Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention is in the field of proteins, more particularly those useful for the study of neuroblastoma and cellular imaging, and those which interact with bacterial toxins.
• Neuroblastoma is the second most common solid tumour in children. It is a neoplasm of the peripheral autonomic nervous system that usually occurs before children are 6 years old. Approaches to its diagnosis are reviewed in reference 1.
• Vimentin is a protein of the intermediate filament (IF) family. Homopolymers of the protein form class-Ill intermediate filaments in various non-epithelial cells, particularly those of the mesenchyma.
• the human and murine genes have been characterised. Vimentin and other IF proteins have been used in the histological classification of human tumours [ref. 2]. Vimentin in particular has been used as a marker for de-differentiation in several types of tumour.
• cytotoxin The vacuolating cytotoxin (VacA) of Helicobacter pylori is a major virulence factor [3,4,5]. Cells exposed to the toxin vacuolate following extensive fusion and swelling of late endosomal/ lysosomal compartments. Cellular trafficking is thereby disrupted. It is believed that VacA interacts with cytosolic host proteins. VacA is a 953aa protein that is cleaved after residue 311 to give two fragments (p37 & p58) which remain non-covalently associated [6].
• the invention is based on the discovery of a protein referred to hereinafter as 'VIP54'.
• the amino acid sequence of human VIP54 is shown in Figure 2 ('hVIP54'; SEQ ID 1) together with the sequence of the murine ortholog ('mVIP54'; SEQ ID 2).
• VIP54 is not detected in neurons, but is detected in neuroblastoma cells removed from a tumour. Furthermore, VIP54 co-distributes with vimentin and decorates intermediate filaments with a dotted pattern.
• VIP54 was identified by yeast two-hybrid screening using VacA as bait.
• the invention therefore provides a protein comprising the amino acid sequence of hVIP54 shown in Figure 2.
• the invention also provides proteins comprising sequences homologous (i.e. having sequence identity) to the hVIP54 amino acid sequence.
• the degree of sequence identity is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more).
• These proteins include homologs, orthologs, allelic variants and functional mutants of hVIP54.
• the invention further provides proteins comprising fragments of the hVIP54 and mVIP54 amino acid sequences shown in Figure 2.
• the fragments should comprise at least n consecutive amino acids from the sequences, where n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20 or more).
• Preferably the fragments comprise an epitope from the sequence.
• Preferred fragments are (a) SEQ IDs 4, 5 and 6 (b) amino acids 9 to 500 of SEQ ID 1.
• the proteins of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (e.g. native, fusions etc.). They are preferably prepared in substantially pure or isolated form (i.e. substantially free from other host cell proteins with which it is normally associated in nature)
• the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means.
• the antibodies may include a detectable label.
• These antibodies can be used as diagnostic reagents (e.g. for tumours such as neuroblastoma), as markers for intermediate filaments, and for the inhibition of the VIP54-VacA interaction.
• the invention provides nucleic acid encoding VIP54 protein, and in particular nucleic acid encoding hVIP54 (e.g. nucleotides 1-1500 of SEQ ID 3) or mV ⁇ P54.
• the invention provides nucleic acid comprising sequences homologous (i.e. having sequence identity) to sequences that encode VIP54 protein. The degree of sequence identity is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more).
• the invention provides nucleic acid which can hybridise to said nucleic acid, preferably under "high stringency" conditions (eg. 65°C, O.lxSSC, 0.5% SDS).
• Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the VIP54-encoding sequences where n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
• the invention provides nucleic acid encoding the proteins and protein fragments of the invention.
• nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing purposes).
• Nucleic acid according to the invention can, of course, be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes etc.).
• nucleic acid includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
• the invention provides vectors comprising nucleotide sequences of the invention (e.g. expression vectors) and host cells transformed with such vectors.
• vectors comprising nucleotide sequences of the invention (e.g. expression vectors) and host cells transformed with such vectors.
• compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as diagnostic reagents, for instance, or as immunogenic compositions.
• the invention also provides nucleic acid, protein, or antibody according to the invention for use as diagnostic reagents or medicaments (e.g. as immunogenic compositions, such as vaccines). It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a reagent for diagnosing tumours (e.g. neuroblastoma), and (ii) a medicament for inhibiting the interaction between VacA and VIP54.
• a reagent for diagnosing tumours e.g. neuroblastoma
• a medicament for inhibiting the interaction between VacA and VIP54 e.g. VIP54.
• the invention also provides a reagent that can inhibit the VacA-VIP54 interaction.
• the invention provides various processes.
• a process for producing proteins of the invention comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.
• a process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.
• a process for detecting polynucleotides of the invention comprising the steps of: (a) contacting a nucleic acid probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting said duplexes.
• a process for detecting proteins of the invention comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes. This process may be carried out in situ in a cell.
• a composition containing X is "substantially free of Y when at least 85 % by weight of the total X +Y in the composition is X .
• X comprises at least about 90% by weight of the total of X+Y in the com position, more preferably at least about 95% or even 99% by weight.
• heterologous refers to two biological com ponents that are not found together in nature.
• the components m ay be host cells, genes, or regulatory regions, such as promoters.
• the heterologous components are not found together in nature, they can function together, as w hen a promoter heterologous to a gene is operably linked to the gene.
• Another exam ple is w here a bacterial sequence is heterologous to a mouse host cell.
• a further examples would be two epitopes from the same or different proteins w hich have been assem bled in a single protein in an arrangement not found in nature.
• An "origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector.
• the origin of replication behaves as an autonom ous unit of polynucleotide replication within a cell, capable of replication under its ow n control.
• An origin of replication may be needed for a vector to replicate in a particular host cell. W ith certain origins of replication, an expression vector can be reproduced at a high copy num ber in the presence of the appropriate proteins within the cell. Examples of origins are the autonom ously replicating sequences, w hich are effective in yeast; and the viral T-antigen, effective in C0 S-7 cells.
• a "m utant" sequence is defined as DNA , RN A or am ino acid sequence differing from but having sequence identity with the native or disclosed sequence.
• the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60% , 70% , 80% , 90% , 95% , 99% or more, calculated using the Sm ith-W aterm an algorithm as described above).
• an "allelic variant" of a nucleic acid m olecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a sim ilar but not identical nucleic acid sequence.
• a coding region allelic variant typically encodes a protein having sim ilar activity to that of the protein encoded by the gene to which it is being com pared.
• a n allelic variant can also com prise an alteration in the 5 ' or 3' untranslated regions of the gene, such as in regulatory control regions ⁇ eg. see U S patent 5 ,753 ,235).
• Nucleotide sequences can be expressed in a variety of different expression system s; for exam ple those used with mam m alian cells, baculoviruses, plants, bacteria, and yeast. i. M am malian System s
• a m am m alian promoter is any DNA sequence capable of binding m am malian RNA polym erase and initiating the dow nstream (3') transcription of a coding sequence (eg. structural gene) into m RN A .
• a promoter w ill have a transcription initiating region, w hich is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
• a mam m alian prom oter w ill also contain an upstream promoter element, usually located w ithin 100 to 200 bp upstream of the TATA box.
• An upstream promoter element determ ines the rate at w hich transcription is initiated and can act in either orientation [Sam brook et al. ( 1989) "Expression of Cloned Genes in M am m alian Cells" in Molecular Cloning: A Laboratory Manual, 2nd ed.].
• Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences.
• Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (AdMLP), and herpes simplex virus promoter.
• sequences derived from non-viral genes e.g. the murine metallotheionein gene
• Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
• Enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 256:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range.
• Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci.79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet.2:215; Maniatis etal.
• an inducer such as a hormone or metal ion
• a DNA molecule may be expressed intracellularly in mammalian cells.
• a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
• foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells.
• a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells.
• processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro.
• the leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
• the adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
• transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
• the 3' terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 47:349; Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukaryotic RNA. In Transcription and splicing (ed. B.D. Hames and D.M. Glover); Proudfoot (1989) Trends Biochem.
• transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].
• the above described components comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.
• Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.
• Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.
• Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.
• plasmids containing the replication systems of papovaviruses such as SV40 [Gluzman (1981) Cell23: ⁇ 15] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen.
• mammalian replicons include those derived from bovine papillomavirus and Epstein- Barr virus.
• the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification.
• mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol.6:1074].
• the transformation procedure used depends upon the host to be transformed.
• Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
• Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.
• ATCC American Type Culture Collection
• CHO Chinese hamster ovary
• HeLa cells HeLa cells
• BHK baby hamster kidney cells
• COS monkey kidney cells
• human hepatocellular carcinoma cells eg. Hep G2
• the polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector.
• Vector construction employs techniques which are known in the art.
• the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
• the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine.
• the packaged recombinant virus is expressed and recombinant plaques are identified and purified.
• Kit form Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from , inter alia, Invitrogen, San Diego CA ("M axBac” kit). These techniques are generally know n to those skilled in the art and fully described in Sum mers and Sm ith, Texas Agricultural Experiment Station Bulletin No. 1555 ( 1987) (hereinafter "Sum mers and Sm ith”).
• transplacement construct Prior to inserting the DNA sequence encoding the protein into the baculovirus genom e, the above described components, com prising a prom oter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assem bled into an interm ediate transplacement construct (transfer vector).
• This construct m ay contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or m ultiple genes, regulated by the same set of regulatory elements.
• Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal elem ent (e.g.
• a plasm id capable of stable m aintenance in a host, such as a bacterium .
• the replicon w ill have a replication system , allowing it to be m aintained in a host for cloning and amplification.
• the plasm id usually also contains the polyhedrin polyadenylation signal (M iller et al. (1988) Ann. Rev. MicrobioL, 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.
• polyhedrin polyadenylation signal M iller et al. (1988) Ann. Rev. MicrobioL, 42:177
• amp prokaryotic ampicillin-resistance
• Baculovirus transfer vectors usually contain a baculovirus prom oter.
• a baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polym erase and initiating the dow nstream (5' to 3 ') transcription of a coding sequence (eg. structural gene) into mRNA .
• a prom oter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.
• a baculovirus transfer vector m ay also have a second dom ain called an enhancer, which, if present, is usually distal to the structural gene. Expression m ay be either regulated or constitutive.
• Structural genes abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) "The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. W alter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p l O protein, Vlak et al., ( 1988), 7. Gen. Virol. 69:765.
• DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 75:409).
• the signals for m am malian cell posttranslational m odifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accum ulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human ⁇ -interferon, M aeda et al., (1985), Nature 5/5:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec.
• a recombinant polypeptide or polyprotein m ay be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted.
• Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader " sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N -term inus m ay be cleaved from the mature protein by in vitro incubation with cyanogen bromide.
• recombinant polyproteins or proteins w hich are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.
• the leader sequence fragment usually encodes a signal peptide comprised of hydrophobic am ino acids which direct the translocation of the protein into the endoplasmic reticulum .
• an insect cell host is co-transformed w ith the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus - usually by co-transfection.
• the promoter and transcription termination sequence of the construct will usually comprise a 2-5kb section of the baculovirus genome.
• the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recom bination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. M iller et al., ( 1989), Bioessays 4:91 .
• the DNA sequence when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5' and 3 ' by polyhedrin-specific sequences and is positioned dow nstream of the polyhedrin promoter.
• the new ly formed baculovirus expression vector is subsequently packaged into an infectious recom binant baculovirus.
• Homologous recom bination occurs at low frequency (between - 1 % and -5% ); thus, the m ajority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses.
• An advantage of the expression system is a visual screen allowing recom binant viruses to be distinguished.
• the polyhedrin protein, w hich is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accum ulated polyhedrin protein form s occlusion bodies that also contain em bedded particles.
• occlusion bodies up to 15 ⁇ m in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope.
• Cells infected w ith recombinant viruses lack occlusion bodies.
• the transfection supernatant is plaqued onto a m onolayer of insect cells by techniques known to those skilled in the art - the plaques are screened under the light microscope for the presence (indicative of wild- type virus) or absence (recom binant virus) of occlusion bodies.
• Current Protocols in Microbiology Vol. 2 (Ausubel e. ⁇ /. eds) at 16.8 (Supp.
• Recombinant baculovirus expression vectors have been developed for infection into several insect cells.
• recombinant baculoviruses have been developed for, inter alia: Aedes aegypti , Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol.56:153; Wright (1986) Nature 527:718; Smith et al., (1983) Mol. Cell. Biol.5:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol.25:225).
• Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.
• the modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients.
• the product may be purified by such techniques as chromatography (e.g.
• the product may be further purified, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris e.g. proteins, lipids and polysaccharides.
• recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art. iii. Plant Systems
• a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants.
• the expression cassette is inserted into a desired expression vector w ith com panion sequences upstream and downstream from the expression cassette suitable for expression in a plant host.
• the com panion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to perm it the vectors to m ove D NA from an original cloning host, such as bacteria, to the desired plant host.
• the basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin ; a prokaryote selectable m arker; and, for Agrobacterium transform ations, T DNA sequences for Agrobacterium -mediated transfer to plant chromosomes.
• W the heterologous gene is not readily am enable to detection, the construct will preferably also have a selectable m arker gene suitable for determ ining if a plant cell has been transformed.
• suitable m arkers for exam ple for the mem bers of the grass fam ily, is found in W ilm ink and Dons, 1993, Nan. Mol. Biol. Reptr, 1 1 (2): 165- 1 85.
• Sequences suitable for perm itting integration of the heterologous sequence into the plant genom e are also recom mended. These m ight include transposon sequences and the like for hom ologous recom bination as well as Ti sequences w hich perm it random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable m arkers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
• the nucleic acid molecules of the subject invention m ay be included into an expression cassette for expression of the protein(s) of interest.
• the recom binant expression cassette w ill contain in addition to the heterologous protein encoding sequence the follow ing elem ents, a promoter region, plant 5' untranslated sequences, initiation codon depending upon w hether or not the structural gene com es equipped with one, and a transcription and translation term ination sequence.
• Unique restriction enzym e sites at the 5' and 3 ' ends of the cassette allow for easy insertion into a pre-existing vector.
• a heterologous coding sequence m ay be for any protein relating to the present invention.
• the sequence encoding the protein of interest will encode a signal peptide w hich allow s processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region w ill be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in w hich they are expressed and may be efficiently harvested.
• site-directed mutagenesis of the intron region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed & M aniatis, Cell 41 :95-105, 1985.
• the vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recom binant DNA . Crossway, Mol. Gen. Genet, 202: 179- 185 , 1985.
• the genetic m aterial may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982.
• nucleic acid segments Another method of introduction of nucleic acid segments is high velocity ballistic penetration by sm all particles with the nucleic acid either w ithin the matrix of small beads or particles, or on the surface, K lein, et al., Nature, 327, 70-73 , 1987 and Knudsen and M uller, 1991 , Planta, 185 :330-336 teaching particle bombardment of barley endosperm to create transgenic barley.
• Yet another method of introduction w ould be fusion of protoplasts w ith other entities, either m inicells, cells, lysosom es or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci.
• the vector m ay also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985).
• plant protoplasts are electroporated in the presence of plasm ids containing the gene construct. Electrical im pulses of high field strength reversibly permeabilize biomem branes allow ing the introduction of the plasm ids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus. All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene.
• plants can be regenerated from cultured cells or tissues, including but not lim ited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
• Some suitable plants include, for exam ple, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium , Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum , Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum , Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum , Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum , Ranunculus, Senecio, Salpiglossis, Cucumis, Brow
• Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. A lternatively, embryo form ation can be induced from the protoplast suspension. These embryos germinate as natural em bryos to form plants.
• the culture media will generally contain various am ino acids and horm ones, such as auxin and cytokinins. It is also advantageous to add glutam ic acid and proline to the m edium , especially for such species as corn and alfalfa. Shoots and roots norm ally develop simultaneously. Efficient regeneration w ill depend on the medium , on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
• the desired protein of the invention may be excreted or, alternatively, the protein m ay be extracted from the whole plant. W here the desired protein of the invention is secreted into the medium , it may be collected. Alternatively, the em bryos and em bryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues.
• the m ixture m ay be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the protein. Parameters of tim e, tem perature, pH, oxygen, and volum es will be adjusted through routine m ethods to optim ize expression and recovery of heterologous protein . iv. Bacterial System s
• a bacterial prom oter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3' ) transcription of a coding sequence (eg. structural gene) into m RNA .
• a prom oter w ill have a transcription initiation region w hich is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.
• a bacterial promoter may also have a second domain called an operator, that m ay overlap an adjacent RNA polym erase binding site at which RNA synthesis begins.
• the operator perm its negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.
• Constitutive expression m ay occur in the absence of negative regulatory elements, such as the operator.
• positive regulation may be achieved by a gene activator protein binding sequence, w hich, if present is usually proxim al (5') to the RNA polymerase binding sequence.
• An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. ( 1984) An ⁇ u. Rev. Genet. 78: 173].
• Regulated expression m ay therefore be either positive or negative, thereby either enhancing or reducing transcription.
• Sequences encoding metabolic pathway enzymes provide particularly useful prom oter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 798: 1056], and m altose. Additional examples include prom oter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057 ; Yelverton et al. ( 1981 ) Nucl. Acids Res. 9:731 ; U S patent 4,738,921 ; EP-A-0036776 and EP-A-0121775].
• trp tryptophan
• synthetic promoters which do not occur in nature also function as bacterial promoters.
• the tac prom oter is a hybrid trp-lac prom oter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. ( 1983) PNAS USA 80:21 ].
• a bacterial prom oter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
• a naturally occurring prom oter of non-bacterial origin can also be coupled w ith a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
• the bacteriophage T7 RNA polymerase/prom oter system is an example of a coupled promoter system [Studier et al. ( 1986) J. Mol. Biol. 189:1 13; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074].
• a hybrid promoter can also be comprised of a bacteriophage promoter and an E.coli operator region (EPO-A-O 267 851 ).
• EPO-A-O 267 851 E.coli operator region
• an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.
• the ribosom e binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-1 1 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34].
• the SD sequence is thought to prom ote binding of m RNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of £. coli 16S rRNA [Steitz et al. ( 1979) "Genetic signals and nucleotide sequences in messenger R NA .” In Biological Regulation and Development: Gene Expression (ed. R .F. Goldberger)].
• To express eukaryotic genes and prokaryotic genes w ith weak ribosome-binding site [Sam brook et al. ( 1989) "Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].
• a DNA m olecule may be expressed intracellularly.
• a prom oter sequence may be directly linked w ith the DNA m olecule, in which case the first am ino acid at the N -term inus will always be a m ethionine, w hich is encoded by the ATG start codon.
• methionine at the N-term inus may be cleaved from the protein by in vitro incubation w ith cyanogen brom ide or by either in vivo on in vitro incubation w ith a bacterial methionine N -term inal peptidase (EPO-A -0 219 237). Fusion proteins provide an alternative to direct expression.
• this construct will provide a fusion of the two amino acid sequences.
• the bacteriophage lam bda cell gene can be linked at the 5 ' terminus of a foreign gene and expressed in bacteria.
• the resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. ( 1984) Nature 509:810].
• Fusion proteins can also be m ade w ith sequences from the lacl [Jia et al. (1987) Gene 60: 197], trpE [Allen et al. (1987) J. B iotechnol. 5 :93 ; M akoff et al. ( 1989) J. Gen. Microbiol. 755: 1 1 ], and Chey [EP-A-0 324 647] genes.
• the DNA sequence at the junction of the two amino acid sequences m ay or may not encode a cleavable site.
• Another example is a ubiquitin fusion protein.
• Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein.
• a processing enzyme eg. ubiquitin specific processing-protease
• foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein com prised of a signal peptide sequence fragm ent that provides for secretion of the foreign protein in bacteria [U S patent 4,336,336].
• the signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
• the protein is either secreted into the grow th media (Gram -positive bacteria) or into the periplasm ic space, located between inner and outer mem branes (Gra -negative bacteria).
• w hich can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
• DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer m em brane protein gene (ompA) [M asui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. ( 1984) EMBO J. 5:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. ( 1985 ) Proc. Natl. Acad. Sci. 82:7212].
• the signal sequence of the alpha-am ylase gene from various B acillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. ( 1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A -0 244 042].
• transcription termination sequences recognized by bacteria are regulatory regions located 3' to the translation stop codon, and thus together with the prom oter flank the coding sequence. These sequences direct the transcription of an mR N A w hich can be translated into the polypeptide encoded by the DNA .
• Transcription term ination sequences frequently include DN A sequences of ⁇ 50nt capable of forming stem loop structures that aid in term inating transcription.
• Exam ples include transcription term ination sequences derived from genes with strong prom oters, such as the trp gene in E.coli as well as other biosynthetic genes.
• Expression constructs are often m aintained in a replicon, such as an extrachromosom al element (eg. plasmids) capable of stable m aintenance in a host, such as bacteria.
• a replicon will have a replication system , thus allow ing it to be m aintained in a prokaryotic host either for expression or for cloning and amplification.
• a replicon may be either a high or low copy num ber plasm id.
• a high copy num ber plasm id will generally have a copy num ber ranging from about 5 to about 200, and usually about 10 to about 150.
• a host containing a high copy num ber plasmid w ill preferably contain at least about 10, and m ore preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
• the expression constructs can be integrated into the bacterial genome with an integrating vector.
• Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DN A in the vector and the bacterial chromosom e.
• integrating vectors constructed w ith DNA from various Bacillus strains integrate into the B acillus chrom osom e (EP-A- 0 127 328).
• Integrating vectors m ay also be comprised of bacteriophage or transposon sequences.
• extrachrom osom al and integrating expression constructs may contain selectable m arkers to allow for the selection of bacterial strains that have been transformed.
• Selectable m arkers can be expressed in the bacterial host and m ay include genes w hich render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythrom ycin, kana ycin (neom ycin), and tetracycline [Davies et al. ( 1978) Annu. Rev. Microbiol. 52:469].
• Selectable markers m ay also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
• Transformation vectors are usually com prised of a selectable market that is either m aintained in a replicon or developed into an integrating vector, as described above.
• Expression and transformation vectors either extra-chrom osom al replicons or integrating vectors, have been developed for transform ation into m any bacteria.
• expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. ( 1982) Proc. Natl. Acad. Sci. USA 79:5582 ;
• Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCli or other agents, such as divalent cations and DM SO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS MicrobioL Lett.60:273; Palva etal. (1982) Proc. Natl. Acad. Sci.
• a yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (eg. structural gene) into mRNA.
• a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site.
• a yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene.
• the UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
• Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3- phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-O 329203).
• the yeast PH05 gene encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) PNAS USA 80:1].
• synthetic promoters which do not occur in nature also function as yeast promoters.
• UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast prom oter, creating a synthetic hybrid prom oter.
• Exam ples of such hybrid promoters include the ADH regulatory sequence linked to the GA P transcription activation region (U S Patents 4,876,197 and 4,880,734).
• Other examples of hybrid prom oters include prom oters w hich consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PH05 genes, com bined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A -0 164 556).
• a yeast prom oter can include naturally occurring prom oters of non-yeast origin that have the ability to bind yeast RNA polym erase and initiate transcription. Examples of such prom oters include, inter alia, [Cohen et al. (1980) PNAS USA 77:1078 ; Henikoff et al. (1981 ) Nature 285:835 ; H ollenberg et al. (1981 ) Curr. Topics MicrobioL Immunol. 96: 1 19; Hollenberg et al. ( 1979) The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae in: Plasmids of Medical, Environmental and Commercial Importance (eds.
• a DN A molecule m ay be expressed intracellularly in yeast.
• a prom oter sequence m ay be directly linked w ith the DN A m olecule, in w hich case the first am ino acid at the N -term inus of the recom binant protein w ill always be a methionine, w hich is encoded by the A TG start codon. If desired, methionine at the N -term inus may be cleaved from the protein by in vitro incubation w ith cyanogen bromide.
• Fusion proteins provide an alternative for yeast expression system s, as well as in m am malian, baculovirus, and bacterial expression systems.
• a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein is fused to the 5' end of heterologous coding sequences.
• this construct w ill provide a fusion of the two am ino acid sequences.
• the yeast or hum an superoxide dism utase (SOD ) gene can be linked at the 5' term inus of a foreign gene and expressed in yeast.
• the DNA sequence at the junction of the tw o amino acid sequences may or m ay not encode a cleavable site.
• a ubiquitin fusion protein is m ade with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein.
• a processing enzyme eg. ubiquitin-specific processing protease
• native foreign protein can be isolated (eg. W O88/024066).
• foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein.
• a leader sequence fragment that provide for secretion in yeast of the foreign protein.
• processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro.
• the leader sequence fragment usually encodes a signal peptide com prised of hydrophobic am ino acids w hich direct the secretion of the protein from the cell.
• DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A -0012873 ; JPO . 62,096,086) and the A -factor gene (US patent 4,588,684).
• yeast invertase gene EP-A -0012873 ; JPO . 62,096,086) and the A -factor gene (US patent 4,588,684).
• leaders of non-yeast origin such as an interferon leader, exist that also provide for secretion in yeast (EP-A -0060057).
• a preferred class of secretion leaders are those that em ploy a fragment of the yeast alpha-factor gene, w hich contains both a "pre" signal sequence, and a "pro” region .
• the types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 am ino acid residues) (U S Patents 4,546,083 and 4,870,008; EP-A -0 324 274).
• Additional leaders em ploying an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see W O 89/02463.)
• transcription term ination sequences recognized by yeast are regulatory regions located 3' to the translation stop codon, and thus together w ith the prom oter flank the coding sequence. These sequences direct the transcription of an mRNA w hich can be translated into the polypeptide encoded by the DNA. Examples of transcription term inator sequence and other yeast-recognized term ination sequences, such as those coding for glycolytic enzymes.
• Expression constructs are often m aintained in a replicon, such as an extrachromosomal element (eg. plasm ids) capable of stable m aintenance in a host, such as yeast or bacteria.
• the replicon may have two replication system s, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al.
• a replicon m ay be either a high or low copy number plasmid.
• a high copy num ber plasm id w ill generally have a copy num ber ranging from about 5 to about 200, and usually about 10 to about 150.
• a host containing a high copy num ber plasm id w ill preferably have at least about 10, and m ore preferably at least about 20. Enter a high or low copy num ber vector m ay be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. B rake et al., supra.
• the expression constructs can be integrated into the yeast genome with an integrating vector.
• Integrating vectors usually contain at least one sequence hom ologous to a yeast chrom osome that allow s the vector to integrate, and preferably contain two hom ologous sequences flanking the expression construct. Integrations appear to result from recom binations between hom ologous DNA in the vector and the yeast chromosome [Orr-W eaver et al. (1983) Methods in Enzymol. 707 :228-245].
• An integrating vector m ay be directed to a specific locus in yeast by selecting the appropriate hom ologous sequence for inclusion in the vector. See Orr-W eaver et al., supra.
• One or more expression construct may integrate, possibly affecting levels of recom binant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750].
• the chromosomal sequences included in the vector can occur either as a single segment in the vector, w hich results in the integration of the entire vector, or two segm ents hom ologous to adjacent segm ents in the chrom osome and flanking the expression construct in the vector, w hich can result in the stable integration of only the expression construct.
• extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.
• Selectable m arkers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, 77754, LEU2, TRP1, and ALG7, and the G418 resistance gene, w hich confer resistance in yeast cells to tunicam ycin and G418, respectively.
• a suitable selectable m arker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal.
• the presence of CUPI allows yeast to grow in the presence of copper ions [Butt etal. (1987) Microbiol, Rev.57:351].
• Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
• Expression and transformation vectors have been developed for transformation into many yeasts.
• expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol.Cell.Biol.6:142], Candida maltosa [Kunze, et al. (1985) J.Basic Microbiol.25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J.Gen. Microbiol.752:3459; Roggenkamp et al. (1986) Mol.Gen.Genet.202:302], Kluyveromyces fragilis [Das, et al.
• Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol.25:141; Candida]; [Gleeson et al. (1986) J. Gen. MicrobioL 752:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.202:302; Hansenula]; [Das et al. (1984) J. Bacteriol.
• antibody refers to a polypeptide or group of polypeptides composed of at least one antibody combining site.
• An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen.
• Antibody includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
• Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying proteins.
• Antibodies to the proteins of the invention may be prepared by conventional methods.
• the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volum e of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies.
• Im m unization is generally perform ed by m ixing or em ulsifying the protein in saline, preferably in an adjuvant such as Freund' s complete adjuvant, and injecting the m ixture or em ulsion parenterally (generally subcutaneously or intramuscularly).
• Im m unization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incom plete adjuvant.
• Polyclonal antisera is obtained by bleeding the im munized anim al into a glass or plastic container, incubating the blood at 25 °C for one hour, followed by incubating at 4°C for 2- 18 hours. The serum is recovered by centrifugation (eg. l ,000g for 10 m inutes). A bout 20-50 m l per bleed m ay be obtained from rabbits.
• M onoclonal antibodies are prepared using the standard method of Kohler & M ilstein [Nature (1975) 256:495-96], or a modification thereof.
• a m ouse or rat is im m unized as described above.
• the spleen (and optionally several large lym ph nodes) is removed and dissociated into single cells.
• B -cells expressing mem brane-bound im munoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension.
• Resulting B -ceils, or all dissociated spleen cells are then induced to fuse with m yelom a cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium , "HAT").
• the resulting hybridom as are plated by limiting dilution, and are assayed for the production of antibodies w hich bind specifically to the im m unizing antigen (and which do not bind to unrelated antigens).
• the selected M Ab-secreting hybridom as are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
• the antibodies (whether polyclonal or monoclonal) m ay be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atom s (particularly 32 P and l25 I), electron-dense reagents, enzym es, and ligands having specific binding partners. Enzym es are typically detected by their activity. For exam ple, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TM B ) to a blue pigment, quantifiable w ith a spectrophotometer.
• TM B 3,3',5,5'-tetramethylbenzidine
• Specific binding partner refers to a protein capable of binding a ligand m olecule w ith high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor.
• Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-Iigand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different m odes. For example, l 25 I m ay serve as a radioactive label or as an electron-dense reagent. HRP m ay serve as enzyme or as antigen for a M Ab.
• M Abs and avidin also require labels in the practice of this invention: thus, one m ight label a M Ab w ith biotin, and detect its presence with avidin labeled with l 25 I, or with an anti-biotin M Ab labeled w ith HR P. O ther perm utations and possibilities w ill be readily apparent to those skilled in the art, and are considered as equivalents within the scope of the invention.
• Pharm aceutical com positions can com prise either polypeptides, antibodies, or nucleic acid of the invention.
• the pharm aceutical compositions w ill com prise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.
• the term "therapeutically effective am ount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chem ical m arkers or antigen levels. Therapeutic effects also include reduction in physical symptom s, such as decreased body temperature.
• the precise effective amount for a subject w ill depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or com bination of therapeutics selected for adm inistration. Thus, it is not useful to specify an exact effective am ount in advance. However, the effective amount for a given situation can be determ ined by routine experimentation and is within the judgement of the clinician.
• an effective dose w ill be from about 0.01 m g/ kg to 50 mg/kg or 0.05 mg/kg to about 10 m g/kg of the DNA constructs in the individual to w hich it is adm inistered.
• a pharmaceutical composition can also contain a pharm aceutically acceptable carrier.
• pharmaceutically acceptable carrier refers to a carrier for adm inistration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any phar aceutical carrier that does not itself induce the production of antibodies harm ful to the individual receiving the composition, and which may be administered without undue toxicity.
• Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric am ino acids, am ino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
• salts can be used therein, for example, m ineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
• m ineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
• organic acids such as acetates, propionates, malonates, benzoates, and the like.
• Pharmaceutically acceptable carriers in therapeutic com positions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, m ay be present in such vehicles.
• the therapeutic com positions are prepared as injectables, either as liquid solutions or suspensions; solid form s suitable for solution in, or suspension in, liquid vehicles prior to injection m ay also be prepared. Liposom es are included w ithin the definition of a pharm aceutically acceptable carrier.
• compositions of the invention can be administered directly to the subject.
• the subjects to be treated can be animals; in particular, human subjects can be treated.
• Direct delivery of the com positions w ill generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intram uscularly or delivered to the interstitial space of a tissue.
• the compositions can also be administered into a lesion.
• Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays.
• Dosage treatment may be a single dose schedule or a multiple dose schedule.
• Vaccines may be a single dose schedule or a multiple dose schedule.
• Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
• Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with "pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
• Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
• Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
• the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, 77. pylori, etc. pathogens.
• a bacterial toxoid such as a toxoid from diphtheria, tetanus, cholera, 77. pylori, etc. pathogens.
• Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59TM (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
• aluminum salts alum
• oil-in-water emulsion formulations with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components
• MF59TM WO 90/14837
• Span 85 containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RibiTM adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (MPL), trehalose dimycolate (TDM), and cell wall skeleton (MPL), trehalose dimycol
• interferons eg. gamma interferon
• M-CSF macrophage colony stimulating factor
• TNF tumor necrosis factor
• other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
• Alum and M F59TM are preferred.
• muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L- alanyl-D-isoglu tarn inyl-L-alanine-2-(l '-2'-dipalm itoyl---n-glycero-3-hydroxyphosphoryloxy)-ethy lam ine (M TP-PE), e.c.
• thr-MDP N-acetyl-muramyl-L-threonyl-D- isoglutamine
• nor-MDP N-acetyl-normuramyl-L-alanyl-D-isoglutamine
• M TP-PE N-acetylmuramy
• the im m unogenic compositions typically w ill contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or em ulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
• the im m unogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid form s suitable for solution in, or suspension in, liquid vehicles prior to injection m ay also be prepared.
• the preparation also m ay be em ulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
• Im munogenic com positions used as vaccines comprise an im m unologically effective am ount of the antigenic or im m unogenic polypeptides, as well as any other of the above-mentioned com ponents, as needed.
• B y "immunologically effective amount” it is meant that the adm inistration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This am ount varies depending upon the health and physical condition of the individual to be treated, the taxonom ic group of individual to be treated (eg.
• the im m unogenic compositions are conventionally adm inistered parenterally, eg. by injection, either subcu- taneously, intramuscularly, or transdermally/transcutaneously (eg. W O98/20734). Additional formulations suitable for other modes of adm inistration include oral and pulm onary formulations, suppositories, and transderm al applications.
• Dosage treatment m ay be a single dose schedule or a m ultiple dose schedule.
• the vaccine m ay be adm inistered in conjunction w ith other im m unoregulatory agents.
• DNA vaccination may be used [eg. Robinson & Torres (1997) Seminars in Immunology 9:271 -283; Donnelly et al. ( 1997) Annu Rev Immunol 15 :617-648 ; see later herein].
• polynucleotide compositions of the invention can be adm inistered (1 ) directly to a subject; (2) delivered ex vivo, to cells derived from a subject; or (3) in vitro for expression of recom binant proteins.
• the subjects to be treated can be mam m als or birds. Also, hum an subjects can be treated.
• Direct delivery of the compositions w ill generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue.
• the compositions can also be adm inistered into a lesion .
• Other modes of administration include oral and pulmonary administration, suppositories, and transderm al or transcutaneous applications (eg. see W O98/20734), needles, and gene guns or hyposprays.
• Dosage treatm ent may be a single dose schedule or a m ultiple dose schedule.
• M ethods for the ex vivo delivery and reimplantation of transformed cells into a subject are know n in the art and described in eg. W 093/14778.
• Exam ples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lym ph cells, macrophages, dendritic cells, or tumor cells.
• nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-m ediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposom es, and direct microinjection of the DNA into nuclei, all well known in the art.
• the following additional agents can be used w ith polynucleotide and/or polypeptide com positions.
• polypeptides w hich include, without lim itation : asioloorosom ucoid (ASO R); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stim ulating factor (GM -CSF), granulocyte colony stim ulating factor (G-CSF), macrophage colony stimulating factor (M -C SF), stem cell factor and erythropoietin.
• Viral antigens such as envelope proteins, can also be used.
• proteins from other invasive organism s such as the 17 amino acid peptide from the circu sporozoite protein of plasm odium falciparum known as RII.
• hormones for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitam ins, folic acid.
• polyalkylene glycol can be included with the desired polynucleotides/polypeptides.
• the polyalkylene glycol is polyethlylene glycol.
• mono-, di-, or polysaccharides can be included.
• the polysaccharide is dextran or DEAE-dextran.
• the desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom .
• Lipid encapsulation is generally accomplished using liposomes w hich are able to stably bind or entrap and retain nucleic acid.
• the ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1 : 1 (m g DNA :m icromoles lipid), or m ore of lipid.
• liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight ( 1991 ) Biochim. Biophys. Acta. 1097: 1 -17 ; Straubinger ( 1983) Meth. Enzymol. 101 :512-527.
• Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations.
• Cationic liposomes have been shown to mediate intracellular delivery of plasm id DN A (Feigner ( 1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (M alone ( 1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.
• Cationic liposomes are readily available.
• N[l-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. (See, also, Feigner supra).
• Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
• Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP
• anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
• Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
• the liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
• MLVs multilammelar vesicles
• SUVs small unilamellar vesicles
• LUVs large unilamellar vesicles
• the various liposome-nucleic acid complexes are prepared using methods known in the art. See eg. Straubinger (1983) Meth. Immunol.101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Ada 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys.
• lipoproteins can be included with the polynucleotide/polypeptide to be delivered.
• lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.
• Naturally occurring lipoproteins comprise a lipid and a protein portion.
• the protein portion are known as apoproteins.
• apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, Al, All, AIV; CI, C II, CHI.
• a lipoprotein can comprise more than one apoprotein.
• naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E apoproteins.
• VLDL comprises A, B, C & E apoproteins
• LDL comprises apoprotein B
• HDL comprises apoproteins A, C, & E.
• the am ino acid of these apoproteins are known and are described in, for exam ple, B reslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp M ed. B iol.
• Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. Lipid compositions vary in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 ( 1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association w ith the polynucleotide binding m olecule. Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance.
• Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for exam ple, A tkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys A a 30: 443. Lipoproteins can also be purchased from com flareal suppliers, such as Biomedical Techniologies, Inc., Stoughton, M assachusetts, U SA . Further description of lipoproteins can be found in Zuckerm ann et al. PCT/U S97/14465.
• Polycationic agents can be included, w ith or w ithout lipoprotein, in a com position with the desired polynucleotide/polypeptide to be delivered.
• Polycationic agents typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location . These agents have both in vitro, ex vivo, and in vivo applications.
• Polycationic agents can be used to deliver nucleic acids to a living subject either intram uscularly, subcutaneously, etc.
• polylysine polyarginine, polyornithine, and protamine.
• Other examples include histones, protam ines, hum an serum album in, DNA binding proteins, non-histone chromosom al proteins, coat proteins from DNA viruses, such as (X 174, transcriptional factors also contain domains that bind DNA and therefore m ay be useful as nucleic aid condensing agents.
• transcriptional factors such as C/CEB P, c-jun, c-fos, AP- 1 , AP-2, A P-3, CPF, Prot-1 , Sp- 1 , Oct- 1 , Oct-2, CREP, and TFIID contain basic dom ains that bind DN A sequences.
• Organic polycationic agents include: sperm ine, spermidine, and purtrescine.
• polycationic agent The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.
• Synthetic polycationic agents w hich are useful include, for example, DEAE-dextran, polybrene.
• LipofectinTM, and lipofectAM INETM are monomers that form polycationic complexes w hen combined with polynucleotides/polypeptides.
• Antigens of the invention can be used in im m unoassays to detect antibody levels (or, conversely, anti-protein antibodies can be used to detect antigen levels).
• Im m unoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics m ethods.
• Antibodies to proteins within biological samples, including for example, blood or serum sam ples, can be detected.
• Design of the im munoassays is subject to a great deal of variation, and a variety of these are know n in the art. Protocols for the im m unoassay m ay be based, for exam ple, upon competition, or direct reaction, or sandw ich type assays.
• Protocols may also, for exam ple, use solid supports, or m ay be by im m unoprecipitation.
• M ost assays involve the use of labeled antibody or polypeptide; the labels m ay be, for exam ple, fluorescent, chem ilum inescent, radioactive, or dye molecules.
• a ssays w hich amplify the signals from the probe are also known ; examples of w hich are assays w hich utilize biotin and avidin, and enzyme-labeled and mediated im m unoassays, such as ELISA assays.
• Kits suitable for im munodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the com positions of the invention, in suitable containers, along with the rem aining reagents and m aterials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.
• Hybridization refers to the association of tw o nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction tem perature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or B LOTTO); concentration of the sequences; use of com pounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization.
• Stringency refers to conditions in a hybridization reaction that favor association of very sim ilar sequences over sequences that differ.
• the combination of temperature and salt concentration should be chosen that is approximately 120 to 200°C below the calculated Tm of the hybrid under study.
• the tem perature and salt conditions can often be determ ined empirically in preliminary experiments in w hich samples of genom ic DN A im mobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sam brook et al. at page 9.50.
• Variables to consider when perform ing are ( 1 ) the complexity of the DNA being blotted and (2) the hom ology between the probe and the sequences being detected.
• the total amount of the fragment(s) to be studied can vary a m agnitude of 10, from 0.1 to I ⁇ g for a plasm id or phage digest to I O "9 to I O '8 g for a single copy gene in a highly complex eukaryotic genom e.
• substantially shorter blotting, hybridization, and exposure tim es a sm aller am ount of starting polynucleotides, and lower specific activity of probes can be used.
• a single-copy yeast gene can be detected w ith an exposure time of only 1 hour starting w ith 1 ⁇ g of yeast D N A , blotting for two hours, and hybridizing for 4-8 hours w ith a probe of I O 8 cpm/ ⁇ g.
• a single-copy m am m alian gene a conservative approach would start w ith 10 ⁇ g of DN A , blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10 8 cpm/ ⁇ g, resulting in an exposure time of -24 hours.
• Tm melting temperature
• Tm 81 + 1 6.6(log
• Ci is the salt concentration (m onovalent ions)
• n is the length of the hybrid in base pairs (slightly modified from M einkoth & W ahl ( 1984) Anal. Biochem. 138: 267-284).
• the temperature of the hybridization and washes and the salt concentration during the washes are the sim plest to adjust.
• the radiolabeled probe is not com pletely hom ologous with the im mobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background w ill increase.
• the temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner.
• the stringency of the washes is also increased with decreasing salt concentrations.
• Nucleic Acid Probe Assays M ethods such as PCR , branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determ ine the presence of cDNA or m RNA .
• a probe is said to "hybridize" with a sequence if it can form a duplex or double stranded complex, w hich is stable enough to be detected.
• nucleic acid probes will hybridize to the nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences w ill encode the am ino acid sequence, the native sequence is preferred because it is the actual sequence present in cells.
• m RN A represents a coding sequence and so a probe should be com plem entary to the coding sequence; single-stranded cDNA is complem entary to m R NA , and so a cDNA probe should be com plem entary to the non-coding sequence.
• the probe sequence need not be identical to the sequence (or its com plem ent) - some variation in sequence and length can lead to increased assay sensitivity if the probe can form a duplex with target nucleotides, which can be detected. Also, the probe can include additional nucleotides to stabilize the formed duplex. Additional sequence m ay also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5' end of the probe, with the remainder of the probe sequence being complem entary to a sequence.
• non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a sequence in order to hybridize therewith and thereby form a duplex w hich can be detected.
• the exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like.
• the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 1 5-25, and more preferably at least 30 nucleotides, although it m ay be shorter than this. Short primers generally require cooler tem peratures to form sufficiently stable hybrid complexes w ith the template.
• Probes may be produced by synthetic procedures, such as the triester method of M atteucci et al. [J. Am. Chem. Soc. (1981 ) 103 :3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461 ], or using commercially available automated oligonucleotide synthesizers.
• the chem ical nature of the probe can be selected according to preference.
• DNA or RNA are appropriate.
• m odifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see A grawal & Iyer ( 1995) Curr Opin Biotechnol 6: 12-19; Agraw al (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey ( 1997) TIBTECH 15 :224-229; B uchardt et al. (1993) TIBTECH 1 1 :384-386],
• PCR polymerase chain reaction
• the assay is described in: M ullis et al. [Meth. Enzymol. ( 1987) 155: 335-350]; U S patents 4,683,195 and 4,683,202.
• Two "primer" nucleotides hybridize with the target nucleic acids and are used to prime the reaction.
• the primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence w ill flank the desired sequence.
• a therm ostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a tem plate. After a threshold am ount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. W hen using the Southern blot method, the labelled probe will hybridize to the sequence (or its complement).
• mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra].
• mRNA , or cDNA generated from mRNA using a polymerase enzyme can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled w ith a radioactive m oiety.
• Figure 1 demonstrates the interaction of VIP54 with VacA.
• Figure 1A shows the results of a yeast two-hybrid screen, specifically H1S3 reporter gene activation (growth on His "ve medium), for interactions between V1P54 and VacA.
• Four forms of VacA were tested: full-length, amino acids 1-672, p58 domain, or p37 domain.
• Figure IB shows an immunoblot using anti- Vac A.
• Figure 2A shows the sequences of human (SEQ ID 1) and murine (SEQ ID 2) VIP54. Different amino acids in the mouse sequence are indicated. Identical amino acid residues (459/500) are shown as dots, whereas the similar ones are shadowed. Peptides used to produce anti-VIP54 antisera are underlined.
• Figure 2B shows a secondary structure prediction for hVIP54 - large bars indicate ⁇ -helix, small bars indicate ⁇ -strand, and spaces between bars are predicted to be structureless.
• Figure 3 is a Northern blot showing tissue distribution of hVIP54 expression.
• Figure 4 shows immunofluorescence results using, demonstrating the co-localisation of VIP54 with vimentin in MDCK cells (A, B, C) and BHK cells (D, E, F).
• Figures 4A & 4D are stained with polyclonal anti-VIP54; 4B & 4E are stained with monoclonal anti-vimentin.
• Figure 4C is the overlay of Figures 4A & 4B;
• Figure 4F is the overlay of 4D & 4E.
• the inserts in 4A & 4B are enlargements to show the different fine staining patterns of VIP54 and vimentin.
• Figure 5 shows how vimentin and VIP54 distribution is affected in HeLa cells after 16 hours treatment with lO ⁇ g/ml colchicine.
• Figure 5 A & 5D are stained with polyclonal anti-VIP54;
• Figure 5B is stained with monoclonal anti-vimentin;
• Figure 5E is stained with monoclonal anti-cytocheratin.
• Figure 6 is a Western blot showing the co-distribution of VIP54 and vimentin in a variety of cells lines. Blots were labelled using monoclonal anti-vimentin or polyclonal anti-VIP54.
• Figure 7 shows Western blots to demonstrate that VIP54 and vimentin remain associated after extraction of filaments.
• P indicates the insoluble fraction after cell lysis and
• S indicates the soluble fraction.
• cell lysates were incubated with (+) or without (-) monoclonal anti-vimentin. Blots were labelled with the same antibodies as in Figure 6.
• FIG. 8 shows that VIP54 is not expressed in neurons (A, B, C), but is expressed in neuroblastoma (D, E, F).
• Cells were labelled with polyclonal anti-VIP54 (A, D), monoclonal anti-neurofilaments (B), or polyclonal anti-vimentin (E).
• the overlays (C & F) show that VIP54 is not expressed in neurons, but is expressed in neuroblastoma cells.
• Y2H Yeast two-hybrid
• the two-hybrid L40 yeast report strain was first transformed with a plasmid encoding a fusion between full-length VacA and the bacterial protein LexA, which recognizes specific DNA sequences upstream of the two reporter genes HIS3 and LacZ [9].
• This screening strain was subsequently transformed with a ⁇ eLa cDNA plasmid library encoding C-terminal fusion proteins with the transcriptional activation domain of Gal4 (GAD).
• the cD ⁇ A encoding for the protein fragment identified by Y2 ⁇ screening was cloned from the pGAD vector in a pGEX vector, in frame with the sequence encoding for the protein GST and expressed in E.coli.
• the GST fusion protein was bound to a matrix of GSH-sepharose and VacA was loaded on the column. After extensive washings, the bound material was analysed by immunoblot staining using an anti-VacA polyclonal antibody. This assay shows that VacA is capable of binding the polypeptide (Figure IB), with high specificity.
• the murine sequence was obtained by screening a cD ⁇ A mouse phage library using the cDNA of the human protein excised from the pGAD construct as a probe. With this method, a clone containing the 5' region, but lacking the 3' region, was obtained. The sequence of the mouse protein was completed using EST data and a working draft sequence
• the VacA interacting protein consists of 500 residues, corresponding to a MW of 54,133 with a predicted isolelectric point of 5.0.
• the protein is referred to as 'VIP54' for VacA interacting protein, 54kDa.
• the protein is highly conserved between human and mouse, suggesting that it plays an essential role in the cell. No parts of it match any sequence present in databases.
• VIP54 co-localises with vimentin-containing intermediate filaments
• KLH-conjugated VIP54 peptides SEQ ID 4, 5 or 6, each with an additional N-terminal cysteine. These peptides were chosen because of their hydrophilicity and their absence in sequence databases.
• Immune antisera were affinity-purified on peptide bound to columns and were tested on a variety of cultured cells. Antisera raised against SEQ ID 4 gave good staining and were chosen for immunofluorescence experiments.
• VIP54 is highly expressed in astrocytes and neuroblastoma, but not CNS neurons
• Vimentin is frequently used as a marker for de-differentiation in several types of tumour [2, 20]. Given the high expression of VIP54 in brain, its distribution in brain was investigated further. Figure 8 compares the VIP54 distribution in the mixed cell population isolated from cerebellum with that in neuroblastoma cells (HTB11).
• Astrocytes show a marked staining, indicating large amounts of VIP54, which defines the same network seen in other non-neuronal cells (Figure 8A).
• VIP54 is not present in neurons, clearly identified by anti-neurofilaments mAb, but is present in large amount in primary neuroblastoma cells (Figure 8D) established in culture after surgical removal of the tumour. VIP54 and vimentin also co-localise in these tumour cells. VIP54 is thus highly expressed upon neuron transformation, and can thus be used in CNS studies and as a marker of tumours originating from CNS neurons.
• IFs were traditionally thought as fixed structural bystanders around which the lively activity of the cell is distributed, but IFs and their associated proteins have now been firmly established as dynamic components of the cytoplasmic and nuclear cytoskeleton.
• EF-associated proteins expands the repertoire of possible interactions permitted to IFs and may add another layer of regulatory complexity.
• the capacity of IFs to associate with the nuclear membrane and with the plasmalemma is clearly documented [21,22]. Although the exact nature of such membrane-IFs interactions are not established, IFs clearly possess elements capable of mediating membrane interactions. IFs may therefore modulate the structure/ dynamics of late endosomal/lysosomal compartments, possibly via VIP54.
• vacuolated cells In vacuolated cells, VIP54 immunofluorescent staining is largely lost, but the protein is present in an unaltered form, as judged from immunoblotting. This is compatible with the idea that a toxin-induced If alteration is involved in vacuolisation, although the alternative possibility that vacuolisation affects the physiological organisation of IFs cannot be dismissed. Both explanations implicate a relation between EFs and late endosomal compartments.

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