hic base of a bialle c marker of the present invention to hybndize therewith and to initiate the amplification
  • Such primers are able to disc ⁇ minate between the two alleles of a biallehc marker.
  • Oligonucleotide Ligation Assay uses two oligonucleotides which are designed to be capable of hybndizing to abutting sequences of a single strand of a target molecules.
  • One of the oligonucleotides is biotmylated, and the other is detectably labeled If the precise complementary sequence is found m a target molecule, the oligonucleotides will hybndize such that their termini abut, and create a hgation substrate that can be captured and detected.
  • OLA is capable of detecting single nucleotide polymo ⁇ hisms and may be advantageously combined with PCR as described by Nickerson et al ( 1990) In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
  • LCR ligase chain reaction
  • GLCR Gap LCR
  • LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase.
  • LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallehc marker site.
  • either oligonucleotide will be designed to include the biallehc marker site.
  • the reaction conditions are selected such that the oligonucleotides can be hgated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallehc marker on the oligonucleotide.
  • the oligonucleotides will not include the biallehc marker, such that when they hybridize to the target molecule, a "gap" is created as described in WO 90/01069. This gap is then "filled" with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides.
  • each single strand has a complement capable of serving as a target dunng the next cycle and exponential allele-specific amplification of the desired sequence is obtained.
  • Ligase/Polymerase-mediated Genetic Bit AnalysisTM is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271). This method involves the co ⁇ oration of a nucleoside t ⁇ phosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a pnmer molecule, and their subsequent ligation to a second oligonucleotide The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution. 5.
  • a preferred method of determining the identity of the nucleotide present at a biallehc marker site involves nucleic acid hybridization.
  • the hybndization probes which can be conveniently used in such reactions, preferably include the probes defined herein Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybndization and solid- phase hybridization (see Sambrook et al , 1989)
  • Specific probes can be designed that hybridize to one form of a biallehc marker and not to the other and therefore are able to discnmmate between different allehc forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybndization conditions should be sufficiently stnngent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
  • Stnngent, sequence specific hybndization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989). Although such hybridization can be performed in solution, it is preferred to employ a solid-phase hybndization assay.
  • the target DNA comprising a biallehc marker of the present invention may be amplified p ⁇ or to the hybridization reaction.
  • the presence of a specific allele m the sample is determined by detecting the presence or the absence of stable hyb ⁇ d duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be earned out by a number of methods.
  • Vanous detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybnd duplexes.
  • hybndization duplexes are separated from unhybndized nucleic acids and the labels bound to the duplexes are then detected.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hyb ⁇ ds using the labels present on the pnmers and probes.
  • the TaqMan assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product.
  • TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase dunng amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.
  • molecular beacons are used for allele discnmmations.
  • Molecular beacons are hai ⁇ in-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., 1998).
  • the polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of bialle c marker alleles in biological samples
  • These probes are characte ⁇ zed in that they preferably comprise between 8 and 50 nucleotides. and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide vanation.
  • a particularly preferred probe is 25 nucleotides m length.
  • the biallehc marker is within 4 nucleotides of the center of the polynucleotide probe.
  • the biallehc marker is at the center of said polynucleotide.
  • Preferred probes comprise a nucleotide sequence selected from the group consisting of amphcons listed in Table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comp ⁇ sing at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymo ⁇ hic base.
  • Preferred probes comprise a nucleotide sequence selected from the group consisting of SEQ ID Nos 5 and 6 and the sequences complementary thereto.
  • the polymo ⁇ hic base(s) are withm 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.
  • the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in “Oligonucleotide Probes and Primers”.
  • the probes can be non-extendable as described in “Oligonucleotide Probes and Primers”.
  • Hybridization assays based on oligonucleotide arrays rely on the differences in hybndization stability of short oligonucleotides to perfectly matched and mismatched target sequence vanants. Efficient access to polymo ⁇ hism information is obtained through a basic structure comp ⁇ sing high- density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions.
  • a solid support e.g., the chip
  • Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged m a grid-like pattern and mmiatunzed to the size of a dime. The chip technology has already been applied with success in numerous cases.
  • Chips of vanous formats for use m detecting biallehc polymo ⁇ hisms can be produced on a customized basis by Affymetnx (GeneChipTM), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.
  • arrays employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymo ⁇ hic marker EP 785280 descnbes a tiling strategy for the detection of single nucleotide polymo ⁇ hisms.
  • arrays may generally be "tiled” for a large number of specific polymorphisms.
  • tilting * is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of nucleotides.
  • the chips may comp ⁇ se an array of nucleic acid sequences of fragments of about 15 nucleotides in length.
  • the chip may compnse an array including at least one of the sequences selected from the group consisting of amphcons listed in table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymo ⁇ hic base.
  • the polymo ⁇ hic base is withm 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.
  • the chip may compnse an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention.
  • Solid supports and polynucleotides of the present invention attached to solid supports are further desc ⁇ bed in "Oligonucleotide Probes And Primers". 7- Integrated Systems
  • Another technique which may be used to analyze polymo ⁇ hisms, includes multicomponent integrated systems, which mmiatunze and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device.
  • An example of such technique is disclosed in US patent 5,589,136, which descnbes the integration of PCR amplification and capillary electrophoresis m chips.
  • Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comp ⁇ se a pattern of microchannel s designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by elect ⁇ c, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.
  • the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser- induced fluorescence detection.
  • Oligonucleotide Probes and primers Polynucleotides de ⁇ ved from the hGGPPS gene are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No 1, or a fragment, complement, or vanant thereof m a test sample Furthermore polynucleotides denved from the hGGPPS gene can be used to generate antisense polynucleotide or polynucleotide for the triple helix strategy
  • probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ LD No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ LD No l : 1-485, 547-632, 827-7291 , 7385-13759, 13831-14062, 14671-15054, and 15252-17131.
  • the invention also relates to nucleic acid probes characte ⁇ zed m that they hybridize specifically, under the stnngent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences 1-485, 547-632, 827-7291, 7385-13759, 13831-14062, 14671-15054, and 15252-17131 of SEQ LD No 1 or a variant thereof or a sequence complementary thereto.
  • probes and primers of the invention include isolated, punfied, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ LD No 2 or the complements thereof, wherein said contiguous span compnses at least 1, 2, 3, 5, or 10 of the nucleotide positions 834-1217 of SEQ ID No 2.
  • Additional preferred probes and p ⁇ mers of the invention include isolated, punfied, or recombinant polynucleotides compnsmg a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ LD No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 967-1351 of SEQ LD No 3.
  • the invention also relates to nucleic acid probes characte ⁇ zed m that they hybridize specifically, under the stnngent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences 834-1217 of SEQ LD No 2 and 967-1351 of SEQ LD No 3, or a variant thereof or a sequence complementary thereto.
  • the invention encompasses isolated, punfied, and recombinant polynucleotides consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ LD Nos 1 -3 and the complement thereof, wherein said span includes a hGGPPS- related biallehc marker in said sequence; optionally, wherein said A G S-related bialle c marker is the biallehc marker 5-187-77, and the complement thereof; optionally, wherein said contiguous span is 18 to 50 nucleotides in length and said biallehc marker is withm 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallehc marker is at the center of said polynucleotide; optionally, wherein the 3' end of said contiguous span is present at the 3' end of said
  • the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 5 nucleotides of SEQ ID Nos 1 -3, or the complements thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located within 20 nucleotides upstream of a hGGPPS -related biallehc marker in said sequence; optionally, wherein said /zGG PS-related biallehc marker is the biallehc marker 5-187-77, and the complement thereof; optionally, wherein the 3' end of said polynucleotide is located 1 nucleotide
  • polynucleotide consists essentially of a sequence of SEQ LD No 7.
  • the invention encompasses isolated, purified, or recombinant polynucleotides comp ⁇ sing, consisting of, or consisting essentially of a sequence selected from the sequences of SEQ LD Nos 8and 9
  • the invention encompasses polynucleotides for use m hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a hGGPPS -related biallehc marker, as well as polynucleotides for use in amplifying segments of nucleotides comprising a hGGPPS -related biallelic marker; optionally, wherein said hGGPPS-related biallehc marker is the biallelic marker 5-
  • a probe or a p ⁇ mer according to the invention has between 8 and 1000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length. More particularly, the length of these probes and pnmers can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30
  • a preferred probe or pnmer consists of a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of SEQ ED Nos 5-9 or a fragment thereof or a complementary sequence thereto.
  • the GC content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %.
  • the pnmers and probes can be prepared by any suitable method, including, for example, cloning and rest ⁇ ction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al (1981) and the solid support method desc ⁇ bed in EP 0 707 592
  • Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed m International Patent Application WO 92/20702, mo ⁇ holmo analogs which are desc ⁇ bed in U.S Patents Numbered 5,185,444. 5,034,506 and 5,142,047
  • the probe may have to be rendered "'non-extendable *" in that additional dNTPs cannot be added to the probe.
  • analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation.
  • the 3' end of the probe can be functionahzed with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
  • the 3' hydroxyl group simply can be cleaved, replaced or modified, U.S. Patent Application Se ⁇ al No. 07/049,061 filed Apnl 19, 1993 describes modifications, which can be used to render a probe non-extendable.
  • any of the polynucleotides of the present invention can be labeled, if desired, by inco ⁇ orating any label known in the art to be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive substances (including, 32 P, 35 S, 3 H, 125 I), fluorescent dyes (including, 5-bromodesoxyu ⁇ dm, fluorescem, acetylammofluorene, digoxigenm) or biotin.
  • polynucleotides are labeled at their 3' and 5' ends. Examples of non-radioactive labeling of nucleic acid fragments are described m the French patent No.
  • the probes according to the present invention may have structural characte ⁇ stics such that they allow the signal amplification, such structural charactenstics being, for example, branched DNA probes as those descnbed by Urdea et al. in 1991 or in the European patent No. EP 0 225 807 (Chiron).
  • a label can also be used to capture the pnmer, so as to facilitate the immobilization of either the primer or a pnmer extension product, such as amplified DNA, on a solid support.
  • a capture label is attached to the pnmers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label earned by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, pnmers or probes provided herein, may, themselves, serve as the capture label.
  • a solid phase reagent's binding member is a nucleic acid sequence
  • it may be selected such that it binds a complementary portion of a pnmer or probe to thereby immobilize the p ⁇ mer or probe to the solid phase.
  • a polynucleotide probe itself serves as the binding member
  • the probe will contain a sequence or "tail" that is not complementary to the target.
  • a polynucleotide p ⁇ mer itself serves as the capture label, at least a portion of the pnmer will be free to hybridize with a nucleic acid on a solid phase.
  • DNA Labeling techniques are well known to the skilled technician
  • the probes of the present invention are useful for a number of pu ⁇ oses They can be notably used in Southern hybndization to genomic DNA.
  • the probes can also be used to detect PCR amplification products They may also be used to detect mismatches in the hGGPPS gene or mRNA using other techniques.
  • any of the polynucleotides, p ⁇ mers and probes of the present invention can be conveniently immobilized on a solid support.
  • Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose st ⁇ ps, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
  • the solid support is not cntical and can be selected by one skilled m the art.
  • Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like.
  • a solid support refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or dunng the performance of the assay.
  • the solid phase thus can be a plastic, denvatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill m the art.
  • the polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support.
  • polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention
  • the invention also deals with a method for detecting the presence of a nucleic acid comprising at least a part of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1-3 in a sample, said method comp ⁇ sing the following steps of : a) b ⁇ ngmg into contact a nucleic acid probe or a plurality of nucleic acid probes, which can hybndize to a nucleotide sequence included in one of the nucleic acids of SEQ ED Nos 1-3, and the sample to be assayed b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample
  • the nucleic acid probe is selected from the group of polynucleotides consisting of the nucleotide sequences SEQ ID Nos 5-9.
  • said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule
  • said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.
  • the invention further concerns a kit for detecting the presence of a nucleic acid comprising at least a part of a nucleotide sequence selected from the group consisting of SEQ ED Nos 1-3 in a sample, said kit comprising .
  • nucleic acid probe or a plurality of nucleic acid probes which can hybndize to a nucleotide sequence included m one of the nucleic acids of SEQ ED Nos 1-3; b) optionally, the reagents necessary for performing the hybridization reaction.
  • the nucleic acid probe or the plurality of nucleic acid probes that are included m the detection kit desc ⁇ bed above may be selected from the group consisting of SEQ ED Nos 5-9.
  • the nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule.
  • the nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.
  • a substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in the hGGPPS gene and may also be used for detecting mutations m the coding or in the non-coding sequences of the hGGPPS gene
  • Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support.
  • the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide.
  • such an ordered array of polynucleotides is designed to be "addressable” where the distinct locations are recorded and can be accessed as part of an assay procedure.
  • Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these "addressable" arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention.
  • VLSIPSTM technologies are provided in US Patents 5.143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, which descnbe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques.
  • further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybndization patterns and sequence information Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.
  • an oligonucleotide probe mat ⁇ x may advantageously be used to detect mutations occurnng m the hGGPPS gene and preferably in its regulatory region.
  • probes are specifically designed to have a nucleotide sequence allowing their hybndization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides).
  • known mutations it is meant, mutations on the hGGPPS gene that have been identified according, for example to the technique used by Huang et al.(1996) or Samson et al.(1996).
  • a high- density DNA array Another technique that is used to detect mutations in the hGGPPS gene is the use of a high- density DNA array.
  • Each oligonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of the hGGPPS genomic DNA or cDNA.
  • an array consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the hGGPPS gene.
  • 4L tiled array is implemented a set of four probes (A, C, G, T), preferably 15-nucleot ⁇ de ohgomers.
  • A, C, G, T the perfect complement will hybndize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence.
  • the hybndization signals of the 15- mer probe set tiled array are perturbed by a single base change m the target sequence. As a consequence, there is a characteristic loss of signal or a "footprint" for the probes flanking a mutation position.
  • the invention concerns an array of nucleic acid molecules comp ⁇ sing at least one polynucleotide desc ⁇ bed above as probes and pnmers.
  • the invention concerns an array of nucleic acid comp ⁇ sing at least two polynucleotides desc ⁇ bed above as probes and pnmers.
  • a further object of the invention consists of an array of nucleic acid sequences compnsmg either at least one of the sequences selected from the group consisting of SEQ ED Nos 5-9, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence comprising the biallehc marker 5-187-77 and the complements thereto
  • the invention also pertains to an array of nucleic acid sequences compnsmg either at least two of the sequences selected from the group consisting of SEQ ED Nos 5-9, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence compnsmg the biallehc marker 5-187-77 and the complements thereto.
  • Vectors for the expression of a regulatory or a coding polynucleotide according to the invention are provided.
  • any of the regulatory polynucleotides or the coding polynucleotides of the invention may be inserted into recombinant vectors for expression in a recombinant host cell or a recombinant host organism.
  • the present invention also encompasses a family of recombinant vectors that contains either a regulatory polynucleotide selected from the group consisting of the regulatory polynucleotides derived from the hGGPS gene, or a polynucleotide comprising the hGGPS coding sequence, or both. More particularly, the present invention relates to expression vectors which include nucleic acids encoding the hGGPS protein of the ammo acid sequence of SEQ ID No 4 descnbed therein under the control of either one regulatory sequence selected among the hGGPS regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence.
  • a recombinant expression vector compnsmg a nucleic acid selected from the group consisting of the 5' or 3' regulatory regions of hGGPPS, or biologically active fragments or vanants thereof, is also part of the present invention.
  • a recombinant vector of the invention may compnse any of the polynucleotides descnbed herein, including regulatory sequences, and coding sequences, as well as any hGGPPS primer or probe as defined above. More particularly, the recombinant vectors of the present invention can comprise any of the polynucleotides described in the "hGGPPS cDNA Sequences" section, the “Coding Regions” section, “Genomic sequences” section and the "Oligonucleotide Probes And Primers" section
  • a recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid. a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non- chromosomal and synthetic DNA.
  • a recombinant vector can comprise a transcnptional unit comprising an assembly of :
  • Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription.
  • a structural or coding sequence which is transc ⁇ bed into mRNA and eventually translated into a polypeptide
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein may include an N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • recombinant expression vectors will include ongms of replication, selectable markers permitting transformation of the host cell, and a promoter de ⁇ ved from a highly expressed gene to direct transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in approp ⁇ ate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of translated protein into the pe ⁇ plasmic space or extracellular medium.
  • the selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycm resistance for eukaryotic cell culture, TRPl for S cerevisiae or tetracychne, ⁇ fampicin or ampicillin resistance in E. coli, or levan saccharase for mycobactena.
  • useful expression vectors for bacterial use can comprise a selectable marker and bactenal ongin of replication denved from commercially available plasmids compnsmg genetic elements of pBR322 (ATCC 37017).
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEMl (Promega Biotec, Madison, WI,
  • bactenal vectors pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescnpt, ps ⁇ X174, pbluescnpt SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pREE5 (Pharmacia); or eukaryotic vectors : pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia), baculovirus transfer vector pVL1392/1393 (Pharmingen);
  • a suitable vector for the expression of the hGGPS polypeptide of SEQ ID No 4 is a baculovirus vector that can be propagated in insect cells and in insect cell lines.
  • a specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 171 1 ) which is derived from Spodoptera frugiperda.
  • Other suitable vectors for the expression of the hGGPS polypeptide of SEQ ID No 4 in a baculovirus expression system include those desc ⁇ bed by Chai et al. (1993), Vlasak et al. (1983) and Lenhard et al. (1996).
  • Mammalian expression vectors will comp ⁇ se an ong of replication, a suitable promoter and enhancer, and also any necessary ⁇ bosome binding sites, polyadenylation signal, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscnbed sequences.
  • DNA sequences derived from the SV40 viral genome for example SV40 ongm, early promoter, enhancer, splice and polyadenylation signals may be used to provide the required nontranscnbed genetic elements.
  • b) Promoters The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host m which the heterologous gene has to be expressed.
  • a suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
  • Preferred bactenal promoters are the Lad, LacZ, the T3 or T7 bactenophage RNA polymerase promoters, the polyhed ⁇ n promoter, or the pi 0 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda P R promoter or also the trc promoter.
  • Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors.
  • Particularly preferred bactenal promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidme kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.
  • the vector containing the appropriate DNA sequence as descnbed above more preferably a hGGPS gene regulatory polynucleotide, a polynucleotide encoding the hGGPS polypeptide of SEQ ID No 4 or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.
  • a hGGPS gene regulatory polynucleotide preferably a polynucleotide encoding the hGGPS polypeptide of SEQ ID No 4 or both of them.
  • hGGPS polypeptide of SEQ ID No 4 may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive hGGPS protein
  • the present invention also deals with recombinant expression vectors mamly designed for the in vivo production of the hGGPS polypeptide of SEQ ED No 4 by the introduction of the appropriate genetic matenal in the organism of the patient to be treated.
  • This genetic material may be introduced in vitro m a cell that has been previously extracted from the organism, the modified cell being subsequently remtroduced in the said organism, directly in vivo into the appropriate tissue, and preferably m the olfactory epithelium
  • a method for delivering a protein or peptide to the te ⁇ or of a cell of a vertebrate in vivo comprises the step of introducing a preparation compnsmg a physiologically acceptable earner and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the mte ⁇ or of the cell and has a physiological effect.
  • the invention provides a composition for the in vivo production of the hGGPS protein or polypeptide described herein.
  • compositions comprising a polynucleotide are described in the PCT application N° WO 90/11092 (Vical Inc.) and also in the PCT application N° WO 95/1 1307 (Instirut Pasteur, INSERM, Universite d'Ottawa) as well as in the articles of Tacson et al. (1996) and of Huygen et al. (1996).
  • the amount of the vector to be injected to the desired host organism vary according to the site of injection.
  • the vector will be injected between 0,1 and 100 ⁇ g of the vector in an animal body, preferably a mammal body, for example a mouse body.
  • it may be introduced m vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.
  • the cell that has been transformed with the vector coding for the desired hGGPS polypeptide or the desired C-termmal fragment thereof is remtroduced into the animal body in order to deliver the recombinant protein withm the body either locally or systemically.
  • the vector is de ⁇ ved from an adenovirus.
  • adenovirus vectors according to the invention are those descnbed by Feldman and Steg (1996) or Ohno et al (1994)
  • Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin ( French patent application N° FR-93.05954)
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous polynucleotides in vivo , particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host
  • retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mmk-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendothehosis virus and Rous Sarcoma virus.
  • retroviruses selected from the group consisting of Mmk-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendothehosis virus and Rous Sarcoma virus.
  • Particularly preferred Munne Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Fnend (ATCC No VR-245), Gross (ATCC No VR- 590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298).
  • Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).
  • Other preferred retroviral vectors are those described m Roth et al. (1996), the PCT Application No WO 93/25234, the PCT Application No WO 94/ 06920, Roux et al., 1989, an et al., 1992 and Neda et al., 1991.
  • the adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a he ⁇ es virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividmg cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989).
  • compositions containing a vector of the invention advantageously comprise an oligonucleotide fragment of a nucleic sequence selected from the group consisting of SEQ LD Nos 2 or 3 as an antisense tool that inhibits the expression of the corresponding hGGPS gene.
  • Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al.
  • the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5'end of the hGGPS mRNAs.
  • a combination of different antisense polynucleotides complementary to different parts of the desired targeted gene are used.
  • Preferred antisense polynucleotides according to the present invention are complementary to a sequence of the mRNAs of hGGPS that contains the translation initiation codon ATG Host cells
  • Another object of the invention consists in cell host that have been transformed or transfected with one of the polynucleotides described therein, and more precisely a polynucleotide either comprising a hGGPS regulatory polynucleotide or the coding sequence of the hGGPS polypeptide having the amino acid sequence of SEQ LD No 4.
  • a polynucleotide either comprising a hGGPS regulatory polynucleotide or the coding sequence of the hGGPS polypeptide having the amino acid sequence of SEQ LD No 4.
  • cell hosts that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as those described above
  • a cell host according to the present invention is characterized in that its genome or genetic background (including chromosome, plasmids) is modified by the heterologous nucleic acid coding for the hGGPS polypeptide of SEQ ED No 4.
  • the cell hosts of the present invention can comprise any of the polynucleotides described in "hGGPPS cDNA Sequences” section, the “Coding Regions” section, “Genomic sequences” section and the “Oligonucleotide Probes And Primers” section.
  • Preferred cell hosts used as recipients for the expression vectors of the invention are the following : a) Prokaryotic host cells : Escherichia coli strains (I.E. DH5- ⁇ strain) or Bacillus subtilis. b) Eukaryotic host cells : HeLa cells (ATCC N°CCL2; N°CCL2.1 ; N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL1650; N°CRL1651), Sf-9 cells (ATCC N°CRL171 1).
  • the constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the selected promoter is induced by appropnate means, such as temperature shift or chemical induction, and cells are cultivated for an additional pe ⁇ od.
  • Cells are typically harvested by centnfugation, disrupted by physical or chemical means, and the resulting crude extract retained for further punfication.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysmg agents. Such methods are well known by the skill artisan.
  • the invention concerns a non-human host animal or mammal comp ⁇ smg a recombinant vector or a host cell according to the invention. More particularly, the invention concerns a mammalian host cell or a non-human host mammal compnsmg a hGGPPS gene disrupted by homologous recombination with a knock out vector and comprising a polynucleotide according to the invention
  • hGGPPS polypeptides is used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides compnsmg such polypeptides.
  • the invention embodies hGGPPS proteins from humans, including isolated or purified hGGPPS proteins consisting, consisting essentially, or comp ⁇ sing the sequence of SEQ LD No 4.
  • hGGPPS proteins of the invention are based on the naturally-occurnng variant of the ammo acid sequence of human hGGPPS, wherein a phenylala ne residue is at positions 204, 257, 295 of SEQ LD No 4, a cysteme residue is at position 205 of SEQ LD No 4, a prohne residue is at position 225 of SEQ ID No 4, and a glutamic acid residue is at position 252 of SEQ ED No 4.
  • the present invention embodies isolated, punfied, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 ammo acids of SEQ ED No 4, wherein said contiguous span includes at least one ammo acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ED No 4, a Pro at position 225 of SEQ LD No 4, and a Glu at position 252 of SEQ ED No 4.
  • the contiguous stretch of ammo acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the ammo acids in the hGGPPS protein sequence.
  • hGGPPS proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes.
  • the hGGPPS polypeptides of the invention can be made using routine expression methods known in the art.
  • the polynucleotide encoding the desired polypeptide is hgated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems is used in forming recombinant polypeptides, and a summary of some of the more common systems.
  • the polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Punfication is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods m Enzymology for a vanety of methods for punfymg proteins.
  • shorter protein fragments is produced by chemical synthesis.
  • the proteins of the invention is extracted from cells or tissues of humans or non-human animals. Methods for punfymg proteins are known m the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.
  • Any hGGPPS cDNA including SEQ LD Nos 2 and 3, is used to express hGGPPS proteins and polypeptides.
  • the nucleic acid encoding the hGGPPS protein or polypeptide to be expressed is operably lmked to a promoter in an expression vector using conventional cloning technology.
  • the hGGPPS insert in the expression vector may compnse the full coding sequence for the hGGPPS protein or a portion thereof.
  • the hGGPPS de ⁇ ved insert may encode a polypeptide compnsmg at least 6 ammo acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 consecutive amino acids of the hGGPPS protein of SEQ LD No 4. wherein said consecutive ammo acids compnse at least one ammo acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ID No 4. a Pro at position 5 225 of SEQ LD No 4, and a Glu at position 252 of SEQ ED No 4
  • the expression vector is any of the mammalian, yeast, insect or bactenal expression systems known in the art.
  • Commercially available vectors and expression systems are available from a vanety of suppliers including Genetics Institute (Cambndge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California).
  • Genetics Institute Cambndge, MA
  • Stratagene La Jolla, California
  • Promega Modison, Wisconsin
  • Invitrogen San Diego, California.
  • the codon context and codon painng of the sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767, the disclosures of which are inco ⁇ orated by reference herein in their entirety.
  • the entire coding sequence of the hGGPPS cDNA through the poly A 5 signal of the cDNA are operably linked to a promoter in the expression vector.
  • an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.
  • this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using 0 Bgll and Sail restnction endonuclease enzymes and lnco ⁇ oratmg it into the mammalian expression vector pXTl (Stratagene).
  • pXTl contains the LTRs and a portion of the gag gene from Moloney Munne Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection.
  • the vector includes the He ⁇ es Simplex Thymidme Kinase promoter and the selectable neomycm gene.
  • the nucleic acid encoding the hGGPPS protein or a portion thereof is obtained by PCR from a bactenal 5 vector containing the hGGPPS cDNA of SEQ ED Nos 2 and 3 using oligonucleotide pnmers complementary to the hGGPPS cDNA or portion thereof and containing restnction endonuclease sequences for Pst I inco ⁇ orated into the 5 'pnmer and BglH at the 5' end of the corresponding cDNA 3' pnmer, taking care to ensure that the sequence encoding the hGGPPS protein or a portion thereof is positioned properly with respect to the poly A signal.
  • the punfied fragment obtained from the resulting 0 PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl El, punfied and hgated to pXTl, now containing a poly A signal and digested with Bglll
  • the hgated product is transfected into mouse NLH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined m the product specification. Positive transfectants are selected after growing the transfected cells m 600ug/ml G418 (Sigma, St. 5 Louis, Missoun).
  • the above procedures may also be used to express a mutant hGGPPS protein responsible for a detectable phenotype or a portion thereof
  • the expressed protein is punfied using conventional punfication techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge
  • the protein encoded by the nucleic acid insert may also be punfied using standard lmmunochromatography techniques
  • a solution containing the expressed hGGPPS protein or portion thereof, such as a cell extract is applied to a column having antibodies against the hGGPPS protein or portion thereof is attached to the chromatography mat ⁇ x
  • the expressed protein is allowed to bind the lmmunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins.
  • the proteins expressed from host cells containing an expression vector containing an insert encoding the hGGPPS protein or a portion thereof can be compared to the proteins expressed m host cells containing the expression vector without an insert.
  • the presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the hGGPPS protein or a portion thereof is being expressed.
  • the band will have the mobility expected for the hGGPPS protein or portion thereof.
  • the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
  • Antibodies capable of specifically recognizing the expressed hGGPPS protein or a portion thereof are descnbed below.
  • the nucleic acids encoding the hGGPPS protein or a portion thereof is inco ⁇ orated into expression vectors designed for use in punfication schemes employing chimenc polypeptides.
  • the nucleic acid encoding the hGGPPS protein or a portion thereof is inserted in frame with the gene encoding the other half of the chimera.
  • the other half of the chimera is ⁇ -globin or a nickel binding polypeptide encoding sequence.
  • a chromatography matrix having antibody to ⁇ -globin or nickel attached thereto is then used to punfy the chimenc protein.
  • Protease cleavage sites is engineered between the ⁇ -globin gene or the nickel binding polypeptide and the hGGPPS protein or portion thereof.
  • the two polypeptides of the chimera is separated from one another by protease digestion.
  • One useful expression vector for generating ⁇ -globin chimenc proteins is pSG5 (Stratagene), which encodes rabbit ⁇ -globin. Intron LI of the rabbit ⁇ -globin gene facilitates splicing of the expressed transcnpt, and the polyadenylation signal inco ⁇ orated into the construct increases the level of expression.
  • Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro ExpressTM Translation Kit (Stratagene) Antibodies That Bind hGGPPS Polypeptides of the Invention
  • Any hGGPPS polypeptide or whole protein may be used to generate antibodies capable of specifically binding to an expressed hGGPPS protein or fragments thereof as described.
  • One antibody composition of the invention is capable of specifically binding or specifically bind to the variant of the hGGPPS protein of SEQ ED No 4.
  • an antibody composition to specifically bind to a first vanant of hGGPPS it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for a full length first vanant of the hGGPPS protein than for a full length second vanant of the hGGPPS protein in an ELISA, RIA, or other antibody- based binding assay.
  • polyclonal or monoclonal antibodies of the invention consists in antibodies raised against a C-termmal portion of the hGGPS polypeptide of the ammo acid sequence of SEQ ED No 4, more preferably antibodies raise against a peptide fragment of the hGGPS polypeptide having the amino acid sequence starting from the amino acid at position 200 and ending at the amino acid in position 300 of the hGGPS polypeptide of SEQ ED No 4, or peptide fragments thereof.
  • the invention concerns antibody compositions, either polyclonal or monoclonal, capable of selectively binding, or selectively bind to an epitope-contaming a polypeptide comprising a contiguous span of at least 6 ammo acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ED No 4 , wherein said epitope comp ⁇ ses at least one amino acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ED No 4, a Pro at position 225 of SEQ ED No 4, and a Glu at position 252 of SEQ ED No 4.
  • the invention also concerns a punfied or isolated antibody capable of specifically binding to a mutated hGGPPS protein or to a fragment or vanant thereof comprising an epitope of the mutated hGGPPS protein.
  • the invention concerns the use in the manufacture of antibodies of a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 ammo acids of SEQ ED No 4 , wherein said epitope comprises at least one ammo acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ED No 4, a Pro at position 225 of SEQ ID No 4, and a Glu at position 252 of SEQ ED No 4.
  • Non-human animals or mammals whether wild-type or transgenic, which express a different species of hGGPPS than the one to which antibody binding is desired, and animals which do not express hGGPPS (i.e. a hGGPPS knock out animal as descnbed herein) are particularly useful for preparing antibodies.
  • hGGPPS knock out animals will recognize all or most of the exposed regions of a hGGPPS protein as foreign antigens, and therefore produce antibodies with a wider array of hGGPPS epitopes.
  • polypeptides with only 10 to 30 ammo acids may be useful in obtaining specific binding to any one of the hGGPPS proteins
  • the humoral immune system of animals which produce a species of hGGPPS that resembles the antigenic sequence will preferentially recognize the differences between the animal ' s native hGGPPS species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence
  • Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the hGGPPS proteins
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-beanng substances in biological samples, they are also used semi -quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body
  • the antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art
  • the invention is also directed to a method for detecting specifically the presence of a hGGPPS polypeptide according to the invention in a biological sample, said method comprising the following steps a) bnngmg into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a hGGPPS polypeptide compnsmg an ammo acid sequence of SEQ ED No 4, or to a peptide fragment or variant thereof; and b) detecting the antigen-antibody complex formed
  • the invention also concerns a diagnostic kit for detecting in vitro the presence of a hGGPPS polypeptide according to the present invention in a biological sample, wherein said kit comprises a) a polyclonal or monoclonal antibody that specifically binds a hGGPPS polypeptide comprising an ammo acid sequence of SEQ ED No 4, or to a peptide fragment or vanant thereof, optionally labeled, b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.
  • Another subject of the present invention is a method for screening molecules that modulate the expression of the hGGPPS protein.
  • Such a screening method comprises the steps of: a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the hGGPPS protein or a vanant or a fragment thereof, placed under the control of its own promoter, b) bringing into contact the cultivated cell with a molecule to be tested, c) quantifying the expression of the hGGPPS protein or a variant or a fragment thereof
  • nucleotide sequence encoding the hGGPPS protein or a vanant or a fragment thereof preferably a fragment comprising an allele of the biallehc marker 5-187-77, and the complement thereof.
  • the method for the screening of a candidate substance or molecule modulating the expression of the hGGPS genecomp ses the following steps a) providing a recombinant host cell expressing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ED Nos 1, 2 and 3 or a fragment thereof, b) obtaining a candidate substance, and c) determining the ability of the candidate substance to modulate the expression levels of the nucleotide sequence selected from the group consisting of SEQ ED Nos 1 , 2 and 3 or a fragment thereof.
  • the hGGPPS protein encoding DNA sequence is inserted into an expression vector, downstream from its promoter sequence.
  • the promoter sequence of the hGGPPS gene is contained in the nucleic acid of the 5' regulatory region.
  • the quantification of the expression of the hGGPPS protein may be realized either at the mRNA level or at the protein level. In the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the hGGPPS protein that have been produced, for example m an ELISA or a RIA assay.
  • the quantification of the hGGPPS mRNA is realized by a quantitative PCR amplification of the cDNA obtained by a reverse transcnption of the total mRNA of the cultivated hGGPPS -transfected host cell, using a pair of p ⁇ mers specific for hGGPPS.
  • the present invention also concerns a method for screening substances or molecules that are able to increase, or in contrast to decrease, the level of expression of the hGGPPS gene. Such a method may allow the one skilled in the art to select substances exerting a regulating effect on the expression level of the hGGPPS gene and which may be useful as active ingredients included m pharmaceutical compositions.
  • nucleic acid comprises a nucleotide sequence of the 5' regulatory region or a biologically active fragment or vanant thereof located upstream a polynucleotide encoding a detectable protein
  • the nucleic acid comp ⁇ sing the nucleotide sequence of the 5' regulatory region or a biologically active fragment or variant thereof also includes a 5'UTR region of the hGGPPS cDNA of SEQ ID Nos 2 or 3, or one of its biologically active fragments or vanants thereof.
  • kits useful for performing the herein descnbed screening method comprise a recombinant vector that allows the expression of a nucleotide sequence of the 5' regulatory region or a biologically active fragment or variant thereof located upstream and operably linked to a polynucleotide encoding a detectable protein or the hGGPPS protein or a fragment or a variant thereof.
  • the nucleic acid that compnses a nucleotide sequence selected from the group consisting of the 5'UTR sequence of the hGGPPS cDNA of SEQ ED Nos 2 or 3 or one of its biologically active fragments or variants includes a promoter sequence which is endogenous with respect to the hGGPPS 5'UTR sequence.
  • the nucleic acid that comprises a nucleotide sequence selected from the group consisting of the 5'UTR sequence of the hGGPPS cDNA of SEQ LD Nos 2 or 3 or one of its biologically active fragments or vanants includes a promoter sequence which is exogenous with respect to the hGGPPS 5'UTR sequence defined therein.
  • the nucleic acid comp ⁇ sing the 5'-UTR sequence of the hGGPPS cDNA or SEQ LD Nos 2 or 3 or the biologically active fragments thereof, preferably those including the biallehc marker 5-187-77 or the complement thereof comprises a kit for the screening of a candidate substance modulating the expression of the hGGPPS gene, wherein said kit comprises a recombinant vector that comprises a nucleic acid including a 5"UTR sequence of the hGGPPS cDNA of SEQ ED Nos 2 or 3, or one of their biologically active fragments or variants, the 5'UTR sequence or its biologically active fragment or vanant being operably linked to a polynucleotide encoding a detectable protein
  • hGGPPS expression levels and patterns of hGGPPS may be analyzed by solution hybndization with long probes as descnbed in International Patent Application No. WO 97/05277, the entire contents of which are inco ⁇ orated herein by reference.
  • the hGGPPS cDNA or the hGGPPS genomic DNA desc ⁇ bed above, or fragments thereof is inserted at a cloning site immediately downstream of a bactenophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA.
  • the hGGPPS insert compnses at least 100 or more consecutive nucleotides of the genomic DNA sequence or the cDNA sequences.
  • the plasmid is linearized and transcnbed in the presence of ⁇ bonucleotides compnsmg modified nbonucleotides (i.e. biotm-UTP and DIG-UTP).
  • ⁇ bonucleotides compnsmg modified nbonucleotides i.e. biotm-UTP and DIG-UTP.
  • An excess of this doubly labeled RNA is hybndized in solution with mRNA isolated from cells or tissues of interest
  • the hybridization is performed under standard stringent conditions (40-50°C for 16 hours in an 80% formamide, 0. 4 M NaCl buffer, pH 7-8)
  • the unhybndized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, TI , Phy M, U2 or A).
  • arrays means a one dimensional, two dimensional, or multidimensional arrangement of a plurality of nucleic acids of sufficient length to permit specific detection of expression of mRNAs capable of hybndizing thereto.
  • the arrays may contain a plurality of nucleic acids denved from genes whose expression levels are to be assessed.
  • the arrays may include the hGGPPS genomic DNA, the hGGPPS cDNA sequences or the sequences complementary thereto or fragments thereof, particularly those compnsmg the biallehc marker 5- 187-77.
  • the fragments are at least 15 nucleotides in length. In other embodiments, the fragments are at least 25 nucleotides in length. In some embodiments, the fragments are at least 50 nucleotides in length More preferably, the fragments are at least 100 nucleotides m length. In another preferred embodiment, the fragments are more than 100 nucleotides m length In some embodiments the fragments may be more than 500 nucleotides m length.
  • hGGPPS gene expression may be performed with a complementary DNA microarray as described by Schena et al (1995 and 1996) Full length hGGPPS cDNAs or fragments thereof are amplified by PCR and arrayed from a 96-well microtiter plate onto silylated microscope slides using high-speed robotics.
  • Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and nnsed, once in 0. 2% SDS for 1 mm, twice in water for 1 min and once for 5 min in sodium borohyd ⁇ de solution The arrays are submerged in water for 2 min at 95°C, transferred into 0. 2% SDS for 1 m , rinsed twice with water, air dried and stored in the dark at 25°C.
  • Probes are hyb ⁇ dized to 1 cm " microarrays under a 14 x 14 mm glass coverslip for 6-12 hours at 60°C. Arrays are washed for 5 mm at 25°C in low stringency wash buffer (1 x SSC/0. 2% SDS), then for 10 mm at room temperature in high stringency wash buffer (0 1 x SSC/0. 2% SDS). Arrays are scanned m 0. 1 x SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations
  • Quantitative analysis of hGGPPS gene expression may also be performed with full length hGGPPS cDNAs or fragments thereof m complementary DNA arrays as descnbed by Pietu et al.(1996).
  • the full length hGGPPS cDNA or fragments thereof is PCR amplified and spotted on membranes. Then, mRNAs ongmatmg from vanous tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hyb ⁇ dized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.
  • expression analysis using the hGGPPS genomic DNA, the hGGPPS cDNA, or fragments thereof can be done through high density nucleotide arrays as descnbed by Lockhart et al.(1996) and Sosnowsky et al.(1997).
  • Oligonucleotides of 15-50 nucleotides from the sequences of the hGGPPS genomic DNA, the hGGPPS cDNA sequences particularly those comprising the biallehc marker 5-187-77, or the sequences complementary thereto, are synthesized directly on the chip (Lockhart et al., supra) or synthesized and then addressed to the chip (Sosnowski et al., supra).
  • the oligonucleotides are about 20 nucleotides in length.
  • hGGPPS cDNA probes labeled with an appropnate compound such as biotin, digoxigemn or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hyb ⁇ dized to the chip. After washing as desc ⁇ bed in Lockhart et al., supra and application of different electric fields (Sosnowsky et al., 1997)., the dyes or labeling compounds are detected and quantified.
  • an appropnate compound such as biotin, digoxigemn or fluorescent dye
  • Human GGPS cDNA was obtained as follows : 4 ⁇ l of ethanol suspension containing 1 mg of human prostate total RNA (Clontech laboratories, Inc., Palo Alto, USA; Catalogue N. 64038-1) was cent ⁇ fuged, and the resulting pellet was air dned for 30 minutes at room temperature.
  • First strand cDNA synthesis was performed using the AdvantageTM RT-for- PCR kit (Clontech laboratories Inc., catalogue N. K1402-1). 1 ⁇ l of 20 mM solution of a specific oligo dT primer was added to 12.5 ⁇ l of RNA solution in water, heated at 74°C for 2.5 min and rapidly quenched in an ice bath.
  • the amplification products corresponding to both cDNA strands are partially sequenced in order to ensure the specificity of the amplification reaction
  • Donors were unrelated and healthy. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the detection of the biallehc markers
  • the pellet of white cells was lysed overnight at 42°C with 3.7 ml of lysis solution composed of:
  • TE 10-2 (Tns-HCl 10 mM, EDTA 2 mM) / NaCl 0.4 M - 200 ⁇ l SDS 10% - 500 ⁇ l K-protemase (2 mg K-protemase in TE 10-2 / NaCl 0 4 M).
  • OD 260 / OD 280 ratio was determined. Only DNA preparations having a OD 260 / OD 280 ratio between 1.8 and 2 were used m the subsequent examples desc ⁇ bed below.
  • the pool was constituted by mixing equivalent quantities of DNA from each individual.
  • the amplification of specific genomic sequences of the DNA samples of example 2 was carried out on the pool of DNA obtained previously. In addition, 50 individual samples were similarly amplified.
  • Each pair of first pnmers was designed using the sequence information of the hGGPS gene disclosed herein and the OSP software (Hillier & Green, 1991). This first pair of pnmers was about 20 nucleotides in length and had the sequences disclosed in Table 1 in the columns labeled PU and RP.
  • the p ⁇ mers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing.
  • Primers PU contain the following additional PU 5' sequence : TGTAAAACGACGGCCAGT (SEQ ID No 10); primers RP contain the 15 following RP 5' sequence : CAGGAAACAGCTATGACC (SEQ ID No 11).
  • DNA amplification was performed on a Genius II thermocycler. After heating at 95°C for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95°C. 54°C for 1 min, and 30 sec at 20 72°C. For final elongation, 10 min at 72°C ended the amplification.
  • the quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as mtercalant agent (Molecular Probes).
  • Detection of the biallelic markers sequencing of amplified genomic DNA and 25 identification of polymorphisms.
  • the sequencing of the amplified DNA obtained in example 3 was earned out on ABI 377 sequencers.
  • the sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol.
  • the products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel 30 image analysis.
  • sequence data were further evaluated to detect the presence of biallehc markers among the pooled amplified fragments.
  • the polymo ⁇ hism search was based on the presence of supenmposed peaks in the electrophoresis pattern resulting from different bases occurnng at the same position as described previously.
  • Table 2 shows the biallehc marker that has been detected after the sequence analysis of the amplification fragments generated by PCR.
  • the two alleles of the biallehc marker 5-187-77 can be defined by an oligonucleotide comprising the polymo ⁇ hic base.
  • the sequence of such oligonucleotides are disclosed in SEQ ED Nos 5 and 6.
  • the biallehc marker identified in example 4 was further confirmed through microsequencing. Microsequencing was earned out for each individual DNA sample desc ⁇ bed in Example 2.
  • Amplification from genomic DNA of individuals was performed by PCR as descnbed above for the detection of the biallehc markers with the same set of PCR pnmers (Table 1).
  • the prefened pnmers used in microsequencing were about 20 nucleotides in length and hybridized just upstream of the considered polymo ⁇ hic base. According to the invention, the pnmer used in microsequencing is detailed in Table 3.
  • microsequencing reaction was performed as follows :
  • the microsequencing reaction mixture was prepared by adding, in a 20 ⁇ l final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G), 1.25 ⁇ l Thermosequenase buffer (260 mM T ⁇ s HCl pH 9.5, 65 mM MgCl 2 ), and the two appropnate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to the nucleotides at the polymo ⁇ hic site of each bialle c marker tested, following the manufacturer's recommendations.
  • the software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous.
  • the software identifies significant peaks (according to shape and height criteria) Among the significant peaks, peaks corresponding to the targeted site are identified based on their position When two significant peaks are detected for the same position, each sample is catego ⁇ zed classification as homozygous or heterozygous type based on the height ratio.
  • Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding the hGGPPS protein or a portion thereof
  • concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml.
  • Monoclonal or polyclonal antibody to the protein can then be prepared as follows: A. Monoclonal Antibody Production by Hybndoma Fusion
  • Monoclonal antibody to epitopes in the hGGPPS protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C, (1975) or derivative methods thereof. Also see Harlow, E., and D. Lane. 1988..
  • a mouse is repetitively inoculated with a few micrograms of the hGGPPS protein or a portion thereof over a penod of a few weeks. The mouse is then sacnficed, and the antibody producmg cells of the spleen isolated. T e spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media compnsmg ammoptenn (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • HAT media selective media compnsmg ammoptenn
  • Antibody- producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally descnbed by Engvall, (1980), and denvative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are descnbed in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2 B Polyclonal Antibody Production by Immunization
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes m the hGGPPS protein or a portion thereof can be prepared by immunizing suitable non-human animal with the hGGPPS protein or a portion thereof, which can be unmodified or modified to enhance lmmunogenicity
  • suitable non-human animal is preferably a non-human mammal is selected, usually a mouse, rat, rabbit, goat, or horse.
  • a crude preparation which has been enriched for hGGPPS concentration can be used to generate antibodies.
  • Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropnate adjuvant (e.g.
  • the protein, fragment or preparation can be pretreated with an agent which will increase antigenicity, such agents are known m the art and include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanm (KLH).
  • agents include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanm (KLH).
  • mBSA methylated bovine serum albumin
  • BSA bovine serum albumin
  • Hepatitis B surface antigen Hepatitis B surface antigen
  • KLH keyhole limpet hemocyanm
  • Serum from the immunized animal is collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies can be purified by lmmunoaffinity chromatography.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple mtradermal sites appears to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987). An effective immunization protocol for rabbits can be found m Vaitukaitis, J. et al. (1971).
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double lmmunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., (1973) Plateau concentration of antibody is usually m the range of 0 1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by prepanng competitive binding curves, as descnbed, for example, by Fisher, D., (1980).
  • Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful m quantitative immunoassays which determine concentrations of antigen-beanng substances m biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

Abstract

The present invention relates to a purified or isolated polynucleotide encoding human geranylgeranyl pyrophosphate synthetase, the regulatory nucleic acids contained therein, a polymorphic marker thereof and the resulting encoded protein, as well as to methods and kits for detecting this polynucleotide and this protein. The present invention also pertains to a polynucleotide carrying the natural regulatory regions of the hGGPS gene which is useful, for example, to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region. The invention also consists in genetic markers, namely biallelic markers, which may be useful for the diagnosis of diseases related to an alteration in the regulatory or coding regions of hGGPS, such as pathologies related to a defect in the mevalonic biosynthetic pathway.

Description

A nucleic acid encoding a geranyl-geranyl pyrophosphate synthetase (GGPPS) and polymorphic markers associated with said nucleic acid.
FIELD OF THE INVENTION
The present invention relates to a purified or isolated polynucleotide encoding human geranylgeranyl pyrophosphate synthetase, the regulatory nucleic acids contained therein, a polymorphic marker thereof and the resulting encoded protein, as well as to methods and kits for detecting this polynucleotide and this protein. The present invention also pertains to a polynucleotide carrying the natural regulatory regions of the hGGPS gene which is useful, for example, to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region. The invention also consists in genetic markers, namely bialle c markers, which may be useful for the diagnosis of diseases related to an alteration in the regulatory or coding regions of hGGPS, such as pathologies related to a defect in the mevalomc biosynthetic pathway
BACKGROUND OF THE INVENTION Prenylation is the least common known lipid modification. Other lipid modifications include palmitylation, myπstylation and glycophosphohpidation. However, prenylation is a surprisingly common form of post-translational protein modification with an occurrence of 0.5 % of all cellular proteins. Prenylation is a covalent modification which involves the attachment of either a C15 farnesyl or a C20 geranylgeranyl isoprenoid, both being products of the mevalomc acid biosynthetic pathway, to one or more cysteme residues at the carboxyl terminus of the protein via a thioether bond The C20 geranylgeranyl modification predominates over the C15 farnesyl modification in terms of frequency of occurrence The structural environment of the cysteme residue determines the specific type and number of isoprenoid groups that attach to each cysteme. The covalent modification resulting from prenylation renders proteins more hydrophobic and, together with a subsequent modification cascade, facilitates their association with membranes. Protein prenylation also mediates protein-protein interactions. Prenylated proteins can be involved in signal transduction, intracellular vesicular transport, cytoskeletal organization, cell growth control and polarity, viral replication and protein folding/assembly. In mammals, prenylated proteins are more frequently modified by one or more geranylgeranyl groups. Farnesylation has only been found to occur in the retinal heterotπmeπc G protein transducin, in retinal rhodopsm kinase, m ras proteins, in nuclear lamins, and in yeast mating factors. Geranylgeranylation is found m all of the remaining heterotπmeπc G proteins and small G proteins.
Heterotπmeπc G-protems which are required for intracellular signal transduction between receptors and effector enzymes present one or two prenylated subumts This modification is often required for association of the functional complex with the membrane Among small G proteins, Ras proteins, which comprise oncogemc forms, regulate signal transduction pathways controlling cell proliferation and differentiation. All ras proteins are prenylated and this modification is critical for their transport to the inner surface of the plasma membrane and their biological functions Other prenylated proteins belonging to the ras protein superfamily are involved in the regulation of intracellular vesicular transport (Rab/YPTl), in the cytoskeletal organization of polymerized actin to produce stress fibers (Rho) or membrane ruffling (Rac), in the oxydative burst of phagocytic cells (Rac), m the control of the cell cycle and polaπty (cdc24Hs/G25K), and in negative growth control (Rap/Krev-1). Prenylation is important to these activities. For example, Rab/YPT prenylation is cπtical for the association of these proteins with specific intracellular compartments and in their regulation of intracellular transport processes.
One hypothesis is that rather than providing only an increase in hydrophobicity, the isoprenoid acts as part of a recognition unit for specific receptors that interact with either farnesylated or geranylgeranylated proteins. The recent observations that geranylgeranyl-modified forms of K-Ras4B or H-Ras proteins exhibit intracellular localizations which are different from those of their authentic farnesylated counterparts is consistent with this possibility.
Moreover, prenylation of nuclear lamins, which are involved in the mitotic control of membrane assembly, is necessary for the proper assembly of these proteins into the nuclear lamina. Indeed, prenylation is necessary to the maturation by cleavage of prelamm A m lamin A and to obtain functional lamm B.
Geranylgeranyl pyrophosphate synthetase (GGPS) is involved in the mevalomc acid biosynthetic pathway and is located in the cytosol. It catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce GGPP. This biosynthesis of GGPPS is regulated according to requirements for protein prenylation. GGPS has been found to be expressed in human fetal heart, as descπbed in the PCT Application No WO 96/21736.
SUMMARY OF THE INVENTION
The present invention pertains to nucleic acid molecules compπsmg the genomic sequence of a novel human gene which encodes a hGGPPS protein. The hGGPPS genomic sequence comprises regulatory sequence located upstream (5 '-end) and downstream (3 '-end) of the transcribed portion of said gene, these regulatory sequences being also part of the invention
The invention also deals with the complete sequence of two cDNAs encoding the hGGPPS protein, as well as with the corresponding translation product
Oligonucleotide probes or pπmers hybridizing specifically with a hGGPPS genomic or cDNA sequences are also part of the present invention, as well as DNA amplification and detection methods using said pπmers and probes.
A further object of the invention consists of recombinant vectors compπsmg any of the nucleic acid sequences descπbed above, and in particular of recombinant vectors compπsmg a h GGPPS regulatory sequence or a sequence encoding a hGGPPS protein, as well as of cell hosts and transgenic non human animals compπsmg said nucleic acid sequences or recombinant vectors The invention also concerns a ΛGG S-related biallehc marker
Finally, the invention is directed to methods for the screening of substances or molecules that modify or inhibit the expression of hGGPPS
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 : Map of the genomic, cDNA and coding (CDS) sequences of hGGPS : (1) upper line, genomic sequence; (2) cDNA sequence of SEQ ID No 2; (3) coding sequence (CDS).
Figure 2 : Map of the genomic, cDNA and coding (CDS) sequences of hGGPS : (1) upper line, genomic sequence; (2) cDNA sequence of SEQ LD No 3; (3) coding sequence (CDS).
Brief Description of the sequences provided in the Sequence Listing
SEQ ID No 1 contains a genomic sequence of hGGPPS comprising the 5' regulatory region (upstream untranscπbed region), the exons and introns, and the 3' regulatory region (downstream untranscπbed region). SEQ LD No 2 contains a cDNA sequence of hGGPPS compπsmg the exons 1, 2, 3, and 4.
SEQ ID No 3 contains a cDNA sequence of hGGPPS compπsmg the exons Ibis, 2, 3, and 4. SEQ ID No 4 contains the ammo acid sequence encoded by the cDNA of SEQ ID No 2 or 3. SEQ LD Nos 5 and 6 contain the fragments containing a polymorphic base of the biallehc marker 5-187-77. SEQ LD No 7 contains the microsequencing pπmer of the biallehc marker 5-187-77.
SEQ LD Nos 8 and 9 contain the amplification pπmers of the biallehc marker 5-187-77. SEQ ID No 10 contains a pπmer containing the additional PU 5' sequence described further in Example 3.
SEQ ID No 1 1 contains a primer containing the additional RP 5' sequence described further in Example 3.
DETAILED DESCRIPTION OF THE INVENTION
The hGGPS gene of the invention is located on chromosome 1, and more precisely on the Iq42-lq43 locus of this chromosome. This chromosome 1 locus has been shown to carry a predisposing gene for prostate cancer (Berthon et al., 1998). The hGGPS gene of the invention is located in the vicinity of a retinoblastoma binding protein gene. Indeed, the coding sequence of this latter gene is on a strand which is opposite to the strand carrying the hGGPS Open Reading Frame.
The aim of the present invention is to provide polynucleotides deπved from the hGGPS gene, particularly those useful to design suitable means for detecting the presence of this gene in a test sample or alternatively to discπminate between the hGGPS mRNA molecules that are present in a test sample Other polynucleotides of the invention are useful to design suitable means to express a desired polynucleotide of interest The invention also relates to the hGGPS polypeptide having the ammo acid sequence of SEQ ID No 4.
Definitions Before descnbmg the invention m greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to descπbe the invention herein
The term " hGGPPS gene", when used herein, encompasses mRNA and cDNA sequences encoding the hGGPPS protein. In the case of a genomic sequence, the hGGPPS gene also includes native regulatory regions which control the expression of the coding sequence of the hGGPPS gene. The term "functionally active fragment" of the hGGPPS protein is intended to designate a polypeptide carrying at least one of the structural features of the hGGPPS protein involved in at least one of the biological functions and/or activity of the hGGPPS protein
A "heterologous" or "exogenous" polynucleotide designates a purified or isolated nucleic acid that has been placed, by genetic engineeπng techniques, in the environment of unrelated nucleotide sequences, such as the final polynucleotide construct does not occur naturally. An illustrative, but not limitative, embodiment of such a polynucleotide construct may be represented by a polynucleotide comprising ( 1 ) a regulatory polynucleotide deπved from the hGGPPS gene sequence and (2) a polynucleotide encoding a cytokme, for example GM-CSF. The polypeptide encoded by the heterologous polynucleotide will be termed an heterologous polypeptide for the purpose of the present invention.
By a "biologically active fragment or vanant" of a regulatory polynucleotide according to the present invention is intended a polynucleotide compπsing or alternatively consisting m a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host. For the purpose of the invention, a nucleic acid or polynucleotide is "functional" as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcπptional and translational regulatory information, and such sequences are "operatively linked" to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide. An operable linkage is a linkage in which the regulatory nucleic acid and the DNA sequence sought to be expressed are linked in such a way as to permit gene expression.
As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be "operably linked" if the nature of the linkage between the two polynucleotides does not (1) result m the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide The promoter polynucleotide would be operably linked to a polynucleotide encoding a desired polypeptide or a desired polynucleotide if the promoter is capable of effecting transcription of the polynucleotide of interest
The terms "sample" or "matenal sample" are used herein to designate a solid or a liquid material suspected to contain a polynucleotide or a polypeptide of the invention A solid matenal may be, for example, a tissue slice or biopsy withm which is searched the presence of a polynucleotide encoding a hGGPPS protein, either a DNA or RNA molecule or withm which is searched the presence of a native or a mutated hGGPPS protein, or alternatively the presence of a desired protein of interest the expression of which has been placed under the control of a hGGPPS regulatory polynucleotide. A liquid matenal may be, for example, any body fluid like serum, urine etc , or a liquid solution resulting from the extraction of nucleic acid or protein matenal of interest from a cell suspension or from cells in a tissue slice or biopsy. The term "biological sample" is also used and is more precisely defined withm the Section dealing with DNA extraction
As used herein, the term "purified" does not require absolute purity; rather, it is intended as a relative definition. Puπfication if starting material or natural matenal to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, punfication from 0.1% concentration to 10% concentration is two orders of magnitude.
The term "isolated" requires that the material be removed from its original environment (e.g. the natural environment if it is naturally occurring) For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting mateπals in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition and still be isolated m that the vector or composition is not part of its natural environment
The term "polypeptide" refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, ohgopeptides, and proteins are included withm the definition of polypeptide This term also does not specify or exclude post-expression modifications of polypeptides, for example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide Also included within the definition are polypeptides which contain one or more analogs of an ammo acid (including, for example, non-naturally occurnng ammo acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurnng and non-naturally occurnng. The term "recombinant polypeptide" is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide. 5 The term "purified" is used herein to describe a polypeptide of the invention which has been separated from other compounds including, but not limited to nucleic acids, lipids, carbohydrates and other proteins. A polypeptide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially pure polypeptide typically comprises about 50%, preferably 60 to 90% weight weight of a protein sample, more usually about
10 95%, and preferably is over about 99% pure. Polypeptide punty or homogeneity is indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.
As used herein, the term "non-human animal" refers to any non-human vertebrate, birds and
15 more usually mammals, preferably primates, farm animals such as swme, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term "animal" is used to refer to any vertebrate, preferable a mammal. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".
As used herein, the term "antibody" refers to a polypeptide or group of polypeptides which
20 are compπsed of at least one binding domain, where an antibody binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distnbution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen. Antibodies include recombinant proteins comprising the binding domains, as wells as fragments, including Fab,
25 Fab', F(ab) , and F(ab')2 fragments
As used herein, an "antigenic determinant" is the portion of an antigen molecule, in this case a hGGPPS polypeptide, that determines the specificity of the antigen-antibody reaction. An "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise as few as 3 ammo acids in a spatial conformation which is unique to the epitope. Generally an epitope consists
30 of at least 6 such amino acids, and more usually at least 8-10 such ammo acids. Methods for determining the ammo acids which make up an epitope include x-ray crystallography, 2-dιmensιonal nuclear magnetic resonance, and epitope mapping e.g. the Pepscan method descnbed by Geysen et al. 1984; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506.
Throughout the present specification, the expression "nucleotide sequence" may be
35 employed to designate indifferently a polynucleotide or an oligonucleotide or a nucleic acid. More precisely, the expression "nucleotide sequence" encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically charactenzes a specific DNA or RNA molecule.
As used interchangeably herein, the term "oligonucleotides", and "polynucleotides" include RNA, DNA. or RNA/DNA hybrid sequences of more than one nucleotide m either single chain or duplex form. The term "nucleotide" as used herein as an adjective to descnbe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term "nucleotide" is also used herein as a noun to refer to individual nucleotides or vaπeties of nucleotides, meaning a molecule, or individual unit a larger nucleic acid molecule, comprising a punne or pyπmidine, a nbose or deoxynbose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides withm an oligonucleotide or polynucleotide. Although the term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of punne, (c) an analogous form of pyπmidme, or (d) an analogous sugar, for examples of analogous linking groups, punne, pyπmidines, and sugars see for example PCT publication No WO 95/04064. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxynbose nucleotides, and most preferably greater than 90% conventional deoxynbose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any punfication methods known m the art. The term "heterozygosity rate" is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallehc system, the heterozygosity rate is on average equal to 2Pa(l-Pa), where Pa is the frequency of the least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous. The term "genotype" as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention a genotype preferably refers to the descπption of the biallehc marker alleles present m an individual or a sample. The term "genotypmg" a sample or an individual for a biallehc marker consists of determining the specific allele or the specific nucleotide earned by an individual at a biallehc marker. The term "polymorphism" as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. "Polymoφhic" refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A "polymorphic site" is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention "single nucleotide polymorphism" preferably refers to a single nucleotide substitution Typically, between different genomes or between different individuals, the polymoφhic site may be occupied by two different nucleotides
The term "biallehc polymoφhism" and "biallehc marker" are used interchangeably herein to refer to a single nucleotide polymoφhism having two alleles at a fairly high frequency in the population. A "biallelic marker allele" refers to the nucleotide variants present at a biallehc marker site Typically, the frequency of the less common allele of the bialle c markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (I e. heterozygosity rate of at least 0.42) A biallehc marker wherein the frequency of the less common allele is 30% or more is termed a "high quality biallelic marker"
The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are descπbed herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3' and 5' ends of the polynucleotide is considered to be "at the center" of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be "within 1 nucleotide of the center." With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be withm 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be "within 1 nucleotide of the center" and any of the four nucleotides in the middle of the polynucleotide would be considered to be "within 2 nucleotides of the center", and so on.
As used herein the terminology "defining a biallehc marker" means that a sequence includes a polymoφhic base from a biallehc marker. The sequences defining a biallelic marker may be of any length consistent with their intended use, provided that they contain a polymoφhic base from a bialle c marker. The sequence has between 1 and 500 nucleotides in length, preferably between 5, 10 , 15, 20, 25, or 40 and 200 nucleotides and more preferably between 30 and 50 nucleotides in length. Each biallehc marker therefore corresponds to two forms of a polynucleotide sequence included in a gene, which, when compared with one another, present a nucleotide modification at one position. Preferably, the sequences defining a bialle c marker include a polymoφhic base of the biallehc marker 5-187-77. In some embodiments the sequences defining a bialle c marker comprise one of the sequences selected from the group consisting of SEQ ID Nos 5 and 6. Likewise, the term "marker" or "biallehc marker" requires that the sequence is of sufficient length to practically (although not necessaπly unambiguously) identify the polymoφhic allele, which usually implies a length of at least 4, 5, 6, 10, 15, 20, 25, or 40 nucleotides. The terms "base paired" and "Watson & Crick base paired" are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymme or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanme residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4th edition, 1995)
The terms "complementary" or "complement thereof are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Cπck base painng with another specified polynucleotide throughout the entirety of the complementary region. For the puφose of the present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base m the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. "Complement" is used herein as a synonym from "complementary polynucleotide", "complementary nucleic acid" and "complementary nucleotide sequence". These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.
Variants and fragments
1. Polynucleotides
The invention also relates to vanants and fragments of the polynucleotides descπbed herein, particularly of a hGGPPS gene containing one or more biallehc markers according to the invention. Vanants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurnng variant such as a naturally occurring allehc variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurnng variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the vanant are closely similar overall and, in many regions, identical.
Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences that are at least 95% identical to any of SEQ LD Nos 1-3 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ LD Nos 1-3 or the sequences complementary thereto, and preferably at least 98% identical, more particularly at least 99.5% identical, and most preferably at least 99.9% identical to any of SEQ ID Nos 1 -3 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ LD Nos 1 -3 or the sequences complementary thereto. Changes in the nucleotide of a vanant may be silent, which means that they do not alter the ammo acids encoded by the polynucleotide. However, nucleotide changes may also result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence The substitutions, deletions or additions may involve one or more nucleotides The vanants may be altered m coding or non-coding regions or both Alterations m the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions
In the context of the present invention, particularly preferred embodiments are those m which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature hGGPPS protein
A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a hGGPPS gene, and variants thereof. The fragment can be a portion of an exon or of an intron of a hGGPPS gene. It can also be a portion of the regulatory sequences of the hGGPPS gene. Preferably, such fragments comprise the polymoφhic base of the biallelic marker 5-187-77 of SEQ LD Nos 5-6.
Such fragments may be "free-standing", I e not part of or fused to other polynucleotides, or they may be compnsed within a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide
As representative examples of polynucleotide fragments of the invention, there may be mentioned those which have from about 4, 6, 8, 15, 20, 25, 40, 10 to 20, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200 nucleotides m length. Preferred are those fragments having about 49 nucleotides in length, such as those of SEQ ID Nos 5-6 or the sequences complementary thereto and containing at least one of the biallehc markers of a hGGPPS gene which are descnbed herein.
2. Polypeptides
The invention also relates to vanants, fragments, analogs and denvatives of the polypeptides described herein, including mutated hGGPPS proteins The vanant may be 1) one in which one or more of the ammo acid residues are substituted with a conserved or non-conserved ammo acid residue (preferably a conserved ammo acid residue) and such substituted ammo acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the ammo acid residues includes a substituent group, or 3) one in which the mutated hGGPPS is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the mutated hGGPPS, such as a leader or secretory sequence or a sequence which is employed for puπfication of the mutated hGGPPS or a preprotem sequence. Such vanants are deemed to be within the scope of those skilled the art
More particularly, a variant hGGPPS polypeptide compnses ammo acid changes ranging from 1, 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of one ammoacid, preferably from 1 to 10, more preferably from 1 to 5 and most preferably from 1 to 3 substitutions, additions or deletions of one ammo acid. The preferred ammo acid changes are those which have little or no influence on the biological activity or the capacity of the vanant hGGPPS polypeptide to be recognized by antibodies raised against a native hGGPPS protein
By homologous peptide according to the present invention is meant a polypeptide containing one or several ammoacid additions, deletions and/or substitutions m the amino acid sequence of a 5 hGGPPS polypeptide. In the case of an ammoacid substitution, one or several -consecutive or non- consecutive- aminoacids are replaced by « equivalent » ammoacids
The expression "equivalent" amino acid is used herein to designate any amino acid that may be substituted for one of the amino acids having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to 10 be substantially unchanged Generally, the following groups of amino acids represent equivalent changes: (1) Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, lie, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Tφ, His.
By an equivalent ammoacid according to the present invention is also meant the replacement of a residue the L-form by a residue in the D form or the replacement of a Glutamic acid (E) 15 residue by a Pyro-glutamic acid compound. The synthesis of peptides containing at least one residue m the D-form is, for example, descnbed by Koch (1977).
A specific, but not restrictive, embodiment of a modified peptide molecule of interest according to the present invention, which consists in a peptide molecule which is resistant to proteolysis, is a peptide in which the -CONH- peptide bond is modified and replaced by a (CH2NH) 0 reduced bond, a (NHCO) retro inverso bond, a (CH2-O) methylene-oxy bond, a (CH2-S) thiomethylene bond, a (CH2CH2) carba bond, a (CO-CH2) cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (N-N) bound, a E-alcene bond or also a -CH=CH- bond.
The polypeptide accoding to the invention could have post-translational modifications. For example, it can present the following modifications: acylation, disulfide bond formation, 25 prenylation, carboxymethylation and phosphorylation.
A polypeptide fragment is a polypeptide having a sequence that entirely is the same as part but not all of a given polypeptide sequence, preferably a polypeptide encoded by a hGGPPS gene and vanants thereof. Preferred fragments include those regions possessing antigenic properties and which can be used to raise antibodies against the hGGPPS protein. 30 Such fragments may be "free-standing", i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger polypeptide of which they form a part or region. However, several fragments may be compnsed withm a single larger polypeptide.
As representative examples of polypeptide fragments of the invention, there may be mentioned those which comprise at least about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 35 55 ammo acids of the hGGPPS In some embodiments, the fragments contain at least one ammo acid mutation in the hGGPPS protein Identity Between Nucleic Acids Or Polypeptides
The terms ""percentage of sequence identity " and '"percentage homolog\ " are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence m the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the vaπety of sequence comparison algoπthms and programs known in the art. Such algonthms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996, Altschul et al., 1993). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see, e.g., Karhn and Altschul, 1990; Altschul et al., 1990, 1993, 1997). In particular, five specific BLAST programs are used to perform the following task:
(1) BLASTP and BLAST3 compare an ammo acid query sequence against a protein sequence database;
(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;
(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and
(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoπng matrix, many of which are known in the art. Preferably, the scoπng matrix used is the BLOSUM62 matnx (Gonnet et al., 1992; Hemkoff and Hemkoff, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karhn (see, e.g., Karhn and Altschul, 1990)
Stringent Hybridization Conditions
By way of example and not limitation, procedures using conditions of high stringency are as 5 follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65°C in buffer composed of 6X SSC, 50 mM Tns-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65°C, the preferred hybndization temperature, m prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe. Alternatively, the hybndization step
10 can be performed at 65°C in the presence of SSC buffer, 1 x SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37°C for 1 h in a solution containing 2 x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1 X SSC at 50°C for 45 mm. Alternatively, filter washes can be performed in a solution containing 2 x SSC and 0.1% SDS, or 0 5 x SSC and 0.1% SDS, or 0.1 x SSC and 0.1% SDS at 68°C for 15 minute intervals.
15 Following the wash steps, the hybπdized probes are detectable by autoradiography. Other conditions of high stnngency which may be used are well known in the art and as cited m Sambrook et al., 1989; and Ausubel et al., 1989, are mcoφorated herein in their entirety. These hybndization conditions are suitable for a nucleic acid molecule of about 20 nucleotides m length. There is no need to say that the hybridization conditions descπbed above are to be adapted according to the 0 length of the desired nucleic acid, following techniques well known to the one skilled m the art. The suitable hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgms (1985) or in Sambrook et al.(1989)
hGGPS gene polynucleotide, cDNAs and associated regulatory regions.
Genomic sequences
25 The invention concerns a purified or isolated nucleic acid encoding the hGGPS polypeptide, wherein said nucleic acid compπses the nucleotide sequence of SEQ LD No 1.
The present invention concerns a punfied or isolated nucleic acid comprising a nucleotide sequence of SEQ LD No 1, or a nucleotide sequence complementary thereto or a fragment or a vanant thereof.
30 Particularly preferred nucleic acids of the invention include isolated, punfied, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No 1 : 1-485, 547-632, 827-7291, 7385-13759, 13831-14062, 14671-15054, and
35 15252-17131. The invention also encompasses a punfied or isolated nucleic acid having at least 95% nucleotide identity with the nucleotide sequence of SEQ ID No 1 or a complementary sequence thereto.
A further object of the invention consists in a purified or isolated nucleic acid of at least 12 nucleotides in length, wherein said nucleic acid hybridizes under stnngent hybndization conditions with a polynucleotide sequence of SEQ LD No 1 or a complementary sequence thereto.
The hGGPS genomic nucleic acid sequence comprises five exons. These five exons are descπbed m Table A.
Table A
Figure imgf000016_0001
The hGGPS mtrons defined hereinafter for the puφose of the present invention are not exactly what is generally understood as "introns" by the one skilled in the art and will consequently be defined below.
Generally, an mtron is defined as a nucleotide sequence that is present both in the genomic DNA and in the unsphced mRNA molecule, and which is absent from the mRNA molecule which has undergone the splicing events. In the case of the hGGPS gene, the inventors have found that at least two different spliced mRNA molecules are produced when this gene is transcribed, as it will be described m detail m a further section of the specification The first spliced mRNA molecule comprises Exons 1, 2, 3 and 4, as shown in Figure 1. Thus, the genomic nucleotide sequence comprised between Exon 1 and Exon 2 is an lntronic sequence as regards to this first mRNA molecule, despite the fact that this lntronic sequence contains Exon Ibis. In contrast, Exon Ibis is of course an exomc nucleotide sequence as regards to the second hGGPS mRNA molecule shown in Figure 2.
For the puφose of the present invention and in order to make a clear and unique designation of the different nucleic acids of the invention, it has been postulated that the polynucleotides contained both in the nucleotide sequence of SEQ LD No 1 and in any of the nucleotide sequences of SEQ LD Nos 2 or 3 are considered as exomc sequences. Conversely, the polynucleotides contained in the nucleotide sequence of SEQ ID No 1 and located between Exon 1 and Exon 4, but which are absent both from the nucleotide sequence of SEQ ID No 2 and from the nucleotide sequence of SEQ ID No 3 are considered as lntronic sequences.
Thus, the invention embodies punfied, isolated, or recombinant polynucleotides compnsing a nucleotide sequence selected from the group consisting of the exons of the hGGPPS gene, or a sequence complementary thereto. The invention also deals with punfied, isolated, or recombinant nucleic acids comprising a combination of at least two exons of the hGGPPS gene, wherein the polynucleotides are arranged within the nucleic acid, from the 5"-end to the 3 "-end of said nucleic acid, in the same order as in SEQ ID No 1
The nucleic acids defining the hGGPS introns descnbed above, as well as their fragments 5 and variants, may be used as oligonucleotide primers or probes in order to detect the presence of a copy of the hGGPS m a test sample, or alternatively in order to amplify a target nucleotide sequence within the hGGPS lntronic sequences hGGPS cD As
The inventors have discovered that the expression of the hGGPS gene leads to the
10 production of at least two mRNA molecules, respectively a first and a second hGGPS transcnption product
The first transcnption product comprises Exons 1, 2, 3 and 4. This cDNA of SEQ ID No 2 includes a 5"-UTR region, spanning the whole Exon 1 and part of Exon 2 This 5'-UTR region starts from the nucleotide at position 1 and ends at the nucleotide in position 84 of SEQ LD No 2. The
15 cDNA of SEQ ID No 2 includes a 3'-UTR region starting from the nucleotide at position 988 and ending at the nucleotide at position 1414 of SEQ ID No 2. The 3'UTR carries a potential polyadenylation signal located between the nucleotide m position 1289 and the nucleotide in position 1294 of the nucleic acid of SEQ LD No 2. The ORF encoding hGGPS is comprised between the nucleotide in position 85 and the nucleotide in position 987 of SEQ LD No 2. 0 The second transcnption product comprises Exons ibis, 2, 3 and 4. This cDNA of SEQ LD
No 3 includes a 5'-UTR region starting from the nucleotide at position 1 and ending at the nucleotide in position 217 of SEQ ID No 3. The cDNA of SEQ ID No 3 includes a 3'-UTR region starting from the nucleotide at position 1121 and ending at the nucleotide at position 1547 of SEQ ID No 3. The 3'UTR carries a potential polyadenylation signal located between the nucleotide in 5 position 1422 and the nucleotide in position 1427 of the nucleic acid of SEQ LD No 3. The ORF encoding hGGPS is compπsed between the nucleotide in position 218 and the nucleotide in position 1120 of the nucleotide sequence of SEQ LD No 3.
Another object of the invention consists of a punfied or isolated nucleic acid selected from the group consisting of the nucleotide sequences of SEQ ID Nos 2 and 3 or a complementary
30 sequence thereto or a fragment thereof
Particularly preferred nucleic acids of the invention include isolated, punfied, or recombinant polynucleotides compπsmg a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70. 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions
35 834-1217 of SEQ LD No 2. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides compπsmg a contiguous span of at least 12, 15, 18, 20, 25, 30, 35. 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3. 5, or 10 of the nucleotide positions 967- 1351 of SEQ LD No 3
The invention also pertains to a purified or isolated nucleic acid having at least 95% of nucleotide identity with any of the nucleotide sequences of SEQ ID Nos 2 and 3 or a complementary sequence thereto
A further object of the invention consists m a punfied or isolated nucleic acid of at least 12 nucleotides in length, wherein said nucleic acid hybridizes under stnngent hybndization conditions with a polynucleotide selected from the group consisting of the nucleotide sequences of SEQ LD Nos 2 and 3, or a sequence complementary thereto. Another object of the invention consists in a punfied or isolated nucleic acid compπsmg a nucleic acid fragment of a nucleotide sequence selected from the group consisting of SEQ LD Nos 2 and 3, wherein this nucleic acid fragment encodes a polypeptide having an ammo acid sequence beginning at the ammo acid in position 200 and ending at the amino acid m position 300 of the hGGPS polypeptide of SEQ ID No 4, or a nucleic acid encoding a peptide fragment thereof. Regulatory sequences
As already mentioned hereinbefore, the polynucleotide of SEQ LD No 1 contains regulatory sequences both in the non-coding 5 "-flanking region and in the non-coding 3 '-flanking region that border the hGGPS coding region.
The longest 5 '-regulatory sequence of the hGGPS gene is localized between the nucleotide in position 1 and the nucleotide in position 632 of SEQ ID Nol . However, a shorter 5 '-regulatory sequence of the hGGPS gene is localized between the nucleotide m position 1 and the nucleotide in position 485 of SEQ LD Nol .
The hGGPS 3 '-regulatory region, as shown in Figure 1, comprises a nucleotide sequence starting from the nucleotide in position 15252 of SEQ LD No 1 and ending at the nucleotide in position 17131 of SEQ ID No 1
Polynucleotides denved from the hGGPS regulatory regions described above are useful m order to detect the presence of at least a copy of the nucleotide sequence of SEQ ID No 1 in a test sample.
The promoter activity of the regulatory regions contained m the h GGPS nucleotide sequence of SEQ LD No 1 can be assessed as descnbed below.
Genomic sequences located upstream of the hGGPS gene are cloned into a suitable promoter reporter vector, such as the pSEAP -Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech. Briefly, each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, beta galactosidase, or green fluorescent protein. The sequences upstream the hGGPS coding region are inserted into the cloning sites upstream of the reporter gene m both onentations and introduced into an appropnate host cell The level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert If necessary, the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcnption levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present m the inserted upstream sequence.
Promoter sequences within the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as Exonuclease III digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundanes of the promoters may be defined If desired, potential individual regulatory sites withm the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcnption factor binding sites within the promoter individually or m combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into cloning sites in promoter reporter vectors
Polynucleotides carrying the regulatory elements located both at the 5' end and at the 3' end of the hGGPS coding region may be advantageously used to control the transcnptional and translational activity of an heterologous polynucleotide of interest. Thus, the present invention also concerns a purified or isolated nucleic acid comprising a polynucleotide which is selected from the group consisting of the 5' and 3' regulatory regions, or a sequence complementary thereto or a biologically active fragment or variant thereof "5' regulatory region" refers to the nucleotide sequence located between positions 1 and 632 of SEQ ID No 1. "3' regulatory region" refers to the nucleotide sequence located between positions 15252 and 17131 of SEQ LD No l
The present invention is also directed to a polynucleotide compπsmg a functional portion of a regulatory region contained m the contemplated hGGPS gene and to its use in a recombinant expression vector carrying a polynucleotide encoding a polypeptide or a nucleic acid of interest.
Preferred fragments of the 5' regulatory region have a length of about 400 nucleotides, more particularly about 300 nucleotides, more preferably 200 nucleotides and most preferably about 100 nucleotides
Preferred fragments of the 3' regulatory region have a length of about 600 nucleotides, more particularly about 300 nucleotides, more preferably 200 nucleotides and most preferably about 100 nucleotides In order, to identify the relevant biologically active polynucleotide derivatives of the 5' and
3* regulatory regions, the one skill in the art will refer to the book of Sambrook et al (1989) which describes the use of a recombinant vector carrying a marker gene (i.e. beta galactosidase. chloramphenicol acetyl transferase. etc.) the expression of which will be detected when placed under the control of a biologically active derivative polynucleotide of the 5' and 3' regulatory regions.
The regulatory polynucleotides of the invention may be prepared from a polynucleotide of the nucleotide sequence SEQ ID No 1 by cleavage using suitable restriction enzymes, as descπbed for example m the book of Sambrook et al. (1989). The regulatory polynucleotides may also be prepared by digestion of a polynucleotide of the nucleotide sequence SEQ LD No 1 by an exonuclease enzyme, such as for example Bal31 (Wabiko et al., 1986). These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification. The regulatory polynucleotides according to the invention may be advantageously part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism. The recombinant expression vectors according to the invention are described elsewhere in the specification.
A preferred 5'-regulatory polynucleotide of the invention includes the 5 '-untranslated region (5'-UTR) located between the nucleotide at position 1 and the nucleotide at position 84 of SEQ ID No 2, or a biologically active fragment or vanant thereof.
Another preferred 5'-regulatory polynucleotide of the invention includes the 5 '-untranslated region (5'-UTR) located between the nucleotide at position 1 and the nucleotide at position 217 of SEQ D No 3, or a biologically active fragment or vanant thereof. A preferred 3 '-regulatory polynucleotide of the invention includes the 3 '-untranslated region
(3'-UTR) consisting in the nucleotide sequence starting from the nucleotide in position 988 and ending a the nucleotide in position 1414 of the nucleic acid of SEQ LD No 2.
A further object of the invention consists of a purified or isolated nucleic acid comprising : a) a nucleic acid comprising the 5' regulatory region or a biologically active fragment or vanant thereof; b) a polynucleotide encoding a desired polypeptide or nucleic acid operably linked to the 5' regulatory region or its biologically active fragment or vanant thereof; c) optionally, a nucleic acid comprising the 3' regulatory region or a biologically active fragment or vanant thereof. The desired polypeptide encoded by the above descnbed nucleic acid may be of vanous nature or ongm, encompassing proteins of prokaryotic or eukaryotic ongin. Among the polypeptides expressed under the control of a hGGPS regulatory region, there may be cited bactenal, fungal or viral antigens. Also encompassed are eukaryotic proteins such as intracellular proteins, like "house keeping" proteins, membrane-bound proteins, like receptors, and secreted proteins like the numerous endogenous mediators such as cytokines. The desired nucleic acids encoded by the above described polynucleotide, usually a RNA molecule, may be complementary to a desired coding polynucleotide, for example to the hGGPS coding sequence, and thus useful as an antisense polynucleotide
Such a polynucleotide may be included a recombinant expression vector in order to express the desired polypeptide or the desired nucleic acid in host cell or in a host organism. Suitable recombinant vectors that contain a polynucleotide such as descπbed hereinbefore are disclosed elsewhere in the specification.
Coding regions
The hGGPS open reading frame is contained in the corresponding mRNAs of SEQ ID Nos 2 and 3.
More precisely, the effective hGGPS coding sequence (CDS) is compπsed between the nucleotide at position 85 (first nucleotide of the ATG codon) and the nucleotide at position 987 (end nucleotide of the TAA codon) of SEQ LD No 2. A purified or isolated polynucleotide comprising the hGGPS coding region defined above is another object of the invention The above disclosed polynucleotide that contains the coding sequence of the hGGPS gene of the invention may be expressed m a desired host cell or a desired host organism, when this polynucleotide is placed under the control of suitable expression signals. The expression signals may be either the expression signals contained m the regulatory regions in the hGGPS gene of the invention or in contrast be exogenous regulatory nucleic sequences. Such a polynucleotide, when placed under the suitable expression signals, may also be inserted m a vector for its expression
Biallelic Markers
The inventors have discovered nucleotide polymoφhisms located withm the genomic DNA containing the hGGPS gene, and among them SNP that are also termed biallehc markers. The biallehc markers of the invention can be used for example for the generation of genetic map, the linkage analysis, the association studies.
A) Identification Of Biallelic Markers
There are two preferred methods through which the biallehc markers of the present invention can be generated. In a first method, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymoφhisms.
One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions which must be carried out. Moreover, this method is sufficiently sensitive so that a biallehc marker obtained therewith usually shows a sufficient degree of informativeness for conducting association studies. In a second method for generating biallehc markers, the DNA samples are not pooled and are therefore amplified and sequenced individually. The resulting nucleotide sequences obtained are then also analyzed to identify significant polymoφhisms
The following is a descnption of the various parameters of a preferred method used by the inventors to generate the markers of the present invention
1 DNA extraction
The genomic DNA samples from which the biallehc markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually preferred to collect DNA samples from at least about 100 individuals order to have sufficient polymoφhic diversity in a given population to identify as many markers as possible and to generate statistically significant results. As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples which can be tested by the methods of the present invention descnbed herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, unne, lymph fluids, and various external secretions of the respiratory, intestinal and genitouπnary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the context of the present invention is from peπpheral venous blood of each donor.
The techniques of DNA extraction are well-known to the skilled technician. Such techniques are described notably by Lin et al. (1998) and by Mackey et al. (1998). Details of a preferred embodiment are provided in Example 2
2. DNA amplification
The identification of biallehc markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art.
Amplification techniques that can be used in the context of the present invention include, but are not limited to, the hgase chain reaction (LCR) described in EP-A- 320 308, WO 9320227 and
EP-A-439 182, the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) descnbed in Guatelli J.C., et al.(1990) and m Compton J.(l 991 ), Q-beta amplification as descnbed in European Patent Application No 4544610, strand displacement amplification as descnbed in Walker et al.(1996) and EP A 684 315 and, target mediated amplification as descπbed m PCT Publication WO 9322461. LCR and Gap LCR are exponential amplification techniques, both depend on DNA hgase to join adjacent primers annealed to a DNA molecule In Ligase Cham Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target The first probe hybridizes to a first segment of the target strand and the second probe hybndizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate- 3 'hydroxyl relationship, and so that a ligase can covalently fuse or hgate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybndize to a portion of the second probe m a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance Once the hgated strand of pnmary probes is separated from the target strand, it will hybndize with the third and fourth probes, which can be hgated to form a complementary, secondary hgated product. It is important to realize that the hgated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and hgation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227) Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.
For amplification of mRNAs, it is withm the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as descπbed in U.S. Patent No. 5,322,770 or, to use Asymmetnc Gap LCR (RT-AGLCR) as described by Marshall et al.(1994). AGLCR is a modification of GLCR that allows the amplification of RNA.
The PCR technology is the preferred amplification technique used m the present invention A vanety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White ( 1997) and the publication entitled "PCR Methods and Applications" ( 1991 , Cold Spπng Harbor Laboratory Press). In each of these PCR procedures, PCR pnmers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybndized pπmers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the pnmer sites. PCR has further been descnbed in several patents including US Patents 4,683,195, 4,683,202; and 4,965,188 The PCR technology is the preferred amplification technique used to identify new biallehc markers. A typical example of a PCR reaction suitable for the puφoses of the present invention is provided m Example 3 One of the aspects of the present invention is a method for the amplification of the human hGGPPS gene, particularly of a fragment of the genomic sequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID No 2 or 3, or a fragment or a variant thereof in a test sample, preferably using the PCR technology This method comprises the steps of a) contacting a test sample with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side of the polynucleotide region to be amplified, and b) optionally, detecting the amplification products.
The invention also concerns a kit for the amplification of a hGGPPS gene sequence, particularly of a portion of the genomic sequence of SEQ LD No 1 or of the cDNA sequence of SEQ ID No 2 or 3, or a variant thereof in a test sample, wherein said kit compnses: a) a pair of oligonucleotide pnmers located on either side of the hGGPPS region to be amplified; b) optionally, the reagents necessary for performing the amplification reaction. In one embodiment of the above amplification method and kit, the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region. In another embodiment of the above amplification method and kit, pnmers comprise a sequence which is selected from the group consisting of SEQ ID Nos 7-9.
In a first embodiment of the present invention, bialle c markers are identified using genomic sequence information generated by the inventors. Sequenced genomic DNA fragments are used to design pnmers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallehc markers. Pnmers may be designed using the OSP software (Hilher L. and Green P., 1991). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing pnmer. Those skilled in the art are familiar with pnmer extensions, which can be used for these puφoses.
Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes. A biallehc marker presents a higher probability to be an eventual causal mutation if it is located in these functional regions of the gene. Preferred amplification pnmers of the invention include the nucleotide sequences of SEQ LD Nos 8 and 9
Other preferred pnmers according to the invention allow the amplification of vaπous fragments of the punfied or isolated nucleic acid of SEQ LD No 1. These pnmers are presented below as couples of forward and reverse pnmers that may be used together to amplify a desired nucleotide sequence.
Figure imgf000024_0001
Figure imgf000025_0001
The pnmers descnbed above are individually useful as oligonucleotide probes in order to detect the corresponding hGGPS nucleotide sequence in a sample, and more preferably to detect the presence of a hGGPS DNA molecule in a sample suspected to contain it 3 Sequencing of amplified genomic DNA and identification of polymoφhisms
The amplification products generated as descπbed above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Sambrook et al.(1989). Alternative approaches include hybridization to high-density DNA probe arrays as descπbed in Chee et al.(1996).
Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-pnmer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymoφhism search is based on the presence of supenmposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallehc site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a compaπson between the peaks is earned out. In order to be registered as a polymoφhic sequence, the polymoφhism has to be detected on both strands.
The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallehc polymoφhisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known alle c frequencies. However, more than 90% of the biallehc polymoφhisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallehc markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.
In another embodiment, biallehc markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallehc marker may be less than 0.1. In a particular embodiment of the invention, the test samples are a pool of 100 individuals and 50 individual samples. This is the methodology used in the preferred embodiment of the present invention, in which 1 biallehc marker has been identified in a genomic region containing the hGGPS gene This bialle c marker is called 5-187-77 and is located in intron 3 of hGGPPS gene The biallehc marker consists in an insertion of a nucleotide T
The polymoφhisms identified above can be further confirmed and their respective frequencies can be determined through various methods using the previously descnbed primers and probes as descπbed herein. These methods can also be useful for genotypmg either new populations in association studies or linkage analysis or individuals in the context of detection of alleles of biallehc markers which are known to be associated with a given trait. The genotypmg of the biallehc markers is also important for the mapping. It will be appreciated that the methods described below can be equally performed on individual or pooled DNA samples. b) Genotyping Of Biallelic Markers
Once a given polymoφhic site has been found and characterized as a bialle c marker as described above, several methods can be used in order to determine the specific allele earned by an individual at the given polymoφhic base. The identification of biallehc markers descπbed previously allows the design of appropπate oligonucleotides, which can be used as probes and pπmers, to amplify a hGGPS gene containing the polymoφhic site of interest and for the detection of such polymoφhisms.
In one embodiment the invention encompasses methods of genotyping comprising determining the identity of a nucleotide at a ΛGGP S-related biallelic marker or the complement thereof in a biological sample; optionally, wherein said hGGPPS -related biallehc marker is the biallehc marker 5-187-77, and the complement thereof; optionally, wherein said biological sample is derived from a single subject; optionally, wherein the identity of the nucleotides at said bialle c marker is determined for both copies of said biallehc marker present in said individual's genome, optionally, wherein said biological sample is deπved from multiple subjects; Optionally, the genotyping methods of the invention encompass methods with any further limitation descπbed in this disclosure, or those following, specified alone or in any combination; Optionally, said method is performed in vitro; optionally, further comprising amplifying a portion of said sequence comprising the biallehc marker pnor to said determining step; Optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector compπsmg an ongin of replication and said fragment m a host cell; optionally, wherein said determining is performed by a hybridization assay, a sequencing assay, a microsequencmg assay, or an enzyme-based mismatch detection assay
1) Amplification
Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallehc marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallehc markers may be used in vaπous methods and for vanous puφoses and is not restπcted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention. Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above in the section entitled, "DNA amplification."
Some of these amplification methods are particularly suited for the detection of single nucleotide polymoφhisms and allow the simultaneous amplification of a target sequence and the identification of the polymoφhic nucleotide as it is further described below. The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention. In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in Example 3. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention are also of use. The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention, amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in "Oligonucleotide probes and primers".
2) Sequencing
The nucleotide present at a polymoφhic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in "Sequencing Of Amplified Genomic DNA And Identification Of Polymoφhisms".
Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site. 3) Microsequencing
In microsequencing methods, the nucleotide at a polymoφhic site in a target DNA is detected by a single nucleotide primer extension reaction This method involves appropnate microsequencing primers which, hybndize just upstream of the polymoφhic base of interest m the target nucleic acid. A polymerase is used to specifically extend the 3' end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymoφhic site. Next the identity of the incoφorated nucleotide is determined m any suitable way.
Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incoφorated nucleotide as descnbed in EP 412 883, the disclosure of which is incoφorated herein by reference m its entirety. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used m the context of the present invention is provided in Example 5. Different approaches can be used for the labeling and detection of ddNTPs. A homogeneous phase detection method based on fluorescence resonance energy transfer has been descπbed by Chen and Kwok (1997) and Chen et al.(1997). Alternatively, the extended pπmer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymoφhic site is identified by the mass added onto the microsequencing primer (see Haff and Smirnov, 1997). Microsequencing may be achieved by the established microsequencing method or by developments or deπvatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogeneous phase assay, in which the pπmer or the target molecule is immobilized or captured onto a solid support. For example, immobilization can be earned out via an interaction between biotmylated DNA and streptavidm-coated microtitration wells or avidm-coated polystyrene particles. In the same manner, oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incoφorated ddNTPs can be radiolabeled (Syvanen, 1994) or linked to fluorescem (Livak and Hainer, 1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescem-hnked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromoge c substrate (such as^-nitrophenyl phosphate). Other possible reporter- detection pairs include: ddNTP linked to dmitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotmylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712). As yet another alternative solid-phase microsequencing procedure, Nyren et al.(1993) descπbed a method relying on the detection of DNA polymerase activity by an enzymatic luminometπc inorganic pyrophosphate detection assay (ELIDA).
Past en et al.(1997) descnbe a method for multiplex detection of single nucleotide polymoφhism in which the solid phase m isequencing principle is applied to an oligonucleotide array format High-density arrays of DNA probes attached to a solid support (DNA chips) are further descπbed below.
In one aspect the present invention provides polynucleotides and methods to genotype one or more biallehc markers of the present invention by performing a microsequencing assay. Preferred microsequencing pπmers include the nucleotide sequence of SEQ ID No 7. It will be appreciated that the microsequencing primer of SEQ ID No 7 is merely exemplary and that, any pπmer having a 3' end immediately adjacent to the polymoφhic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallehc marker or any combination of biallehc markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing pπmers for determining the identity of a nucleotide at a biallehc marker site.
4. Mismatch detection assays based on polymerases and ligases
In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallehc markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymeπzation reactions places particularly stnngent requirements on correct base pairing of the 3' end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the hgation site, especially at the 3' end. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in "DNA amplification".
Allele Specific Amplification Primers
Discnmination between the two alleles of a biallehc marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. For allele specific amplification, at least one member of the pair of pπmers is sufficiently complementary with a region of a hGGPPS gene comprising the polymoφhic base of a bialle c marker of the present invention to hybndize therewith and to initiate the amplification Such primers are able to discπminate between the two alleles of a biallehc marker.
This is accomplished by placing the polymoφhic base at the 3' end of one of the amplification primers. Because the extension forms from the 3 'end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropπate amplification conditions, these pπmers only direct amplification on their complementary allele. Determining the precise location of the mismatch and the corresponding assay conditions are well withm the ordinary skill in the art Ligation/ Amplification Based Methods
The "Oligonucleotide Ligation Assay" (OLA) uses two oligonucleotides which are designed to be capable of hybndizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotmylated, and the other is detectably labeled If the precise complementary sequence is found m a target molecule, the oligonucleotides will hybndize such that their termini abut, and create a hgation substrate that can be captured and detected. OLA is capable of detecting single nucleotide polymoφhisms and may be advantageously combined with PCR as described by Nickerson et al ( 1990) In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
Other amplification methods which are particularly suited for the detection of single nucleotide polymoφhism include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in "DNA Amplification". LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallehc marker site. In one embodiment, either oligonucleotide will be designed to include the biallehc marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be hgated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallehc marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallehc marker, such that when they hybridize to the target molecule, a "gap" is created as described in WO 90/01069. This gap is then "filled" with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target dunng the next cycle and exponential allele-specific amplification of the desired sequence is obtained.
Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271). This method involves the coφoration of a nucleoside tπphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a pnmer molecule, and their subsequent ligation to a second oligonucleotide The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution. 5. Hybridization Assay Methods
A preferred method of determining the identity of the nucleotide present at a biallehc marker site involves nucleic acid hybridization. The hybndization probes, which can be conveniently used in such reactions, preferably include the probes defined herein Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybndization and solid- phase hybridization (see Sambrook et al , 1989)
Specific probes can be designed that hybridize to one form of a biallehc marker and not to the other and therefore are able to discnmmate between different allehc forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybndization conditions should be sufficiently stnngent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stnngent, sequence specific hybndization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989). Although such hybridization can be performed in solution, it is preferred to employ a solid-phase hybndization assay. The target DNA comprising a biallehc marker of the present invention may be amplified pπor to the hybridization reaction. The presence of a specific allele m the sample is determined by detecting the presence or the absence of stable hybπd duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be earned out by a number of methods. Vanous detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybnd duplexes. Typically, hybndization duplexes are separated from unhybndized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybπds using the labels present on the pnmers and probes.
Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., 1998). The TaqMan assay takes advantage of the 5' nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase dunng amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allehc variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., 1995). In an alternative homogeneous hybridization based procedure, molecular beacons are used for allele discnmmations. Molecular beacons are haiφin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., 1998). The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of bialle c marker alleles in biological samples These probes are characteπzed in that they preferably comprise between 8 and 50 nucleotides. and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide vanation. A particularly preferred probe is 25 nucleotides m length. Preferably the biallehc marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes, the biallehc marker is at the center of said polynucleotide. Preferred probes comprise a nucleotide sequence selected from the group consisting of amphcons listed in Table 1 and the sequences complementary thereto, or a fragment thereof, said fragment compπsing at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymoφhic base. Preferred probes comprise a nucleotide sequence selected from the group consisting of SEQ ID Nos 5 and 6 and the sequences complementary thereto. In preferred embodiments the polymoφhic base(s) are withm 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide.
Preferably the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in "Oligonucleotide Probes and Primers". The probes can be non-extendable as described in "Oligonucleotide Probes and Primers". By assaying the hybndization to an allele specific probe, one can detect the presence or absence of a biallehc marker allele in a given sample. High-Throughput parallel hybridization in array format is specifically encompassed within "hybridization assays" and are descπbed below.
6- Hybridization To Addressable Arrays Of Oligonucleotides
Hybridization assays based on oligonucleotide arrays rely on the differences in hybndization stability of short oligonucleotides to perfectly matched and mismatched target sequence vanants. Efficient access to polymoφhism information is obtained through a basic structure compπsing high- density arrays of oligonucleotide probes attached to a solid support (e.g., the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged m a grid-like pattern and mmiatunzed to the size of a dime. The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S cerevisiae mutant strains, and in the protease gene of HLV-1 virus (Hacia et al, 1996; Shoemaker et al., 1996, Kozal et al., 1996) Chips of vanous formats for use m detecting biallehc polymoφhisms can be produced on a customized basis by Affymetnx (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.
In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymoφhic marker EP 785280 descnbes a tiling strategy for the detection of single nucleotide polymoφhisms. Briefly, arrays may generally be "tiled" for a large number of specific polymorphisms. By "tiling*" is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of nucleotides. Tiling strategies are further descnbed in PCT application No. WO 95/11995. Hybndization and scanning may be earned out as described in PCT application No. WO 92/10092 and WO 95/11995 and US patent No. 5,424,186.
Thus, in some embodiments, the chips may compπse an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may compnse an array including at least one of the sequences selected from the group consisting of amphcons listed in table 1 and the sequences complementary thereto, or a fragment thereof, said fragment comprising at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50 consecutive nucleotides and containing a polymoφhic base. In preferred embodiments the polymoφhic base is withm 5, 4, 3, 2, 1, nucleotides of the center of the said polynucleotide, more preferably at the center of said polynucleotide. In some embodiments, the chip may compnse an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further descπbed in "Oligonucleotide Probes And Primers". 7- Integrated Systems
Another technique, which may be used to analyze polymoφhisms, includes multicomponent integrated systems, which mmiatunze and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in US patent 5,589,136, which descnbes the integration of PCR amplification and capillary electrophoresis m chips.
Integrated systems can be envisaged mainly when microfluidic systems are used. These systems compπse a pattern of microchannel s designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electπc, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.
For genotyping bialle c markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser- induced fluorescence detection.
Oligonucleotide Probes and primers Polynucleotides deπved from the hGGPPS gene are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No 1, or a fragment, complement, or vanant thereof m a test sample Furthermore polynucleotides denved from the hGGPPS gene can be used to generate antisense polynucleotide or polynucleotide for the triple helix strategy
Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ LD No 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ LD No l : 1-485, 547-632, 827-7291 , 7385-13759, 13831-14062, 14671-15054, and 15252-17131.
The invention also relates to nucleic acid probes characteπzed m that they hybridize specifically, under the stnngent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences 1-485, 547-632, 827-7291, 7385-13759, 13831-14062, 14671-15054, and 15252-17131 of SEQ LD No 1 or a variant thereof or a sequence complementary thereto.
Particularly preferred probes and primers of the invention include isolated, punfied, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ LD No 2 or the complements thereof, wherein said contiguous span compnses at least 1, 2, 3, 5, or 10 of the nucleotide positions 834-1217 of SEQ ID No 2. Additional preferred probes and pπmers of the invention include isolated, punfied, or recombinant polynucleotides compnsmg a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ LD No 2 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the nucleotide positions 967-1351 of SEQ LD No 3.
The invention also relates to nucleic acid probes characteπzed m that they hybridize specifically, under the stnngent hybridization conditions defined above, with a nucleic acid selected from the group consisting of the nucleotide sequences 834-1217 of SEQ LD No 2 and 967-1351 of SEQ LD No 3, or a variant thereof or a sequence complementary thereto.
In one embodiment the invention encompasses isolated, punfied, and recombinant polynucleotides consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ LD Nos 1 -3 and the complement thereof, wherein said span includes a hGGPPS- related biallehc marker in said sequence; optionally, wherein said A G S-related bialle c marker is the biallehc marker 5-187-77, and the complement thereof; optionally, wherein said contiguous span is 18 to 50 nucleotides in length and said biallehc marker is withm 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallehc marker is at the center of said polynucleotide; optionally, wherein the 3' end of said contiguous span is present at the 3' end of said polynucleotide; and optionally, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and said biallehc marker is present at the 3' end of said polynucleotide. In a preferred embodiment, said probes comprises, consists of, or consists essentially of a sequence selected from SEQ LD Nos 5 and 6 and the complementary sequences thereto
In another embodiment the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 5 nucleotides of SEQ ID Nos 1 -3, or the complements thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located within 20 nucleotides upstream of a hGGPPS -related biallehc marker in said sequence; optionally, wherein said /zGG PS-related biallehc marker is the biallehc marker 5-187-77, and the complement thereof; optionally, wherein the 3' end of said polynucleotide is located 1 nucleotide
10 upstream of said hGGPPS -related bialle c marker in said sequence; and optionally, wherein said polynucleotide consists essentially of a sequence of SEQ LD No 7.
In a further embodiment, the invention encompasses isolated, purified, or recombinant polynucleotides compπsing, consisting of, or consisting essentially of a sequence selected from the sequences of SEQ LD Nos 8and 9
15 In an additional embodiment, the invention encompasses polynucleotides for use m hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a hGGPPS -related biallehc marker, as well as polynucleotides for use in amplifying segments of nucleotides comprising a hGGPPS -related biallelic marker; optionally, wherein said hGGPPS-related biallehc marker is the biallelic marker 5-
20 187-77, and the complements thereof.
A probe or a pπmer according to the invention has between 8 and 1000 nucleotides in length, or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000 nucleotides in length. More particularly, the length of these probes and pnmers can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30
25 nucleotides. The appropnate length for pπmers and probes under a particular set of assay conditions may be empmcally determined by one of skill m the art. A preferred probe or pnmer consists of a nucleic acid comprising a polynucleotide selected from the group of the nucleotide sequences of SEQ ED Nos 5-9 or a fragment thereof or a complementary sequence thereto.
The formation of stable hybπds depends on the meltmg temperature (Tm) of the DNA. The
30 Tm depends on the length of the pnmer or probe, the ionic strength of the solution and the G+C content. The higher the G+C content of the pπmer or probe, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The GC content in the probes of the invention usually ranges between 10 and 75 %, preferably between 35 and 60 %, and more preferably between 40 and 55 %.
35 The pnmers and probes can be prepared by any suitable method, including, for example, cloning and restπction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al.(1979), the phosphodiester method of Brown et al.(1979), the diethylphosphoramidite method of Beaucage et al (1981) and the solid support method descπbed in EP 0 707 592
Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed m International Patent Application WO 92/20702, moφholmo analogs which are descπbed in U.S Patents Numbered 5,185,444. 5,034,506 and 5,142,047 The probe may have to be rendered "'non-extendable*" in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3' end of the probe can be functionahzed with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3' hydroxyl group simply can be cleaved, replaced or modified, U.S. Patent Application Seπal No. 07/049,061 filed Apnl 19, 1993 describes modifications, which can be used to render a probe non-extendable.
Any of the polynucleotides of the present invention can be labeled, if desired, by incoφorating any label known in the art to be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances (including, 32P, 35S, 3H, 125I), fluorescent dyes (including, 5-bromodesoxyuπdm, fluorescem, acetylammofluorene, digoxigenm) or biotin. Preferably, polynucleotides are labeled at their 3' and 5' ends. Examples of non-radioactive labeling of nucleic acid fragments are described m the French patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988). In addition, the probes according to the present invention may have structural characteπstics such that they allow the signal amplification, such structural charactenstics being, for example, branched DNA probes as those descnbed by Urdea et al. in 1991 or in the European patent No. EP 0 225 807 (Chiron). A label can also be used to capture the pnmer, so as to facilitate the immobilization of either the primer or a pnmer extension product, such as amplified DNA, on a solid support. A capture label is attached to the pnmers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label earned by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, pnmers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a pnmer or probe to thereby immobilize the pπmer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to the target. In the case where a polynucleotide pπmer itself serves as the capture label, at least a portion of the pnmer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician
The probes of the present invention are useful for a number of puφoses They can be notably used in Southern hybndization to genomic DNA. The probes can also be used to detect PCR amplification products They may also be used to detect mismatches in the hGGPPS gene or mRNA using other techniques.
Any of the polynucleotides, pπmers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose stπps, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others. The solid support is not cntical and can be selected by one skilled m the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or dunng the performance of the assay. The solid phase thus can be a plastic, denvatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill m the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support. In addition, polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention
Consequently, the invention also deals with a method for detecting the presence of a nucleic acid comprising at least a part of a nucleotide sequence selected from the group consisting of SEQ ID Nos 1-3 in a sample, said method compπsing the following steps of : a) bπngmg into contact a nucleic acid probe or a plurality of nucleic acid probes, which can hybndize to a nucleotide sequence included in one of the nucleic acids of SEQ ED Nos 1-3, and the sample to be assayed b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample Preferably, the nucleic acid probe is selected from the group of polynucleotides consisting of the nucleotide sequences SEQ ID Nos 5-9. In a first preferred embodiment of this detection method, said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule In a second preferred embodiment of said method, said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate. The invention further concerns a kit for detecting the presence of a nucleic acid comprising at least a part of a nucleotide sequence selected from the group consisting of SEQ ED Nos 1-3 in a sample, said kit comprising . a) a nucleic acid probe or a plurality of nucleic acid probes which can hybndize to a nucleotide sequence included m one of the nucleic acids of SEQ ED Nos 1-3; b) optionally, the reagents necessary for performing the hybridization reaction.
The nucleic acid probe or the plurality of nucleic acid probes that are included m the detection kit descπbed above may be selected from the group consisting of SEQ ED Nos 5-9. In a first preferred embodiment of the detection kit, the nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule. In a second preferred embodiment of the detection kit, the nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.
Oligonucleotide arrays
A substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in the hGGPPS gene and may also be used for detecting mutations m the coding or in the non-coding sequences of the hGGPPS gene Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these "addressable" arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips™, and has been generally descπbed US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays may generally be produced using mechanical synthesis methods or hght directed synthesis methods which incoφorate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al , 1991) The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as "Very Large Scale Immobilized Polymer Synthesis" (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPS™ technologies are provided in US Patents 5.143,854; and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, which descnbe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybndization patterns and sequence information Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.
In another embodiment of the oligonucleotide arrays of the invention, an oligonucleotide probe matπx may advantageously be used to detect mutations occurnng m the hGGPPS gene and preferably in its regulatory region. For this particular puφose, probes are specifically designed to have a nucleotide sequence allowing their hybndization to the genes that carry known mutations (either by deletion, insertion or substitution of one or several nucleotides). By known mutations, it is meant, mutations on the hGGPPS gene that have been identified according, for example to the technique used by Huang et al.(1996) or Samson et al.(1996).
Another technique that is used to detect mutations in the hGGPPS gene is the use of a high- density DNA array. Each oligonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of the hGGPPS genomic DNA or cDNA. Thus, an array consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the hGGPPS gene. In one such design, termed 4L tiled array, is implemented a set of four probes (A, C, G, T), preferably 15-nucleotιde ohgomers. In each set of four probes, the perfect complement will hybndize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence. The hybndization signals of the 15- mer probe set tiled array are perturbed by a single base change m the target sequence. As a consequence, there is a characteristic loss of signal or a "footprint" for the probes flanking a mutation position. This technique was described by Chee et al. in 1996. Consequently, the invention concerns an array of nucleic acid molecules compπsing at least one polynucleotide descπbed above as probes and pnmers. Preferably, the invention concerns an array of nucleic acid compπsing at least two polynucleotides descπbed above as probes and pnmers. A further object of the invention consists of an array of nucleic acid sequences compnsmg either at least one of the sequences selected from the group consisting of SEQ ED Nos 5-9, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence comprising the biallehc marker 5-187-77 and the complements thereto
The invention also pertains to an array of nucleic acid sequences compnsmg either at least two of the sequences selected from the group consisting of SEQ ED Nos 5-9, the sequences complementary thereto, a fragment thereof of at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, and at least one sequence compnsmg the biallehc marker 5-187-77 and the complements thereto.
Vectors for the expression of a regulatory or a coding polynucleotide according to the invention.
Any of the regulatory polynucleotides or the coding polynucleotides of the invention may be inserted into recombinant vectors for expression in a recombinant host cell or a recombinant host organism.
Thus, the present invention also encompasses a family of recombinant vectors that contains either a regulatory polynucleotide selected from the group consisting of the regulatory polynucleotides derived from the hGGPS gene, or a polynucleotide comprising the hGGPS coding sequence, or both. More particularly, the present invention relates to expression vectors which include nucleic acids encoding the hGGPS protein of the ammo acid sequence of SEQ ID No 4 descnbed therein under the control of either one regulatory sequence selected among the hGGPS regulatory polynucleotides, or alternatively under the control of an exogenous regulatory sequence.
A recombinant expression vector compnsmg a nucleic acid selected from the group consisting of the 5' or 3' regulatory regions of hGGPPS, or biologically active fragments or vanants thereof, is also part of the present invention.
Generally, a recombinant vector of the invention may compnse any of the polynucleotides descnbed herein, including regulatory sequences, and coding sequences, as well as any hGGPPS primer or probe as defined above. More particularly, the recombinant vectors of the present invention can comprise any of the polynucleotides described in the "hGGPPS cDNA Sequences" section, the "Coding Regions" section, "Genomic sequences" section and the "Oligonucleotide Probes And Primers" section
Some of the elements which can be found m the vectors of the present invention are descnbed m further detail m the following sections. a) Vectors
A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid. a cosmid, a plasmid or even a linear DNA molecule which may consist of a chromosomal, non- chromosomal and synthetic DNA. Such a recombinant vector can comprise a transcnptional unit comprising an assembly of :
(1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp in length that act on the promoter to increase the transcription. (2) a structural or coding sequence which is transcπbed into mRNA and eventually translated into a polypeptide, and
(3) appropnate transcnption initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
Generally, recombinant expression vectors will include ongms of replication, selectable markers permitting transformation of the host cell, and a promoter deπved from a highly expressed gene to direct transcription of a downstream structural sequence. The heterologous structural sequence is assembled in appropπate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of translated protein into the peπplasmic space or extracellular medium.
The selectable marker genes for selection of transformed host cells are preferably dihydrofolate reductase or neomycm resistance for eukaryotic cell culture, TRPl for S cerevisiae or tetracychne, πfampicin or ampicillin resistance in E. coli, or levan saccharase for mycobactena.
As a representative but non-hmitmg example, useful expression vectors for bacterial use can comprise a selectable marker and bactenal ongin of replication denved from commercially available plasmids compnsmg genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEMl (Promega Biotec, Madison, WI,
USA)
Large numbers of suitable vectors and promoters are known to those of skill m the art, and commercially available, such as bactenal vectors : pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescnpt, psιX174, pbluescnpt SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pREE5 (Pharmacia); or eukaryotic vectors : pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia), baculovirus transfer vector pVL1392/1393 (Pharmingen); pQE-30 (QIAexpress). A suitable vector for the expression of the hGGPS polypeptide of SEQ ID No 4 is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 171 1 ) which is derived from Spodoptera frugiperda. Other suitable vectors for the expression of the hGGPS polypeptide of SEQ ID No 4 in a baculovirus expression system include those descπbed by Chai et al. (1993), Vlasak et al. (1983) and Lenhard et al. (1996).
Mammalian expression vectors will compπse an ong of replication, a suitable promoter and enhancer, and also any necessary πbosome binding sites, polyadenylation signal, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscnbed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 ongm, early promoter, enhancer, splice and polyadenylation signals may be used to provide the required nontranscnbed genetic elements. b) Promoters The suitable promoter regions used in the expression vectors according to the present invention are chosen taking into account the cell host m which the heterologous gene has to be expressed.
A suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
Preferred bactenal promoters are the Lad, LacZ, the T3 or T7 bactenophage RNA polymerase promoters, the polyhedπn promoter, or the pi 0 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.
Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors. Particularly preferred bactenal promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidme kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.
The choice of a promoter is well withm the ability of a person skilled the field of genetic egmeenng. For example, one may refer to the book of Sambrook et al. (1989) or also to the procedures descπbed by Fuller et al . ( 1996)
The vector containing the appropriate DNA sequence as descnbed above, more preferably a hGGPS gene regulatory polynucleotide, a polynucleotide encoding the hGGPS polypeptide of SEQ ID No 4 or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide. c) Other types of vectors
The in vivo expression of a hGGPS polypeptide of SEQ ID No 4 may be useful in order to correct a genetic defect related to the expression of the native gene in a host organism or to the production of a biologically inactive hGGPS protein
Consequently, the present invention also deals with recombinant expression vectors mamly designed for the in vivo production of the hGGPS polypeptide of SEQ ED No 4 by the introduction of the appropriate genetic matenal in the organism of the patient to be treated. This genetic material may be introduced in vitro m a cell that has been previously extracted from the organism, the modified cell being subsequently remtroduced in the said organism, directly in vivo into the appropriate tissue, and preferably m the olfactory epithelium
By « vector » according to this specific embodiment of the invention is intended either a circular or a linear DNA molecule. One specific embodiment for a method for delivering a protein or peptide to the teπor of a cell of a vertebrate in vivo comprises the step of introducing a preparation compnsmg a physiologically acceptable earner and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the mteπor of the cell and has a physiological effect. In a specific embodiment, the invention provides a composition for the in vivo production of the hGGPS protein or polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable earner, and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide. Compositions comprising a polynucleotide are described in the PCT application N° WO 90/11092 (Vical Inc.) and also in the PCT application N° WO 95/1 1307 (Instirut Pasteur, INSERM, Universite d'Ottawa) as well as in the articles of Tacson et al. (1996) and of Huygen et al. (1996). The amount of the vector to be injected to the desired host organism vary according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body. In another embodiment of the vector according to the invention, it may be introduced m vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired hGGPS polypeptide or the desired C-termmal fragment thereof is remtroduced into the animal body in order to deliver the recombinant protein withm the body either locally or systemically.
In one specific embodiment, the vector is deπved from an adenovirus. Preferred adenovirus vectors according to the invention are those descnbed by Feldman and Steg (1996) or Ohno et al (1994) Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin ( French patent application N° FR-93.05954)
Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous polynucleotides in vivo , particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host
Particularly preferred retroviruses for the preparation or construction of retroviral in vitro or in vitro gene delivery vehicles of the present invention include retroviruses selected from the group consisting of Mmk-Cell Focus Inducing Virus, Murine Sarcoma Virus, Reticuloendothehosis virus and Rous Sarcoma virus. Particularly preferred Munne Leukemia Viruses include the 4070A and the 1504A viruses, Abelson (ATCC No VR-999), Fnend (ATCC No VR-245), Gross (ATCC No VR- 590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferred Rous Sarcoma Viruses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728). Other preferred retroviral vectors are those described m Roth et al. (1996), the PCT Application No WO 93/25234, the PCT Application No WO 94/ 06920, Roux et al., 1989, an et al., 1992 and Neda et al., 1991.
Yet another viral vector system that is contemplated by the invention consists m the adeno- associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a heφes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividmg cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989). One advantageous feature of AAV denves from its reduced efficacy for transducing pπmary cells relative to transformed cells. Other compositions containing a vector of the invention advantageously comprise an oligonucleotide fragment of a nucleic sequence selected from the group consisting of SEQ LD Nos 2 or 3 as an antisense tool that inhibits the expression of the corresponding hGGPS gene. Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al. (1995) or also in the PCT Application No WO 95/24223 Preferably, the antisense tools are chosen among the polynucleotides (15-200 bp long) that are complementary to the 5'end of the hGGPS mRNAs. In another embodiment, a combination of different antisense polynucleotides complementary to different parts of the desired targeted gene are used.
Preferred antisense polynucleotides according to the present invention are complementary to a sequence of the mRNAs of hGGPS that contains the translation initiation codon ATG Host cells
Another object of the invention consists in cell host that have been transformed or transfected with one of the polynucleotides described therein, and more precisely a polynucleotide either comprising a hGGPS regulatory polynucleotide or the coding sequence of the hGGPS polypeptide having the amino acid sequence of SEQ LD No 4. Are included cell hosts that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as those described above
A cell host according to the present invention is characterized in that its genome or genetic background (including chromosome, plasmids) is modified by the heterologous nucleic acid coding for the hGGPS polypeptide of SEQ ED No 4.
More particularly, the cell hosts of the present invention can comprise any of the polynucleotides described in "hGGPPS cDNA Sequences" section, the "Coding Regions" section, "Genomic sequences" section and the "Oligonucleotide Probes And Primers" section.
Preferred cell hosts used as recipients for the expression vectors of the invention are the following : a) Prokaryotic host cells : Escherichia coli strains (I.E. DH5-α strain) or Bacillus subtilis. b) Eukaryotic host cells : HeLa cells (ATCC N°CCL2; N°CCL2.1 ; N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL1650; N°CRL1651), Sf-9 cells (ATCC N°CRL171 1).
The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
Following transformation of a suitable host and growth of the host to an appropnate cell density, the selected promoter is induced by appropnate means, such as temperature shift or chemical induction, and cells are cultivated for an additional peπod.
Cells are typically harvested by centnfugation, disrupted by physical or chemical means, and the resulting crude extract retained for further punfication.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysmg agents. Such methods are well known by the skill artisan.
Cell hosts can be used to generate transgenic animals. Therefore, the invention concerns a non-human host animal or mammal compπsmg a recombinant vector or a host cell according to the invention. More particularly, the invention concerns a mammalian host cell or a non-human host mammal compnsmg a hGGPPS gene disrupted by homologous recombination with a knock out vector and comprising a polynucleotide according to the invention
hGGPPS Proteins and Polypeptide Fragments: The term "hGGPPS polypeptides" is used herein to embrace all of the proteins and polypeptides of the present invention. Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides compnsmg such polypeptides The invention embodies hGGPPS proteins from humans, including isolated or purified hGGPPS proteins consisting, consisting essentially, or compπsing the sequence of SEQ LD No 4. It should be noted the hGGPPS proteins of the invention are based on the naturally-occurnng variant of the ammo acid sequence of human hGGPPS, wherein a phenylala ne residue is at positions 204, 257, 295 of SEQ LD No 4, a cysteme residue is at position 205 of SEQ LD No 4, a prohne residue is at position 225 of SEQ ID No 4, and a glutamic acid residue is at position 252 of SEQ ED No 4.
The present invention embodies isolated, punfied, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 ammo acids of SEQ ED No 4, wherein said contiguous span includes at least one ammo acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ED No 4, a Pro at position 225 of SEQ LD No 4, and a Glu at position 252 of SEQ ED No 4. In other preferred embodiments the contiguous stretch of ammo acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the ammo acids in the hGGPPS protein sequence. hGGPPS proteins are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes. The hGGPPS polypeptides of the invention can be made using routine expression methods known in the art. The polynucleotide encoding the desired polypeptide, is hgated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems is used in forming recombinant polypeptides, and a summary of some of the more common systems. The polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Punfication is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like. See, for example, Methods m Enzymology for a vanety of methods for punfymg proteins.
In addition, shorter protein fragments is produced by chemical synthesis. Alternatively the proteins of the invention is extracted from cells or tissues of humans or non-human animals. Methods for punfymg proteins are known m the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.
Any hGGPPS cDNA, including SEQ LD Nos 2 and 3, is used to express hGGPPS proteins and polypeptides. The nucleic acid encoding the hGGPPS protein or polypeptide to be expressed is operably lmked to a promoter in an expression vector using conventional cloning technology. The hGGPPS insert in the expression vector may compnse the full coding sequence for the hGGPPS protein or a portion thereof. For example, the hGGPPS deπved insert may encode a polypeptide compnsmg at least 6 ammo acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 consecutive amino acids of the hGGPPS protein of SEQ LD No 4. wherein said consecutive ammo acids compnse at least one ammo acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ID No 4. a Pro at position 5 225 of SEQ LD No 4, and a Glu at position 252 of SEQ ED No 4
The expression vector is any of the mammalian, yeast, insect or bactenal expression systems known in the art. Commercially available vectors and expression systems are available from a vanety of suppliers including Genetics Institute (Cambndge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and 0 facilitate proper protein folding, the codon context and codon painng of the sequence is optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767, the disclosures of which are incoφorated by reference herein in their entirety.
In one embodiment, the entire coding sequence of the hGGPPS cDNA through the poly A 5 signal of the cDNA are operably linked to a promoter in the expression vector. Alternatively, if the nucleic acid encoding a portion of the hGGPPS protein lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the insert from the hGGPPS cDNA lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using 0 Bgll and Sail restnction endonuclease enzymes and lncoφoratmg it into the mammalian expression vector pXTl (Stratagene). pXTl contains the LTRs and a portion of the gag gene from Moloney Munne Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Heφes Simplex Thymidme Kinase promoter and the selectable neomycm gene. The nucleic acid encoding the hGGPPS protein or a portion thereof is obtained by PCR from a bactenal 5 vector containing the hGGPPS cDNA of SEQ ED Nos 2 and 3 using oligonucleotide pnmers complementary to the hGGPPS cDNA or portion thereof and containing restnction endonuclease sequences for Pst I incoφorated into the 5 'pnmer and BglH at the 5' end of the corresponding cDNA 3' pnmer, taking care to ensure that the sequence encoding the hGGPPS protein or a portion thereof is positioned properly with respect to the poly A signal. The punfied fragment obtained from the resulting 0 PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl El, punfied and hgated to pXTl, now containing a poly A signal and digested with Bglll
The hgated product is transfected into mouse NLH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined m the product specification. Positive transfectants are selected after growing the transfected cells m 600ug/ml G418 (Sigma, St. 5 Louis, Missoun).
The above procedures may also be used to express a mutant hGGPPS protein responsible for a detectable phenotype or a portion thereof The expressed protein is punfied using conventional punfication techniques such as ammonium sulfate precipitation or chromatographic separation based on size or charge The protein encoded by the nucleic acid insert may also be punfied using standard lmmunochromatography techniques In such procedures, a solution containing the expressed hGGPPS protein or portion thereof, such as a cell extract, is applied to a column having antibodies against the hGGPPS protein or portion thereof is attached to the chromatography matπx The expressed protein is allowed to bind the lmmunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound expressed protein is then released from the column and recovered using standard techniques To confirm expression of the hGGPPS protein or a portion thereof, the proteins expressed from host cells containing an expression vector containing an insert encoding the hGGPPS protein or a portion thereof can be compared to the proteins expressed m host cells containing the expression vector without an insert. The presence of a band in samples from cells containing the expression vector with an insert which is absent in samples from cells containing the expression vector without an insert indicates that the hGGPPS protein or a portion thereof is being expressed. Generally, the band will have the mobility expected for the hGGPPS protein or portion thereof. However, the band may have a mobility different than that expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.
Antibodies capable of specifically recognizing the expressed hGGPPS protein or a portion thereof are descnbed below.
If antibody production is not possible, the nucleic acids encoding the hGGPPS protein or a portion thereof is incoφorated into expression vectors designed for use in punfication schemes employing chimenc polypeptides. In such strategies the nucleic acid encoding the hGGPPS protein or a portion thereof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera is β-globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to β-globin or nickel attached thereto is then used to punfy the chimenc protein. Protease cleavage sites is engineered between the β-globin gene or the nickel binding polypeptide and the hGGPPS protein or portion thereof. Thus, the two polypeptides of the chimera is separated from one another by protease digestion. One useful expression vector for generating β-globin chimenc proteins is pSG5 (Stratagene), which encodes rabbit β-globin. Intron LI of the rabbit β-globin gene facilitates splicing of the expressed transcnpt, and the polyadenylation signal incoφorated into the construct increases the level of expression. These techniques are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems such as the In vitro Express™ Translation Kit (Stratagene) Antibodies That Bind hGGPPS Polypeptides of the Invention
Any hGGPPS polypeptide or whole protein may be used to generate antibodies capable of specifically binding to an expressed hGGPPS protein or fragments thereof as described.
One antibody composition of the invention is capable of specifically binding or specifically bind to the variant of the hGGPPS protein of SEQ ED No 4. For an antibody composition to specifically bind to a first vanant of hGGPPS, it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for a full length first vanant of the hGGPPS protein than for a full length second vanant of the hGGPPS protein in an ELISA, RIA, or other antibody- based binding assay. In a preferred embodiment of polyclonal or monoclonal antibodies of the invention consists in antibodies raised against a C-termmal portion of the hGGPS polypeptide of the ammo acid sequence of SEQ ED No 4, more preferably antibodies raise against a peptide fragment of the hGGPS polypeptide having the amino acid sequence starting from the amino acid at position 200 and ending at the amino acid in position 300 of the hGGPS polypeptide of SEQ ED No 4, or peptide fragments thereof.
In a preferred embodiment, the invention concerns antibody compositions, either polyclonal or monoclonal, capable of selectively binding, or selectively bind to an epitope-contaming a polypeptide comprising a contiguous span of at least 6 ammo acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ED No 4 , wherein said epitope compπses at least one amino acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ED No 4, a Pro at position 225 of SEQ ED No 4, and a Glu at position 252 of SEQ ED No 4.
The invention also concerns a punfied or isolated antibody capable of specifically binding to a mutated hGGPPS protein or to a fragment or vanant thereof comprising an epitope of the mutated hGGPPS protein.
In a preferred embodiment, the invention concerns the use in the manufacture of antibodies of a polypeptide comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 ammo acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 ammo acids of SEQ ED No 4 , wherein said epitope comprises at least one ammo acid selected from the group consisting of a Phe at positions 204, 257, 295 of SEQ ED No 4, a Cys at position 205 of SEQ ED No 4, a Pro at position 225 of SEQ ID No 4, and a Glu at position 252 of SEQ ED No 4.
Non-human animals or mammals, whether wild-type or transgenic, which express a different species of hGGPPS than the one to which antibody binding is desired, and animals which do not express hGGPPS (i.e. a hGGPPS knock out animal as descnbed herein) are particularly useful for preparing antibodies. hGGPPS knock out animals will recognize all or most of the exposed regions of a hGGPPS protein as foreign antigens, and therefore produce antibodies with a wider array of hGGPPS epitopes. Moreover, smaller polypeptides with only 10 to 30 ammo acids may be useful in obtaining specific binding to any one of the hGGPPS proteins In addition, the humoral immune system of animals which produce a species of hGGPPS that resembles the antigenic sequence will preferentially recognize the differences between the animal's native hGGPPS species and the antigen sequence, and produce antibodies to these unique sites in the antigen sequence Such a technique will be particularly useful in obtaining antibodies that specifically bind to any one of the hGGPPS proteins
Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-beanng substances in biological samples, they are also used semi -quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body
The antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art
Consequently, the invention is also directed to a method for detecting specifically the presence of a hGGPPS polypeptide according to the invention in a biological sample, said method comprising the following steps a) bnngmg into contact the biological sample with a polyclonal or monoclonal antibody that specifically binds a hGGPPS polypeptide compnsmg an ammo acid sequence of SEQ ED No 4, or to a peptide fragment or variant thereof; and b) detecting the antigen-antibody complex formed
The invention also concerns a diagnostic kit for detecting in vitro the presence of a hGGPPS polypeptide according to the present invention in a biological sample, wherein said kit comprises a) a polyclonal or monoclonal antibody that specifically binds a hGGPPS polypeptide comprising an ammo acid sequence of SEQ ED No 4, or to a peptide fragment or vanant thereof, optionally labeled, b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.
Method For Screening Ligands That Modulate The Expression Of The hGGPPS Gene.
Another subject of the present invention is a method for screening molecules that modulate the expression of the hGGPPS protein. Such a screening method comprises the steps of: a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the hGGPPS protein or a vanant or a fragment thereof, placed under the control of its own promoter, b) bringing into contact the cultivated cell with a molecule to be tested, c) quantifying the expression of the hGGPPS protein or a variant or a fragment thereof
In an embodiment, the nucleotide sequence encoding the hGGPPS protein or a vanant or a fragment thereof, preferably a fragment comprising an allele of the biallehc marker 5-187-77, and the complement thereof.
In one embodiment of the invention, the method for the screening of a candidate substance or molecule modulating the expression of the hGGPS genecompπses the following steps a) providing a recombinant host cell expressing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ED Nos 1, 2 and 3 or a fragment thereof, b) obtaining a candidate substance, and c) determining the ability of the candidate substance to modulate the expression levels of the nucleotide sequence selected from the group consisting of SEQ ED Nos 1 , 2 and 3 or a fragment thereof. Using DNA recombination techniques well known by the one skill m the art, the hGGPPS protein encoding DNA sequence is inserted into an expression vector, downstream from its promoter sequence. As an illustrative example, the promoter sequence of the hGGPPS gene is contained in the nucleic acid of the 5' regulatory region.
The quantification of the expression of the hGGPPS protein may be realized either at the mRNA level or at the protein level. In the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the hGGPPS protein that have been produced, for example m an ELISA or a RIA assay.
In a preferred embodiment, the quantification of the hGGPPS mRNA is realized by a quantitative PCR amplification of the cDNA obtained by a reverse transcnption of the total mRNA of the cultivated hGGPPS -transfected host cell, using a pair of pπmers specific for hGGPPS.
The present invention also concerns a method for screening substances or molecules that are able to increase, or in contrast to decrease, the level of expression of the hGGPPS gene. Such a method may allow the one skilled in the art to select substances exerting a regulating effect on the expression level of the hGGPPS gene and which may be useful as active ingredients included m pharmaceutical compositions.
Thus, is also part of the present invention a method for screening of a candidate substance or molecule that modulated the expression of the hGGPPS gene, this method compπses the following steps:
- providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence of the 5' regulatory region or a biologically active fragment or vanant thereof located upstream a polynucleotide encoding a detectable protein;
- obtaining a candidate substance; and - determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein
In a further embodiment, the nucleic acid compπsing the nucleotide sequence of the 5' regulatory region or a biologically active fragment or variant thereof also includes a 5'UTR region of the hGGPPS cDNA of SEQ ID Nos 2 or 3, or one of its biologically active fragments or vanants thereof.
Among the preferred polynucleotides encoding a detectable protein, there may be cited polynucleotides encoding beta galactosidase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT) The invention also pertains to kits useful for performing the herein descnbed screening method. Preferably, such kits comprise a recombinant vector that allows the expression of a nucleotide sequence of the 5' regulatory region or a biologically active fragment or variant thereof located upstream and operably linked to a polynucleotide encoding a detectable protein or the hGGPPS protein or a fragment or a variant thereof. In another embodiment of a method for the screening of a candidate substance or molecule that modulates the expression of the hGGPPS gene, wherein said method compnses the following steps: a) providing a recombinant host cell containing a nucleic acid, wherein said nucleic acid comprises a 5'UTR sequence of the hGGPPS cDNA of SEQ ED Nos 2 or 3, or one of its biologically active fragments or variants, the 5'UTR sequence or its biologically active fragment or variant being operably linked to a polynucleotide encoding a detectable protein; b) obtaining a candidate substance; and c) determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein. In a specific embodiment of the above screening method, the nucleic acid that compnses a nucleotide sequence selected from the group consisting of the 5'UTR sequence of the hGGPPS cDNA of SEQ ED Nos 2 or 3 or one of its biologically active fragments or variants, includes a promoter sequence which is endogenous with respect to the hGGPPS 5'UTR sequence.
In another specific embodiment of the above screening method, the nucleic acid that comprises a nucleotide sequence selected from the group consisting of the 5'UTR sequence of the hGGPPS cDNA of SEQ LD Nos 2 or 3 or one of its biologically active fragments or vanants, includes a promoter sequence which is exogenous with respect to the hGGPPS 5'UTR sequence defined therein.
In a further preferred embodiment, the nucleic acid compπsing the 5'-UTR sequence of the hGGPPS cDNA or SEQ LD Nos 2 or 3 or the biologically active fragments thereof, preferably those including the biallehc marker 5-187-77 or the complement thereof The invention further deals with a kit for the screening of a candidate substance modulating the expression of the hGGPPS gene, wherein said kit comprises a recombinant vector that comprises a nucleic acid including a 5"UTR sequence of the hGGPPS cDNA of SEQ ED Nos 2 or 3, or one of their biologically active fragments or variants, the 5'UTR sequence or its biologically active fragment or vanant being operably linked to a polynucleotide encoding a detectable protein
For the design of suitable recombinant vectors useful for performing the screening methods described above, it will be referred to the section of the present specification wherein the preferred recombinant vectors of the invention are detailed
Expression levels and patterns of hGGPPS may be analyzed by solution hybndization with long probes as descnbed in International Patent Application No. WO 97/05277, the entire contents of which are incoφorated herein by reference. Bnefly, the hGGPPS cDNA or the hGGPPS genomic DNA descπbed above, or fragments thereof, is inserted at a cloning site immediately downstream of a bactenophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA. Preferably, the hGGPPS insert compnses at least 100 or more consecutive nucleotides of the genomic DNA sequence or the cDNA sequences. The plasmid is linearized and transcnbed in the presence of πbonucleotides compnsmg modified nbonucleotides (i.e. biotm-UTP and DIG-UTP). An excess of this doubly labeled RNA is hybndized in solution with mRNA isolated from cells or tissues of interest The hybridization is performed under standard stringent conditions (40-50°C for 16 hours in an 80% formamide, 0. 4 M NaCl buffer, pH 7-8) The unhybndized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, TI , Phy M, U2 or A). The presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidm The presence of the DIG modification enables the hybπd to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase Quantitative analysis of hGGPPS gene expression may also be performed using arrays As used herein, the term array means a one dimensional, two dimensional, or multidimensional arrangement of a plurality of nucleic acids of sufficient length to permit specific detection of expression of mRNAs capable of hybndizing thereto. For example, the arrays may contain a plurality of nucleic acids denved from genes whose expression levels are to be assessed. The arrays may include the hGGPPS genomic DNA, the hGGPPS cDNA sequences or the sequences complementary thereto or fragments thereof, particularly those compnsmg the biallehc marker 5- 187-77. Preferably, the fragments are at least 15 nucleotides in length. In other embodiments, the fragments are at least 25 nucleotides in length. In some embodiments, the fragments are at least 50 nucleotides in length More preferably, the fragments are at least 100 nucleotides m length. In another preferred embodiment, the fragments are more than 100 nucleotides m length In some embodiments the fragments may be more than 500 nucleotides m length. For example, quantitative analysis of hGGPPS gene expression may be performed with a complementary DNA microarray as described by Schena et al (1995 and 1996) Full length hGGPPS cDNAs or fragments thereof are amplified by PCR and arrayed from a 96-well microtiter plate onto silylated microscope slides using high-speed robotics. Printed arrays are incubated in a humid chamber to allow rehydration of the array elements and nnsed, once in 0. 2% SDS for 1 mm, twice in water for 1 min and once for 5 min in sodium borohydπde solution The arrays are submerged in water for 2 min at 95°C, transferred into 0. 2% SDS for 1 m , rinsed twice with water, air dried and stored in the dark at 25°C.
Cell or tissue mRNA is isolated or commercially obtained and probes are prepared by a single round of reverse transcnption Probes are hybπdized to 1 cm" microarrays under a 14 x 14 mm glass coverslip for 6-12 hours at 60°C. Arrays are washed for 5 mm at 25°C in low stringency wash buffer (1 x SSC/0. 2% SDS), then for 10 mm at room temperature in high stringency wash buffer (0 1 x SSC/0. 2% SDS). Arrays are scanned m 0. 1 x SSC using a fluorescence laser scanning device fitted with a custom filter set. Accurate differential expression measurements are obtained by taking the average of the ratios of two independent hybridizations
Quantitative analysis of hGGPPS gene expression may also be performed with full length hGGPPS cDNAs or fragments thereof m complementary DNA arrays as descnbed by Pietu et al.(1996). The full length hGGPPS cDNA or fragments thereof is PCR amplified and spotted on membranes. Then, mRNAs ongmatmg from vanous tissues or cells are labeled with radioactive nucleotides. After hybridization and washing in controlled conditions, the hybπdized mRNAs are detected by phospho-imaging or autoradiography. Duplicate experiments are performed and a quantitative analysis of differentially expressed mRNAs is then performed.
Alternatively, expression analysis using the hGGPPS genomic DNA, the hGGPPS cDNA, or fragments thereof can be done through high density nucleotide arrays as descnbed by Lockhart et al.(1996) and Sosnowsky et al.(1997). Oligonucleotides of 15-50 nucleotides from the sequences of the hGGPPS genomic DNA, the hGGPPS cDNA sequences particularly those comprising the biallehc marker 5-187-77, or the sequences complementary thereto, are synthesized directly on the chip (Lockhart et al., supra) or synthesized and then addressed to the chip (Sosnowski et al., supra). Preferably, the oligonucleotides are about 20 nucleotides in length. hGGPPS cDNA probes labeled with an appropnate compound, such as biotin, digoxigemn or fluorescent dye, are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50 to 100 nucleotides. The said probes are then hybπdized to the chip. After washing as descπbed in Lockhart et al., supra and application of different electric fields (Sosnowsky et al., 1997)., the dyes or labeling compounds are detected and quantified. Duplicate hybndizations are performed Comparative analysis of the intensity of the signal oπgmating from cDNA probes on the same target oligonucleotide different cDNA samples indicates a differential expression of hGGPPS mRNA. Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specification referenced in this application are hereby incoφorated by reference into the present disclosure to more fully descnbe the sate of the art to which this invention pertains
EXAMPLES
Example 1 :
Analysis of the mRNAs encoding the hGGPS polypeptide of SEQ ID No 4 synthesized by the cells. Human GGPS cDNA was obtained as follows : 4μl of ethanol suspension containing 1 mg of human prostate total RNA (Clontech laboratories, Inc., Palo Alto, USA; Catalogue N. 64038-1) was centπfuged, and the resulting pellet was air dned for 30 minutes at room temperature.
First strand cDNA synthesis was performed using the AdvantageTM RT-for- PCR kit (Clontech laboratories Inc., catalogue N. K1402-1). 1 μl of 20 mM solution of a specific oligo dT primer was added to 12.5 μl of RNA solution in water, heated at 74°C for 2.5 min and rapidly quenched in an ice bath. 10 μl of 5 x RT buffer (50 mM Tns-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 2.5 μl of dNTP mix (10 mM each), 1.25 μl of human recombinant placental RNA inhibitor were mixed with 1 ml of MMLV reverse transcriptase (200 units). 6.5 μl of this solution were added to RNA-primer mix and incubated at 42°C for one hour. 80 μl of water were added and the solution was incubated at 94°C for 5 minutes.
5μl of the resulting solution were used in a Long Range PCR reaction with hot start, in 50 μl final volume, using 2 units of rtTHXL, 20 pmol/μl of each of 5'-
TGGAGAAGACTCAAGAAACAGTCCAAA-3' (from the nucleotide m position 86 to the nucleotide in position 1 12 of SEQ ID No 1) and 5'-CCTGGAAGCAAGTCTTTTTTATTGACG-3' (from the nucleotide in position 1285 to the nucleotide in position 1311 of SEQ ED No 1) primers with 35 cycles of elongation for 6 minutes at 67°C in thermocycler
The amplification products corresponding to both cDNA strands are partially sequenced in order to ensure the specificity of the amplification reaction
Results of Northern blot analysis of prostate mRNAs support the existence of a hGGPS cDNA which corresponds to the nucleotide sequence of SEQ ED No 1. Example 2 :
Detection of hGGPS biallelic markers: DNA extraction
Donors were unrelated and healthy. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the detection of the biallehc markers
30 ml of penpheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centnfugation for 10 minutes at 2000 φm. Red cells were lysed by a lysis solution (50 ml final volume . 10 mM Tns pH7.6; 5 mM MgCl2; 10 mM NaCl). The solution was centπfuged (10 minutes, 2000 φm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution.
The pellet of white cells was lysed overnight at 42°C with 3.7 ml of lysis solution composed of:
- 3 ml TE 10-2 (Tns-HCl 10 mM, EDTA 2 mM) / NaCl 0.4 M - 200 μl SDS 10% - 500 μl K-protemase (2 mg K-protemase in TE 10-2 / NaCl 0 4 M).
For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 φm.
For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 φm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 φm. The pellet was dried at 37°C, and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration was evaluated by measunng the OD at 260 nm (1 unit OD = 50 μg/ml DNA)
To determine the presence of proteins in the DNA solution, the OD 260 / OD 280 ratio was determined. Only DNA preparations having a OD 260 / OD 280 ratio between 1.8 and 2 were used m the subsequent examples descπbed below.
The pool was constituted by mixing equivalent quantities of DNA from each individual.
Example 3 :
Detection of the biallelic markers: amplification of genomic DNA by PCR
The amplification of specific genomic sequences of the DNA samples of example 2 was carried out on the pool of DNA obtained previously. In addition, 50 individual samples were similarly amplified.
PCR assays were performed using the following protocol: Final volume 25 μl
DNA 2 ng/μl MgCl2 2 mM dNTP (each) 200 μM pπmer (each) 2 9 ng/μl
Amph Taq Gold DNA polymerase 0 05 unit/μl
PCR buffer (lOx = 0 1 M TnsHCl pH8.3 0.5M KCl) lx
Each pair of first pnmers was designed using the sequence information of the hGGPS gene disclosed herein and the OSP software (Hillier & Green, 1991). This first pair of pnmers was about 20 nucleotides in length and had the sequences disclosed in Table 1 in the columns labeled PU and RP.
Table 1
Amplicon Position range of the Position range of Complementary position amplicon in SEQ ED amplification pnmer in SEQ range of amplification pπmer genomic ID No genomic in SEQ ED No genomic
5-187 13982-14409 13982-14000 14390-14409
10 The sequences of the amplification pπmers Bl and Cl are respectively disclosed in SEQ ED
Nos 8 and 9.
Preferably, the pπmers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing. Primers PU contain the following additional PU 5' sequence : TGTAAAACGACGGCCAGT (SEQ ID No 10); primers RP contain the 15 following RP 5' sequence : CAGGAAACAGCTATGACC (SEQ ID No 11).
The synthesis of these pπmers was performed following the phosphoramidite method, on a GENSET UFPS 24.1 synthesizer
DNA amplification was performed on a Genius II thermocycler. After heating at 95°C for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95°C. 54°C for 1 min, and 30 sec at 20 72°C. For final elongation, 10 min at 72°C ended the amplification. The quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as mtercalant agent (Molecular Probes).
Example 4 :
Detection of the biallelic markers: sequencing of amplified genomic DNA and 25 identification of polymorphisms.
The sequencing of the amplified DNA obtained in example 3 was earned out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel 30 image analysis.
The sequence data were further evaluated to detect the presence of biallehc markers among the pooled amplified fragments. The polymoφhism search was based on the presence of supenmposed peaks in the electrophoresis pattern resulting from different bases occurnng at the same position as described previously.
Table 2 shows the biallehc marker that has been detected after the sequence analysis of the amplification fragments generated by PCR. Table 2
Figure imgf000058_0001
The two alleles of the biallehc marker 5-187-77 can be defined by an oligonucleotide comprising the polymoφhic base. The sequence of such oligonucleotides are disclosed in SEQ ED Nos 5 and 6.
Example 5 : Validation of the polymorphisms through microsequencing
The biallehc marker identified in example 4 was further confirmed through microsequencing. Microsequencing was earned out for each individual DNA sample descπbed in Example 2.
Amplification from genomic DNA of individuals was performed by PCR as descnbed above for the detection of the biallehc markers with the same set of PCR pnmers (Table 1).
The prefened pnmers used in microsequencing were about 20 nucleotides in length and hybridized just upstream of the considered polymoφhic base. According to the invention, the pnmer used in microsequencing is detailed in Table 3.
Table 3
Marker Name | Microsequencing pnmer
5-187-77 I SEQ ED No 7
The microsequencing reaction was performed as follows :
After punfication of the amplification products, the microsequencing reaction mixture was prepared by adding, in a 20μl final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tπs HCl pH 9.5, 65 mM MgCl2), and the two appropnate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to the nucleotides at the polymoφhic site of each bialle c marker tested, following the manufacturer's recommendations. After 4 minutes at 94°C, 20 PCR cycles of 15 sec at 55°C, 5 sec at 72°C, and 10 sec at 94°C were carried out in a Tetrad PTC-225 thermocycler (MJ Research). The unmcoφorated dye terminators were then removed by ethanol precipitation. Samples were finally resuspended m formamide-EDTA loading buffer and heated for 2 min at 95°C before being loaded on a polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer)
Following gel analysis, data were automatically processed with software that allows the determination of the alleles of biallehc markers present in each amplified fragment. The software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous. In addition, the software identifies significant peaks (according to shape and height criteria) Among the significant peaks, peaks corresponding to the targeted site are identified based on their position When two significant peaks are detected for the same position, each sample is categoπzed classification as homozygous or heterozygous type based on the height ratio.
Example 6 : Preparation of Antibody Compositions to the GENE protein
Substantially pure protein or polypeptide is isolated from transfected or transformed cells containing an expression vector encoding the hGGPPS protein or a portion thereof The concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows: A. Monoclonal Antibody Production by Hybndoma Fusion
Monoclonal antibody to epitopes in the hGGPPS protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C, (1975) or derivative methods thereof. Also see Harlow, E., and D. Lane. 1988..
Bnefly, a mouse is repetitively inoculated with a few micrograms of the hGGPPS protein or a portion thereof over a penod of a few weeks. The mouse is then sacnficed, and the antibody producmg cells of the spleen isolated. T e spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media compnsmg ammoptenn (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody- producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally descnbed by Engvall, (1980), and denvative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are descnbed in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2 B Polyclonal Antibody Production by Immunization
Polyclonal antiserum containing antibodies to heterogeneous epitopes m the hGGPPS protein or a portion thereof can be prepared by immunizing suitable non-human animal with the hGGPPS protein or a portion thereof, which can be unmodified or modified to enhance lmmunogenicity A suitable non-human animal is preferably a non-human mammal is selected, usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crude preparation which has been enriched for hGGPPS concentration can be used to generate antibodies. Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropnate adjuvant (e.g. aluminum hydroxide, RLBI, etc ) which is known m the art In addition the protein, fragment or preparation can be pretreated with an agent which will increase antigenicity, such agents are known m the art and include, for example, methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, and keyhole limpet hemocyanm (KLH). Serum from the immunized animal is collected, treated and tested according to known procedures. If the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies can be purified by lmmunoaffinity chromatography.
Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple mtradermal sites appears to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987). An effective immunization protocol for rabbits can be found m Vaitukaitis, J. et al. (1971).
Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double lmmunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al., (1973) Plateau concentration of antibody is usually m the range of 0 1 to 0.2 mg/ml of serum (about 12 μM). Affinity of the antisera for the antigen is determined by prepanng competitive binding curves, as descnbed, for example, by Fisher, D., (1980). Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful m quantitative immunoassays which determine concentrations of antigen-beanng substances m biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
While the preferred embodiment of the invention has been illustrated and descnbed, it will be appreciated that various changes can be made therein by the one skilled in the art without departing from the spmt and scope of the invention.
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SEQUENCE LISTING FREE TEXT
The following free text appears in the accompanying Sequence Listing : Homology with sequence in ref
Polymoφhic base insertion of
Complement
Diverging ammo acid in ref
Artificial sequence Sequencing oligonucleotide pnmer

Claims

What is claimed:
1 An isolated, punfied, or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ LD No 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ LD No 1 : 1-485, 547-632, 827- 5 7291, 7385-13759, 13831-14062, 14671-15054, and 15252-17131.
2. An isolated, punfied, or recombinant polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ED No 2 or the complements thereof, wherein said contiguous span comprises at least 1 of the nucleotide positions 834-1217 of SEQ ED No 2. 0
3. An isolated, punfied, or recombinant polynucleotide compnsmg a contiguous span of at least 12 nucleotides of SEQ ED No 3 or the complements thereof, wherein said contiguous span comprises at least 1 of the nucleotide positions 967-1351 of SEQ ED No 2.
5 4. An isolated, punfied, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of anyone of SEQ LD Nos 1-3 or the complement thereof, wherein said span includes a hGGPPS-τelated biallehc marker in said sequence.
5. A polynucleotide according to claim 4, wherein said hGGPPS-τelated biallehc marker is 0 the biallehc marker 5-187-77.
6. A polynucleotide according to any one of claims 4 or 5, wherein said contiguous span is 18 to 50 nucleotides in length and said biallehc marker is withm 4 nucleotides of the center of said polynucleotide. 5
7. A polynucleotide according to claim 6, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides m length and said biallehc marker is at the center of said polynucleotide.
0 8 A polynucleotide according to claim 6, wherein said polynucleotide consists essentially of a sequence selected from the group consisting of SEQ ED Nos 5 and 6 and the complementary sequences thereto.
9. A polynucleotide according to any one of claims 1-5, wherein the 3' end of said 5 contiguous span is present at the 3' end of said polynucleotide.
10. An isolated, punfied. or recombinant polynucleotide consisting essentially of a sequence selected from the group consisting of SEQ ED Nos 8-9
11. A polynucleotide according to any one of claims 4 or 5, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide and said biallehc marker is present at the 3' end of said polynucleotide
12. An isolated, punfied, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of anyone of SEQ ED Nos 1-3 or the complement thereof, wherein the 3' end of said contiguous span is located at the 3' end of said polynucleotide, and wherein the 3' end of said polynucleotide is located withm 20 nucleotides upstream of a hGGPPS- related biallehc marker in said sequence
13. A polynucleotide according to claim 12, wherein the 3' end of said polynucleotide is located 1 nucleotide upstream of said hGGPPS -related biallehc marker in said sequence.
14. A polynucleotide according to claim 13, wherein said polynucleotide consists essentially of the sequence of SEQ ED No 7.
15. An isolated, punfied, or recombinant polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 ammo acids of SEQ ED No 4, wherein said contiguous span includes at least one ammo acid selected from the group consisting of a Phe residue at positions 204, 257, 295 of SEQ ED No 4, a Cys residue at position 205 of SEQ ED No 4, a Pro residue at position 225 of SEQ ED No 4, and a Glu residue at position 252 of SEQ ED No 4.
16. A polynucleotide for use in a genotyping assay for determining the identity of the nucleotide at a ΛGG S-related bialle c marker or the complement thereof.
17. A polynucleotide according to claim 16, wherein the polynucleotide is used m a hybridization assay.
18. A polynucleotide according to claim 16, wherein the polynucleotide is used in a sequencing assay.
19. A polynucleotide according to claim 16, wherein the polynucleotide is used m an enzyme-based mismatch detection assay
20 A polynucleotide according to claim 16, wherein the polynucleotide is used in amplifying a segment of nucleotides comprising said biallelic marker.
21. A polynucleotide according to any one of claims 1-20 attached to a solid support
22. An array of polynucleotides comprising at least one polynucleotide according to claim 21.
23. An array according to claim 22, wherein said array is addressable.
24. A polynucleotide according to any one of claims 1-20 further comprising a label.
25. A recombinant vector compnsmg a polynucleotide according to any one of claims 1-20.
26. A host cell compnsmg a recombinant vector according to claim 25.
27. A non-human host animal or mammal comprising a recombinant vector according to claim 25.
28. A method of genotyping compnsmg determining the identity of a nucleotide at a hGGPPS-τe\ated biallehc marker or the complement thereof in a biological sample.
29. A method according to claim 28, wherein said biological sample is deπved from a single subject.
30. A method according to claim 29, wherein the identity of the nucleotides at said biallehc marker is determined for both copies of said biallehc marker present in said individual's genome.
31. A method according to claim 28, wherein said biological sample is derived from multiple subjects.
32. A method according to any one of claims 28, further compnsmg amplifying a portion of said sequence comprising the biallehc marker pπor to said determining step.
33. A method according to claim 32, wherein said amplifying is performed by PCR.
34 A method according to any one of claims 28-33, wherein said determining is performed by a hybridization assay.
35 A method according to any one of claims 28-33, wherein said determining is performed by a sequencing assay
36 A method according to any one of claims 28-33, wherein said determining is performed by a microsequencing assay.
37 A method according to any one of claims 28-33, wherein said determining is performed by an enzyme-based mismatch detection assay
38 An isolated, punfied, or recombinant polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ED No 4, wherein said contiguous span includes at least one ammo acid selected from the group consisting of a Phe residue at positions 204, 257, 295 of SEQ ED No 4, a Cys residue at position 205 of SEQ LD No 4, a Pro residue at position 225 of SEQ ED No 4, and a Glu residue at position 252 of SEQ ED No 4.
39. An isolated or punfied antibody composition are capable of selectively binding to an epitope-contammg fragment of a polypeptide according to claim 38, wherein said epitope compnses at least one ammo acid selected from the group consisting of a Phe residue at positions 204, 257, 295 of SEQ ED No 4, a Cys residue at position 205 of SEQ ED No 4, a Pro residue at position 225 of SEQ ED No 4, and a Glu residue at position 252 of SEQ LD No 4.
40 A method for the screening of a candidate substance or molecule modulating the expression of the hGGPS gene, said method compnsmg the following steps : a) providing a recombinant host cell expressing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ED Nos 1 , 2 and 3 or a fragment thereof; b) obtaining a candidate substance, and c) determining the ability of the candidate substance to modulate the expression levels of the nucleotide sequence selected from the group consisting of SEQ ED Nos 1, 2 and 3 or a fragment thereof
41. A method for the screening of a candidate substance or molecule modulating the expression of the hGGPS gene, said method compnsmg the following steps : - providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence of the 5' regulatory region or a biologically active fragment or vanant thereof located upstream a polynucleotide encoding a detectable protein;
- obtaining a candidate substance; and - determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.
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