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WO1997045535A1 - Phosphate cyclases a terminaison 3' d'arn de recombinaison et procedes de production - Google Patents

Phosphate cyclases a terminaison 3' d'arn de recombinaison et procedes de production Download PDF

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WO1997045535A1
WO1997045535A1 PCT/EP1997/002566 EP9702566W WO9745535A1 WO 1997045535 A1 WO1997045535 A1 WO 1997045535A1 EP 9702566 W EP9702566 W EP 9702566W WO 9745535 A1 WO9745535 A1 WO 9745535A1
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gly
leu
rna
val
ala
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PCT/EP1997/002566
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Pascal Genschik
Witold Filipowicz
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Novartis Ag
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Priority to AU30277/97A priority Critical patent/AU3027797A/en
Publication of WO1997045535A1 publication Critical patent/WO1997045535A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the present invention relates to a ubiquitously distributed RNA 3'-term ⁇ nal phosphate cyclase protein
  • RNA 3'-term ⁇ nal phosphate cyclase catalyses the conversion of 3' phosphate to a 2', 3'-phosphod ⁇ ester at the 3' end of RNA molecules
  • the activity has been previously detected and partial purifications thereof have been prepared (reviewed in Fihpowicz and Vicente, (1990) Meth. Enzymol. 181, 499-510) Its activity results in the activation of the 3' end of RNA molecules.
  • RNA ligases require 3'-term ⁇ nal cyclic phosphate in order to perform the hgation reaction RNA 3'-term ⁇ nal phosphate cyclase may also be involved in the cyclisation of the 3'-term ⁇ nal phosphate of the spliceosomal small nuclear RNA U6
  • RNA 3'-term ⁇ nal phosphate cyclase appears to be a useful tool for the study of RNA synthesis and metabolism in the cell, as well as for in vitro RNA manipulation, it has hitherto not been available except in the form of an activity purified from HeLa cells and also identified in Xenopus oocytes
  • RNA 3'-termmal phosphate cyclase Surprisingly, we have found that one of the open reading frames of unknown function in E.
  • RNA 3'-term ⁇ nal phosphate cyclase activity Sequences with significant similarity to RNA 3'-term ⁇ nal phosphate cyclase tsoalted from human and E coli sources are also found in other organisms, suggesting a key role for this enzyme in cellular metabolism
  • RNA 3'-term ⁇ na! phosphate cyclase comprising expressing the a nucleic acid sequence encoding said cyclase, or a derivative thereof in a recombinant host cell
  • the invention also provides a nucleic acid sequence encoding RNA 3'-term ⁇ nal phosphate cyclase, or a derivative thereof, as well as vectors and host cells containing such nucleic acids
  • the RNA 3'-term ⁇ nal phosphate cyclase of the invention is found to be a conserved enzyme across a wide variety of organisms
  • the invention accodingly provides RNA 3'- terminal phosphate cyclase polypeptides of eukaryotic as well as prokaryotic origin
  • the method of the first aspect of the invention preferably comprises the steps of transfecting a host cell with a vector comprising a nucleic acid sequence encoding RNA 3'- terminal phosphate cyclase, culturing the host cell to express RNA 3'-term ⁇ nal phosphate cyclase and purifying RNA 3'-term ⁇ nal phosphate cyclase from the cell culture
  • RNA 3'-terminal phosphate cyclase polypeptide produced by the method of the invention has homology, as hereinafter defined, with the sequence set forth in SEQ ID No 2 Accordingly, it should be understood that the term "RNA 3'-term ⁇ nal phosphate cyclase" as used henn refers to any polypeptide which is homologous to this sequence It has been determined, and is set forth herein, that RNA 3'-term ⁇ nal phosphate cyclase is a widely conserved polypeptide.
  • RNA 3'-term ⁇ nal phosphate cyclase polypeptides from man and E. coli retain common functional activity, as well as close structural homology.
  • homology means that the entities compared share sufficient characteristics for the skilled person to determine that they are similar in origin and function.
  • Each of the RNA 3'-term ⁇ nal phosphate cyclase polypeptides disclosed herein is homologous to the others.
  • homology is used to refer to sequence identity.
  • the derivatives of RNA 3'-term ⁇ nal phosphate cyclase preferably retain substantial sequence identity with the RNA 3'-term ⁇ nal phosphate cyclase polypeptides disclosed herein.
  • Substantial homology where homology indicates sequence identity, means more than 20% sequence identity, preferably more than 35% sequence identity and most preferably a sequence identity of 30% or more
  • RNA 3'-term ⁇ nal phosphate cyclases in accordance with the present invention are set out herein as follows
  • polypeptides which are derivatives of RNA 3'-term ⁇ nal phosphate cyclase, and share at least one common structural determinant therewith
  • RNA 3'-term ⁇ nal phosphate cyclase as provided by the present invention includes ammo acid mutants, glycosylation variants and other covalent derivatives of RNA 3'-term ⁇ nal phosphate cyclase which retain the physiological and/or physical properties of RNA 3'- terminal phosphate cyclase Further included are naturally occurring variants of RNA 3'- terminal phosphate cyclase found with a particular species, preferably a mammal
  • the invention comprises derivatives of RNA 3 -terminal phosphate cyclase which have been modified by chemical means
  • derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring ammo acid
  • a moiety may be a detectable moiety such as an enzyme or a radioisotope
  • Derivatives can be fragments of RNA 3'-term ⁇ nal phosphate cyclase.
  • Fragments of RNA 3'-term ⁇ nal phosphate cyclase comprise individual domains thereof, as well as smaller polypeptides derived from the domains
  • smaller polypeptides derived from RNA 3'-term ⁇ nal phosphate cyclase according to the invention define a single feature which is characteristic of RNA 3'-terminal phosphate cyclase.
  • Fragments may in theory be almost any size, as long as they retain one feature of RNA 3'-term ⁇ al phosphate cyclase.
  • fragments will be between 5 and 200 ammo acids in length. Longer fragments are regarded as truncations of the full-length RNA 3'-term ⁇ nal phosphate cyclase and generally encompassed by the term "RNA 3'-term ⁇ nal phosphate cyclase".
  • RNA 3'-term ⁇ nal phosphate cyclase also comprise mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain at least one feature characteristic of RNA 3'-term ⁇ nal phosphate cyclase.
  • conservative ammo acid substitutions may be made substantially without altering the nature of RNA 3'-terminal phosphate cyclase, as may truncations from the 5' or 3' ends.
  • Deletions and substitutions may moreover be made to the fragments of RNA 3'- terminal phosphate cyclase comprised by the invention.
  • RNA 3 -term ⁇ nal phosphate cyclase mutants may be produced from a DNA encoding RNA 3'-term ⁇ nal phosphate cyclase which has been subjected to in vitro mutagenesis resulting e.g in an addition, exchange and/or deletion of one or more amino acids.
  • substitutional, deletional or insertional variants of RNA 3'-terminal phosphate cyclase can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of RNA 3'-term ⁇ nal phosphate cyclase.
  • Mutations may be performed by any method known to those of skill in the art. Preferred, however, is site-directed mutagenesis of a nucleic acid sequence encoding the cyclase of interest.
  • a number of methods for site-directed mutagenesis are known in the art, from methods employing single-stranded phage such as M13 to PCR-based techniques (see “PCR Protocols: A guide to methods and applications", M A Innis, D.H Gelfand, J.J. Sninsky, T.J. White (eds.). Academic Press, New York, 1990)
  • the commercially available Altered Site II Mutagenesis System (Promega) may be employed, according to the directions given by the manufacturer
  • RNA 3'-term ⁇ nal phosphate cyclase polypeptides in essentially pure form It has been determined that RNA 3'-terminal phosphate cyclase is highly conserved throughout all organisms which have been tested RNA 3'-terminal phosphate cyclase highly homologous to the cloned human HeLa cell RNA 3'-terminal phosphate cyclase set forth in SEQ ID No 2 has been found in E. coli, D melanogaster, S. pombe and S. cervisiae, as set out hereinbefore.
  • RNA 3'-term ⁇ nal phosphate cyclase polypeptides and derivatives thereof from any source where "derivatives" are as hereinbefore defined
  • a particularly surprising aspect of the invention is the provision of RNA 3'-term ⁇ nal phosphate cyclase from prokaryotic sources
  • E coli RNA 3'-term ⁇ nal phosphate cyclase is encoded by a coding sequence which, due to a sequencing error, has previously been classified as two separate ORFs
  • Re-sequencing of the relevant part of the E coli genome demonstrates that the sequence previously believed to comprise two ORFs of unknown function is actually a single ORF encoding an enzyme which is highly homologous to human RNA 3'-term ⁇ nal phosphate cyclase and is functionally equivalent thereto
  • nucleic acid encoding RNA 3'-term ⁇ nal phosphate cyclase In addition to being useful for the production of recombinant RNA 3'-term ⁇ nal phosphate cyclase protein, these nucleic acids are also useful as probes, thus readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding RNA 3'-term ⁇ nal phosphate cyclase
  • nucleic acid according to the invention is useful in a method determining the presence of RNA 3'-term ⁇ nal phosphate cyclase-specific nucleic acid, said method comprising hybridising the DNA (or RNA) encoding RNA 3'-term ⁇ nal phosphate cyclase (or complementary thereto) to test sample nucleic acid and determining the presence of RNA 3'-term ⁇ nal phosphate cyclase.
  • the invention provides nucleic acid sequence that is complementary to, or hybridises under stringent conditions to, a nucleic acid sequence encoding RNA 3'-term ⁇ nal phosphate cyclase
  • the invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) RNA 3'-termmal phosphate cyclase
  • nucleic acids encoding RNA 3'-term ⁇ nal phosphate cyclase polypeptides may be cloned from tissues according to established procedures using probes derived from sequences identified herein
  • such DNAs can be prepared by a) isolating mRNA from suitable cells, selecting the desired mRNA, for example by hybridisation with a DNA probe or by expression in a suitable expression system and screening for expression of the desired polypeptide, preparing single-stranded cDNA complementary to that mRNA, then double-stranded cDNA therefrom, or b) isolating cDNA from a cDNA library and selecting the desired cDNA, for example using a DNA probe or using a suitable expression system and screening for expression of the desired polypeptide, or c) incorporating the double-stranded DNA of step a) or b) into an appropriate expression vector, d) transforming appropriate host cells with the vector and isolating the desired DNA
  • Polyadenylated messenger RNA (step a) is isolated by known methods. Isolation methods involve, for example, homogenizing cells in the presence of a detergent and a nbonuclease inhibitor, for example hepann, guanidmium isothiocyanate or mercaptoethanol, extracting the mRNA with a chloroform-phenol mixture, optionally in the presence of salt and buffer solutions, detergents and/or cation chelating agents, and precipitating mRNA from the remaining aqueous, salt-containing phase with ethanol, isopropanol or the like
  • the isolated mRNA may be further purified by centnfuging in a caesium chloride gradient followed by ethanol precipitation and/or by chromatographic methods, for example affinity chromatography, for example chromatography on ol ⁇ go(dT) cellulose or on ol ⁇ go(U) sepharose
  • chromatographic methods for example affinity chromatography, for example chromatography on ol ⁇ go(
  • the desired mRNA is selected by screening the mRNA directly with a DNA probe, or by translation in suitable cells or cell-free systems and screening the obtained polypeptides
  • RNA hybridisation probe a DNA hybridisation probe, thereby avoiding the additional step of translation
  • Suitable DNA probes are DNAs of known nucleotide sequence consisting of at least 17 nucleotides derived from DNAs encoding RNA 3'-term ⁇ nal phosphate cyclase
  • Synthetic DNA probes are synthesised according to known methods as detailed hereinbelow, preferably by stepwise condensation using the solid phase phosphot ⁇ ester, phosphite tnester or phosphoramidite method, for example the condensation of dinucleotide coupling units by the phosphotnester method
  • stepwise condensation using the solid phase phosphot ⁇ ester, phosphite tnester or phosphoramidite method, for example the condensation of dinucleotide coupling units by the phosphotnester method
  • These methods are adapted to the synthesis of mixtures of the desired oligonucleotides by using mixtures of two, three or four nucleotides dA, dC, dG and/or dT in protected form or the corresponding dinucleotide coupling units in the appropriate condensation step as described by Y Ike et al (Nucleic Acids Research H, 477, 1983)
  • the DNA probes are labelled, for example radioactively labelled by the well known kinase reaction
  • the hybridisation of the size-fractionated mRNA with the DNA probes containing a label is performed according to known procedures, i e in buffer and salt solutions containing adjuncts, for example calcium chelators, viscosity regulating compounds, proteins, irrelevant DNA and the like, at temperatures favouring selective hybridisation, for example between 0°C and 80°C, for example between 25°C and 50°C or around 65°C, preferably at around 20° lower than the hybrid double-stranded DNA melting temperature.
  • adjuncts for example calcium chelators, viscosity regulating compounds, proteins, irrelevant DNA and the like
  • Fractionated mRNA may be translated in cells, for example frog oocytes, or in cell-free systems, for example in reticulocyte lysates or wheat germ extracts.
  • the obtained polypeptides are screened for RNA 3'-term ⁇ nal phosphate cyclase activity or for reaction with antibodies raised against RNA 3'-terminal phosphate cyclase, for example in an immunoassay, for example radioimmunoassay, enzyme immunoassay or immunoassay with fluorescent markers.
  • an immunoassay for example radioimmunoassay, enzyme immunoassay or immunoassay with fluorescent markers.
  • immunoassays and the preparation of polyclonal and monoclonal antibodies are well known in the art and are applied accordingly.
  • a single-stranded complementary DNA (cDNA) from the selected mRNA template is well known in the art, as is the preparation of a double-stranded DNA from a single-stranded DNA.
  • the mRNA template is incubated with a mixture of deoxynucleoside t ⁇ phosphates, optionally radioactively labelled deoxynucleoside tnphosphates (in order to be able to screen the result of the reaction), a primer sequence such as an ohgo-dT residue hybridising with the poly(A) tail of the mRNA and a suitable enzyme such as a reverse transcriptase for example from avian myeloblastosis virus (AMV).
  • AMV avian myeloblastosis virus
  • the cDNA is incubated with a mixture of deoxynucleoside tnphosphates and a suitable enzyme to give a double-stranded DNA.
  • suitable enzymes are for instance a reverse transcriptase, the Klenow fragment of E. cojj DNA polymerase I or T4 DNA polymerase.
  • a hairpin loop structure formed spontaneously by the single-stranded cDNA acts as a primer for the synthesis of the second strand This hairpin structure is removed by digestion with S1 nuclease.
  • the 3'-end of the single-stranded DNA is first extended by homopolymeric deoxynucleotide tails prior to the hydrolysis of the mRNA template and the subsequent synthesis of the second cDNA strand.
  • double-stranded cDNA is isolated from a cDNA library and screened for the desired cDNA (step b).
  • the cDNA library is constructed by isolating mRNA from suitable cells, for example human mononuclear leukocytes or human embryonic epithelial lung cells, and preparing single-stranded and double-stranded cDNA therefrom as described above.
  • This cDNA is digested with suitable restriction endonucleases and incorporated into ⁇ phage, for example ⁇ charon 4A or ⁇ gt1 1 following established procedures.
  • the cDNA library replicated on nitrocellulose membranes is screened by using a DNA probe as described hereinbefore, or expressed in a suitable expression system and the obtained polypeptides screened for reaction with an antibody specific for the desired RNA 3'-term ⁇ nal phosphate cyclase, for example an antibody specific for RNA 3'-term ⁇ nal phosphate cyclase
  • step c A variety of methods are known in the art for the incorporation of double-stranded cDNA into an appropriate vector (step c).
  • complementary homopolymer tracts may be added to the double-stranded DNA and the vector DNA by incubation in the presence of the corresponding deoxynucleoside tnphosphates and an enzyme such as terminal deoxynucleotidyl transferase
  • the vector and double-stranded DNA are then joined by base pairing between the complementary homopolyme ⁇ c tails and finally ligated by specific joining enzymes such as ligases.
  • Other possibilities are the addition of synthetic linkers to the termini of the double-stranded DNA, or the incorporation of the double- stranded DNA into the vector by blunt- or staggered-end ligation
  • Hybrid vectors and host cells may be particularly suitable for the production of DNA, or for the production of the desired RNA 3'-term ⁇ nal phosphate cyclase
  • the isolation of the desired DNA is achieved by methods known in the art, for example extraction with phenol and/or chloroform or glass beads
  • the DNA can be further manipulated for example by treatment with mutagenic agents to obtain mutants, or by digestion with restriction enzymes to obtain fragments, modify one or both termini to facilitate incorporation into the vector.
  • the nucleic acid is DNA and further comprises a rephcable vector comprising the nucleic acid encoding RNA 3 -terminal phosphate cyclase operably linked to control sequences recognised by a host transformed by the vector
  • the invention provides host cells transformed with such a vector and a method of using a nucleic acid encoding RNA 3'-term ⁇ nal phosphate cyclase to effect the production of RNA 3'-term ⁇ nal phosphate cyclase, comprising expressing RNA 3'-term ⁇ nal phosphate cyclase nucleic acid in a culture of the transformed host cells and if desired, recovering RNA 3'-term ⁇ nal phosphate cyclase from the host cell culture
  • RNA 3'-term ⁇ nal phosphate cyclase nucleic acid includes nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of RNA 3'-term ⁇ nal phosphate cyclase nucleic acid or in crude nucleic acid preparations, such as DNA libraries and the like.
  • isolated nucleic acid thus is present in other than in the form or setting in which it is found in nature
  • isolated RNA 3'- termi ⁇ al phosphate cyclase encoding nucleic acid includes RNA 3'-term ⁇ nal phosphate cyclase nucleic acid in ordinarily RNA 3'-term ⁇ nal phosphate cyclase-expressmg cells where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature
  • RNA 3'-term ⁇ nal phosphate cyclase is that having substantially the same nucleotide sequence as the coding sequences in SEQ ID No 1 , 3, 5, 7, 9 or 1 1 , with the nucleic acid having the same sequence as these coding sequences being most preferred
  • nucleotide sequences which are substantially the same share at least about 90 % identity.
  • splice variants having e.g an additional exon sequence homology may be lower
  • nucleic acid sequences may be substantially altered, by virtue of the degeneracy of the genetic code, without affecting the sequence of the polypeptide encoded thereby Such “degenerate” equivalents, therefore, are included within the scope of the present invention
  • the invention includes all sequences which encode the polypeptides set forth in SEQ ID No 2, 4, 6, 8, 10 or 12
  • nucleic acids of the invention whether used as probes or otherwise, preferably possess substantial homology to the sequences encoding RNA 3'-term ⁇ nal phosphate cyclase as set forth herein
  • the terms "substantial” and “homology” are used as hereinbefore defined with reference to the RNA 3'-term ⁇ nal phosphate cyclase polypeptide
  • nucleic acids according to the invention are fragments of the RNA 3'- termmal phosphate cyclase-encoding sequence, or derivatives thereof as hereinbefore defined in relation to polypeptides Fragments of the nucleic acid sequence of a few nucleotides in length, preferably 5 to 150 nucleotides in length, are especially useful as probes
  • nucleic acids can alternatively be characterised as those nucleotide sequences which encode a RNA 3'-term ⁇ nal phosphate cyclase protein and hybridise to the DNA sequences set forth herein, or a selected fragment of said DNA sequence Preferred are such sequences encoding RNA 3'-term ⁇ nal phosphate cyclase which hybridise to the sequences set foth in SEQ ID No. 1 , 3, 5, 7, 9 or 1 1
  • hybridisation occurs under conditions of medium or high stringency
  • Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable Such conditions are evident to those of ordinary skill in the field As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T m ) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.
  • high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na + at 65-68 °C.
  • High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na + pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor.
  • high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - 0.1 x SSC, 0.1 % SDS.
  • Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridisation temperature in 1x SSC, 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0.1 % SDS.
  • nucleic acids of the invention are obtainable according to methods well known in the art.
  • a DNA of the invention is obtain ⁇ able by chemical synthesis, using polymerase chain reaction (PCR) or by screening a genomic library or a suitable cDNA library prepared from a source believed to possess RNA 3'-terminal phosphate cyclase and to express it at a detectable level.
  • PCR polymerase chain reaction
  • Chemical methods for synthesis of a nucleic acid of interest include tnester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports. These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available Alternatively, if the target ammo acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each ammo acid residue
  • a nucleic acid encoding RNA 3'-term ⁇ na! phosphate cyclase may be isolated by screening suitable cDNA or genomic libraries under suitable hybridisation conditions with a probe, i e a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth herein.
  • Suitable libraries are commercially available or can be prepared e.g. from cell lines, tissue samples, and the like
  • RNA 3'-term ⁇ nal phosphate cyclase oligonucleotides of about 20 to 80 bases in length that encode known or suspected RNA 3'-term ⁇ nal phosphate cyclase cDNA from the same or different species, and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridising gene
  • probes for screening genomic DNA libraries include, but are not limited to oligonucleotides, cDNAs or fragments thereof that encode the same or hybridising DNA, and/or homologous genomic DNAs or fragments thereof
  • RNA 3'-term ⁇ nal phosphate cyclase An alternative means to isolate the gene encoding RNA 3'-term ⁇ nal phosphate cyclase is to use PCR technology as described e.g. in section 14 of Sambrook et al , 1989 This method requires the use of oligonucleotide probes that will hybridise to RNA 3'-term ⁇ nal phosphate cyclase nucleic acid.
  • an oligonucleotide probe is e g a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases set forth in SEQ ID No.
  • nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised
  • the nucleotide sequences are usually based on conserved or highly homologous nucleotide sequences or regions of RNA 3'-term ⁇ nal phosphate cyclase
  • the nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known
  • nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation.
  • suitable label means is a radiolabel.
  • the preferred method of labelling a DNA fragment is by incorporating ⁇ 32P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art.
  • Oligonucleotides are usually end-labelled with ⁇ 32P -labelled ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • RNA 3'-terminal phosphate cyclase-encoding sequence or a suitable oligonucleotide based on a portion of said DNA After screening the library, e.g. with a portion of DNA including substantially the entire RNA 3'-terminal phosphate cyclase-encoding sequence or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified by detecting a hybridisation signal; the identified clones are characterised by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding a complete RNA 3'-terminal phosphate cyclase (i.e., if they include translation initiation and termination codons).
  • the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNAs and deduced amino acid sequences provided herein.
  • RNA 3'-terminal phosphate cyclase In order to detect any abnormality of endogenous RNA 3'-terminal phosphate cyclase, genetic screening may be carried out using the nucleotide sequences of the invention as hybridisation probes. Also, based on the nucleic acid sequences provided herein a ⁇ tisense- type therapeutic agents may be designed. In particular reference thereto, it is to be noted that antisense oligonucleotides are based on oligonucleotide probes as hereinbefore defined, and included within the definition thereof.
  • Such oligonucleotides may comprise modifications to the oligonucleotide, for example by incorporation of un-natural nucleotide analogues and modifications to natural oligonucleotides.
  • the oligonucleotides may encompass an altered backbone, for example in the form of a phosphorothioate, modifications such as 2'-0-Methyl modifications, or may be in the form of peptide nucleic acids. It is envisaged that the nucleic acid of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof.
  • Such mutants can be used e g to produce a RNA 3'- termmal phosphate cyclase mutant that has an ammo acid sequence differing from the RNA 3'-term ⁇ nal phosphate cyclase sequences as found in nature
  • Mutagenesis may be predetermined (site-specific) or random A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridise to produce secondary mRNA structure such as loops or hairpins
  • RNA 3'- terminal phosphate cyclase Suitable prokaryotes include eubacte ⁇ a, such as Gram- negative or Gram-positive organisms, such as E. coli, e g. E coli K-12 strains, DH5a and HB101 , or Bacilli, especially Bacillus subtilis
  • eukaryotic microbes such as filamentous fungi or yeast, e g.
  • Saccharomyces cerevisiae Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells
  • mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells
  • the host cells referred to in this disclosure comprise cells in in vitro culture as well as cells that are within a host animal
  • DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art
  • Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency
  • RNA 3'-term ⁇ nal phosphate cyclase-encodmg nucleic acid to form RNA 3'-term ⁇ nal phosphate cyclase
  • the precise amounts of DNA encoding RNA 3'- terminal phosphate cyclase may be empirically determined and optimised for a particular cell and assay
  • Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.
  • Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby RNA 3'-terminal phosphate cyclase encoded by the DNA is expressed,
  • media and culturing methods known in the art, preferably under conditions, whereby RNA 3'-terminal phosphate cyclase encoded by the DNA is expressed.
  • suitable media is known to those in the art, so that they can be readily prepared.
  • Suitable culturing media are also commercially available.
  • vector refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible.
  • the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.
  • an origin of replication one or more marker genes
  • an enhancer element an enhancer element
  • a promoter a transcription termination sequence
  • a signal sequence a signal sequence that is associated with the expression of cells.
  • Incorporation of cloned DNA into a suitable expression vector, transfection of cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
  • Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of RNA 3'-terminal phosphate cyclase.
  • transient expression systems are useful e.g. for identifying RNA 3'-terminal phosphate cyclase mutants, to identify potential phosphorylation sites, or to characterise functional domains of the protein.
  • Plasmids employs conventional Hgation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing RNA 3'-terminal phosphate cyclase expression and function are known to those skilled in the art.
  • Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.
  • Both expression and cloning vectors generally contain nucleic acid sequence that enable the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.
  • Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression.
  • a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome.
  • DNA may also be replicated by insertion into the host genome.
  • the recovery of genomic DNA encoding RNA 3'-terminal phosphate cyclase is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise RNA 3'-terminal phosphate cyclase DNA.
  • DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.
  • an expression and cloning vector may contain a selection gene also referred to as selectable marker.
  • This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.
  • any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.
  • Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEJJ2, LYS2, TRPL or HIS3 gene.
  • E. co// genetic marker and an E. co//origin of replication are advantageously included.
  • E. coli plasmids such as pBR322, Bluescripf* vector or a pUC plasmid, e.g. pUCl ⁇ or pUCl9, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up RNA 3'-terminal phosphate cyclase nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase genes conferring resistance to G418 or hygromycin.
  • the mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive.
  • selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes RNA 3'-terminal phosphate cyclase.
  • Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesised from thus amplified DNA.
  • Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to RNA 3'-terminal phosphate cyclase nucleic acid. Such a promoter may be inducible or constitutive.
  • the promoters are operably linked to DNA encoding RNA 3'-terminal phosphate cyclase by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native RNA 3'-terminal phosphate cyclase promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of RNA 3'-terminal phosphate cyclase DNA.
  • Promoters suitable for use with prokaryotic hosts include, for example, the ⁇ - lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding RNA 3'-terminal phosphate cyclase, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding RNA 3'- terminal phosphate cyclase.
  • RNA 3'-terminal phosphate cyclase gene preferably includes a secretion sequence in order to facilitate secretion of the polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body.
  • the peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.
  • Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomvces cerevisiae gene.
  • GAP glyceraldehyde-3-phosphate dehydrogenase
  • 3-phospho glycerate kinase PGK
  • hexokinase pyruvate decarboxylase
  • phosphofructokinase glucose-6-phosphate isomerase
  • 3-phosphoglycerate mutase phosphoglucose isomerase
  • glucokinase or glucokinase genes
  • TBP TATA binding protein
  • hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene
  • a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene PH05-GAP hybrid promoter
  • a suitable constitutive PHQ5 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.
  • RNA 3'-terminal phosphate cyclase gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with RNA 3'- terminal phosphate cyclase sequence, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (
  • Enhancers are orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to RNA 3'-terminal phosphate cyclase DNA, but is preferably located at a site 5' from the promoter.
  • the polypeptide according to the invention may advantageously be expressed in insect cell systems.
  • Insect cells suitable for use in the method of the invention include, in principle, any lepidopteran cell which is capable of being transformed with an expression vector and expressing heterologous proteins encoded thereby.
  • Sf cell lines such as the Spodoptera fr ⁇ giperda cell line IPBL-SF-21 AE (Vaughn et al., (1977) In Vitro, 13, 213-217) is preferred.
  • the derivative cell line Sf9 is particularly preferred.
  • other cell lines such as Tricopl ⁇ sia n/368 (Kurstack and Marmorosch, (1976) Invertebrate Tissue Culture Applications in Medicine, Biology and Agriculture. Academic Press, New York, USA) may be employed.
  • These cell lines, as well as other insect cell lines suitable for use in the invention are commercially available (e.g. from Stratagene, La Jolla, CA, USA).
  • the invention also comprises the expression of heterologous proteins in whole insect organisms.
  • virus vectors such as baculovirus allows infection of entire insects, which are in some ways easier to grow than cultured cells as they have fewer requirements for special growth conditions.
  • the protein can be extracted from the insects according to conventional extraction techniques.
  • Expression vectors suitable for use in insect expression systems according to the invention include all vectors which are capable of expressing foreign proteins in insect cell lines.
  • vectors which are useful in mammalian and other eukaryotic cells are also applicable to insect cell culture.
  • Baculovirus vectors, specifically intended for insect cell culture are especially preferred and are widely obtainable commercially (e.g. from Invitrogen and Clontech).
  • Other virus vectors capable of infecting insect cells are known, such as Sindbis virus (Hahn et al., (1992) PNAS (USA) 89, 2679-2683).
  • the baculovirus vector of choice (reviewed by Miller (1988) Ann. Rev. Microbiol. 42, 177-199) is Autographa californica multiple nuclear polyhedrosis virus, AcMNPV.
  • the heterologous gene replaces at least in part the polyhedrin gene of AcMNPV, since polyhedrin is not required for virus production.
  • a transfer vector is advantageously used. Transfer vectors are prepared in E. coli hosts and the DNA insert is then transferred to AcMNPV by a process of homologous recombination.
  • a eukaryotic expression vector encoding RNA 3'-terminal phosphate cyclase may comprise a locus control region (LCR).
  • LCRs are capable of directing high- level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the RNA 3'-terminal phosphate cyclase gene is to be expressed in the context of a permanently-transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, in vectors designed for gene therapy applications or in transgenic animals.
  • the invention also provides a transgenic non-human mammal which has been modified to modulate the expression of endogenous RNA 3'-terminal phosphate cyclase.
  • the transgenic non-human mammal is a transgenic mouse.
  • a transgenic mouse may be designed in which RNA 3'-terminal phosphate cyclase production is greatly reduced or eliminated
  • the transgenic mouse of the invention may express elevated levels of RNA 3'-term ⁇ nal phosphate cyclase or may be subject to regulation of RNA 3'-term ⁇ nal phosphate cyclase expression in a developmentally or tissue-specific manner, or via control by exogenous agents Study of such an animal provides insights into the importance of RNA 3'-term ⁇ nal phosphate cyclase in vivo.
  • antibodies specifically recognising and binding to RNA 3'-term ⁇ nal phosphate cyclase may be generated against the RNA 3'-term ⁇ nal phosphate cyclase having the ammo acid sequences set forth in SEQ ID No 2
  • RNA 3'-term ⁇ nal phosphate cyclase or RNA 3'-term ⁇ nal phosphate cyclase fragments are fused (by recombinant expression or an in vitro peptidyl bond) to an immunogenic polypeptide and this fusion polypeptide, in turn, is used to raise antibodies against an RNA 3'-term ⁇ nal phosphate cyclase epitope
  • Anti-RNA 3'-term ⁇ nal phosphate cyclase antibodies are recovered from the serum of immunised animals Alternatively, monoclonal antibodies are prepared from cells in vitro or from in vivo immunised animals in conventional manner
  • the antibodies of the invention are useful for studying RNA 3 -terminal phosphate cyclase tissue localisation, screening of an expression library to identify nucleic acids encoding RNA 3'-term ⁇ nal phosphate cyclase or the structure of functional domains, as well as for the purification of RNA 3'-term ⁇ nal phosphate cyclase and the like
  • Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies Moreover, the invention includes antibody fragments, such as Fab, F(ab') 2 , Fv and ScFv Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution
  • the antibodies according to the invention are especially indicated for diagnostic and therapeutic applications Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label Especially preferred are labels which allow the imaging of the distribution of the antibody in vivo
  • labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient
  • chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications.
  • immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [see international patent application WO 90/07861 (Protein Design Labs)].
  • Antibodies according to the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferable in mammalian cell culture. The selected cell culture system preferably secretes the antibody product.
  • the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. E. co//or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.
  • a host e.g. E. co//or a mammalian cell
  • a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.
  • Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally replenished by a mammalian serum, e.g. fetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like.
  • suitable culture media which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium
  • a mammalian serum e.g. fetal calf serum
  • trace elements and growth sustaining supplements e.g. feeder cells
  • feeder cells such as normal mouse peritoneal exudate cells, sple
  • Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
  • In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies.
  • Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.
  • the desired antibodies can also be obtained by multiplying mammalian cells in vivo.
  • hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody- producing tumours.
  • the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection.
  • pristane tetramethyl-pentadecane
  • hybridoma cells obtained by fusion of suitable myeloma cells with antibody- producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.
  • the cell culture supematants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing RNA 3'-terminal phosphate cyclase, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • an enzyme immunoassay e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.
  • the immunoglobulins in the culture supematants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like.
  • the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-)affinity chromatography, e.g. affinity chromatography with RNA 3'-terminal phosphate cyclase protein or with Protein-A.
  • the invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention.
  • the preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.
  • the invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed RNA 3'-terminal phosphate cyclase, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with purified RNA 3'- terminal phosphate cyclase protein, an antigenic carrier containing purified RNA 3'-terminal phosphate cyclase or with cells bearing RNA 3'-terminal phosphate cyclase, antibody- producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected.
  • a suitable mammal for example a Balb/c mouse
  • spleen cells of Balb/c mice immunised with cells bearing RNA 3'-terminal phosphate cyclase are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.
  • a fusion promoter preferably polyethylene glycol.
  • the myeloma cells are fused with a three- to twentyfoid excess of spleen cells from the immunised mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000.
  • the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
  • suitable culture media for example HAT medium
  • the invention also concerns recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to RNA 3'-terminal phosphate cyclase as described hereinbefore.
  • DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.
  • DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed RNA 3'-terminal phosphate cyclase can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.
  • a mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids.
  • said modification (s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody.
  • Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s).
  • Such a mutant sequence is also a degenerated sequence.
  • Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded.
  • Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.
  • mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
  • the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.
  • the invention therefore also concerns recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed RNA 3'-terminal phosphate cyclase fused to a human constant domain ⁇ , for example ⁇ 1 , ⁇ 2, ⁇ 3 or ⁇ 4, preferably ⁇ 1 or ⁇ 4.
  • the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to RNA 3'- terminal phosphate cyclase fused to a human constant domain K or ⁇ , preferably K.
  • the invention pertains to recombinant DNAs coding for a recombinant DNA wherein the heavy chain variable domain and the light chain variable domain are linked by way of a DNA insert coding for a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a DNA coding for a cleavage site and/or a DNA coding for a peptide spacer and/or a DNA coding for an effector molecule.
  • the DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications.
  • effector molecules which are toxins or enzymes, especially enzymes capable of catalysing the activation of prodrugs, are particularly indicated.
  • the DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.
  • Antibodies and antibody fragments according to the invention are useful in diagnosis and therapy Accordingly, the invention provides a composition for therapy or diagnosis comprising an antibody according to the invention
  • the antibody is preferably provided together with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotopic or other means
  • means for detecting the antibody which may be enzymatic, fluorescent, radioisotopic or other means
  • the antibody and the detection means may be provided for simultaneous, simultaneous separate or sequential use, in a diagnostic kit intended for diagnosis
  • the cyclase is purified from HeLa cells following an established protocol (Vicente, O and Filipowicz, W (1988) Eur J Biochem 176, 431-439) with some modifications (1)
  • the cyclase is eluted from the Hepa ⁇ n- Sepharose column (step 3) with a linear gradient of 75-450 mM NaCl Fractions corresponding to pool (a) (Filipowicz er a/.
  • Partial sequence of the cyclase cDNA is obtained by two consecutive PCR amplifications, using ⁇ gtl 1 human HeLa cell cDNA library (Clontech) as a template
  • oligonucleotides 1 5'- CACATGGCTGAATATCGAC-3'
  • SEQ ID No 12 corresponding to the border sequence of the EcoRI ⁇ gtl 1 cloning site and 2
  • 5'-TC(C/ ⁇ TG(C/G)ACA/GAr)GG(C/T)TG(A/G)AT (A/G)TT-3' SEQ ID No.
  • PCR reaction contained 1 ⁇ M oligonucleotide 1 , 4 ⁇ M oligonucleotide 2, 200 ng of the library DNA, 250 ⁇ M of each of the four nucleotides, 2.5 U Taq DNA polymerase in the PERKIN ELMER buffer. Thirty PCR cycles consisted of denaturation at 94°C for 40 sec, annealing at 45°C for 1 min, and extension at 70°C for 1.5 min each cycle.
  • oligonucleotides 3 (5'- GA(C/ ⁇ GG(A/C/GAT)TC(A/C/ ⁇ AT(A/C ⁇ " )ATGGA (A/G)GG-3'), SEQ ID No. 14, a mixture of 144 oligomers coding for the sequence DGSIMEG of peptide pepl and 4 (5'-GGCTG(A/G)AT(A/G)TT(A/C/G)AC(A/G)TA(C/G)AG (A/G)TC-3'), SEQ ID No.
  • overlapping with oligonucleotide 2 is a mixture of 96 oligomers coding for the sequence DLYVNIQP of peptide pep4) ate used as primers.
  • the 100 ⁇ l PCR reaction contained 4 ⁇ M of each primer, 250 ⁇ M of each of the four nucleotides, 2.5 U Taq DNA polymerase and 8 ⁇ l of the first PCR reaction. Thirty PCR cycles consisted of denaturation at 94°C for 40 sec, annealing at 52°C for 1 min, and extension at 72°C for 1.5 min each cycle.
  • a 668 bp amplified DNA fragment is subcloned into the Sma ⁇ site of pBluescribe vector (Stratagene) and sequenced by the dideoxy chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74, 5463-5467,1977).
  • the fragment contained a large ORF encoding the peptides pep2 and pep3.
  • the PCR amplification scheme described above represents one of many similar reactions tested with different combination of primers; it is the only one which yielded the cDNA encoding expected peptide sequences.
  • the HeLa ⁇ gtl 1 cDNA library (Clontech) is screened with the PCR amplified fragment as a probe. Hybridizations are performed overnight at 42°C in 5 x SSPE, 50% formamide, 5 x Denhart's solution, 1% SDS and 50 ⁇ g/ml denatured salmon sperm DNA. Eight clones are isolated after screening one million recombinant phages. All clones are subcloned in pBluescribe vector (Stratagene) and analysed by restriction mapping and sequencing of the ends. The longest clones are sequenced on both strands.
  • RNA is blotted onto GeneScreen membranes and UV cross-linked.
  • the blot containing RNAs originating from different human organs is purchased from Clontech.
  • the integrity as well as the amount of RNAs is checked by using a human ⁇ -actin cDNA, furnished by Clontech, as control probe.
  • the probes are labelled with [ ⁇ - 32 P]dCTP (300 Ci/mmol, Amersham) by the random priming method (Feinberg and Vogelstein, 1983).
  • the hybridization is for 16 h at 42°C in 5 x SSPE, 50% formamide, 10% dextran sulfate, 1 % SDS and 50 ⁇ g/ml denatured salmon sperm DNA.
  • the blots are subsequently washed in 2 x SSC and 0.1% SDS for 30 min at 42°C, and 0.2 x SSC and 0.1 % SDS for 30 min at 42°C and then at 60°C.
  • the SamHI sites are introduced 5' and 3' of the cyclase coding sequence using the PCR site-directed mutagenesis.
  • the BamH ⁇ lBamH ⁇ fragment is cloned into pGEX-2T vector (Pharmacia) for expression in the E. coli strain BL21 (DE3).
  • the Schistosoma japonicum glutathione S-transferase is placed in frame at the N-terminus of the fusion protein.
  • the protein remained soluble during expression in E. coli and is purified in the native form under non-denaturing conditions on the Glutathione Sepharose 4B resin (following the manufacturer's protocol; Pharmacia).
  • oligoSY 5'- CCCCACCCCG-3'
  • SEQ ID No. 16 amino acid sequence sequence of SEQ ID No. 16
  • oligoSR 5'-AAAATAAAAG-3'
  • SEQ ID No. 17 are obtained from MWG-BIOTECH (Munich). They are 3'-terminally labelled using [5'- 32 P]pCp (*pCp) and T4 RNA ligase, to produce (Np)gGp*Cp.
  • Reaction mixtures (15 ⁇ l) contained 70 mM Hepes/KOH, pH 8.3, 10 mM MgCl2, 3 mM dithiothreitol, 10% glycerol, 10% dimethylsulfoxide, 40 ⁇ M ATP, 1.1 ⁇ M p*Cp (spec. 3000 Ci/mmol), 0.7 ⁇ g of oligoribonucleotides and 6 units of T4 RNA ligase. After 14 h at 4°C, samples are diluted with 60 ⁇ l of 50 mM Hepes/KOH, pH 7.6, containing 3 mM EDTA and incubated at 37°C for 1 h with 5 U of RNase T1.
  • Cyclase assay The cyclase activity is assayed by the No ⁇ t method as described before (Filipowicz and Vicente; 1990). Analysis of the reaction products by TLC is as described above.
  • pBact-CYC-myc The EcoRI sites are introduced 5' and 3' of the cyclase coding sequence by the PCR based site directed mutagenesis using oligonucleotides 5 (5'-GAGGAGAAAGAATTCATGGCGGGG CCGTGG-3'), SEQ ID No. 18, and 6 (5'-AGTGGTGATGGTGGAATTCCTATAGATTTG-3'), SEQ ID No 19, as upstream and downstream primers, respectively.
  • the EcoRI/EcoRI fragment is cloned into the pBact- myc vector (Cravchik and Matus, 1993) for expression of the myc-epitope-tagged cyclase in transfected HeLa cells.
  • HeLa cells are cultured in the DME medium (Sambrook et al., Molecular Cloning. A Laboratory Manual (2nd ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) with the addition of 10% fetal calf serum in 5% CO2 humidified atmosphere.
  • Cell are transfected with pBact-CYC-myc (2-10 ⁇ g), using the Calcium phosphate transformation method (Chen and Okayama, 1987) and fixed with 3% paraformaldehyde in PBS (Sambrook et al , 1989) at room temperature for 20 min.
  • Cells are permeabilised for 10 min with 0.5% Triton X-100 solution in PBS, and blocked with 5% non immune goat serum in PBS for 20 min.
  • the myc-tagged cyclase is immunoprobed with a mAb GE10 raised to the human c-myc epitope (Evan et al., (1985) Mol. Cell. Biol. 5, 3610-3616), obtained from the European Collection of Cell Cultures (Porton Down, UK).
  • the FITC-conjugated goat anti-mouse antibody (AffiniPure F(ab')2 fragment, Jackson ImmunoResearch Laboratories, Inc.) is used as a secondary antibody.
  • Samples are examined with the Zeiss Axiophot microscop and the Leica confocal scanning laser microscope using a 63X objective. Images are recorded using Leica SCANware 4.2 provided with the system.
  • Nco ⁇ and BamHI sites are introduced 5' and 3', respectively of the cyclase coding sequence by the PCR based site directed mutagenesis using oligonucleotides 1 (5'- TGCGTAAAAGGATCCATGGTGAAAAGGATG-3'), SEQ ID No. 20, and 8 (5'- CTTACATCTCTGGATCCTTCAATGCTCAC-3'), SEQ ID No. 21 , as upstream and downstream primers, respectively.
  • the ⁇ /col/SamHI fragment is cloned into the pET-1 1 d vector (Novagen) modified in order to include 6 histidine residues in frame at the C-terminus of the recombinant protein.
  • the protein overexpression is performed in the E.
  • the purified cyclase is applied to a 10 ml Sephadex G-25 column equilibrated and eluted with 30 mM HEPES-KOH (pH 7.6), 0.1 mM EDTA, 0.5 mM DTT, 5% (v/v) glycerol, 0.01% Triton X-100 and 10 ⁇ M PMSF Protein concentration is measured by the method of Bradford, M.M (1976) Anal. Biochem. 72, 248-254 using the reagent obtained from BioRad and BSA as a standard.
  • Bacterial strain and plasmid The gene replacement is performed in the strain MC1061 [F" araD139 (ara-leu) 7697 (/ac)X74 gahJ gaK sttA]
  • pMAK705 is a chloramphenicol- resistant derivative of pSC101 that contains a temperature-sensitive rep mutation which prevents from the replication at 44°C (Hamilton et al., 1989).
  • pMAK705cyc " ::/ran r plasmid the 1324 bp Sma ⁇ /Hind ⁇ fragment from pREP4 (Qiagen), which includes the kanamycm resistance gene is ligated into Sma ⁇ /H ⁇ nd ⁇ sites of pBluesc ⁇ ptKS (Stratagene), yielding pBSKan r .
  • a Xba ⁇ /BamH ⁇ fragment of 669 bp corresponding to the cyclase gene 3' flanking sequences is obtained using the PCR site- directed mutagenesis with oligonucleotides 9 (5'-ATCTCTAGAGATACCTGTTGCTGCTTA- ATC-3'), SEQ ID No.
  • Cyclase chromosomal gene replacement Chromosomal insertion and excision steps with pMAK705cyc"::/can r , using chloramphenicol as a selection marker, are performed in MC1061 as described by Hamilton et al. (1989), with the following modifications MC1061 is transformed and plated at 30°C Several transformants are grown up at 30°C in a rich medium (2YT) in the presence of 30 ⁇ g/ml chloramphenicol to 10 8 cells/ml. Different dilutions are plated at both 30°C (for viable count) and 44°C (for cointegrates). The observed insertion frequency of 3 x 10 ⁇ 4 is consistent with the results of Hamilton et al.
  • the primary sequence of the human cyclase protein is used for database "homology" searching.
  • ORFs of unknown functions from different species gave high scores over 100, using TBLASTN Program.
  • the highest similarities are found in two adjacent ORFs from E. co// K12.
  • the chromosomal region is re-sequenced and showed several sequencing errors. Based on our sequencing only one large ORF is present in this region, which is 55% similar and 32% identical to the human cyclase.
  • the bacterial protein is overexpressed in E. coli and the RNA 3'-terminal phosphate cyclase activity is detected.
  • the phylogenic relationship of all cyclase and cyclase-like proteins is determined on a cumputer generated dendrogram, using the PileUp GCG program.
  • ORGANISM Homo sapiens
  • C INDIVIDUAL ISOLATE: HeLa
  • IMMEDIATE SOURCE
  • ATT ATT GCT GAG ACC TCC ACT GGC TGT TTG TTT GCT GGA TCA TCG CTT 940 lie lie Ala Glu Thr Ser Thr Gly Cys Leu Phe Ala Gly Ser Ser Leu 250 255 260 GGT AAA CGA GGT GTA AAT GCA GAC AAA GTT GGA ATT GAA GCT GCC GAA 988 Gly Lys Arg Gly Val Asn Ala Asp Lys Val Gly lie Glu Ala Ala Glu 265 270 275
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ORGANISM Saccharomyces cerevisiae
  • GGC GTT AGA GAG TGC GCC CTA CAT ACT TTG AAA
  • AGA GGT TCA CCA CCA 780 Gly Val Arg Glu Cys Ala Leu His Thr Leu Lys Arg Gly Ser Pro Pro 145 150 155 160
  • GTT GGT AGA AAC CAG CTT CCA TTA GCA ATT GTT TAC ATG GTC ATC GGG 1212 Val Gly Arg Asn Gin Leu Pro Leu Ala He Val Tyr Met Val He Gly 290 295 300
  • ORGANISM Escherichia coli
  • ORGANISM Saccharomyces pombe
  • ORGANISM Homo sapiens
  • C INDIVIDUAL ISOLATE: HeLa
  • CAACAAGTTC ATACCTGATA TCTATATTTA CACAGATCAC ATGAAAGGAG TCAACTCTGG 360

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Abstract

L'invention concerne un procédé de production de la phosphate cyclase à terminaison 3' d'ARN. Ce procédé consiste à exprimer une séquence d'acides nucléiques codant cette cyclase, ou un dérivé de cette dernière, dans une cellule hôte de recombinaison. L'invention a aussi pour objet une séquence d'acides nucléiques codant la phosphate cyclase à terminaison 3' d'ARN, ou un dérivé de cette dernière, ainsi que des vecteurs et des cellules hôtes contenant ces acides nucléiques. Selon les résultats des études, la phosphate cyclase à terminaison 3' d'ARN est une enzyme conservée dans une grande variété d'organismes. L'invention traite également de polypeptides de phosphate cyclase à terminaison 3' d'ARN d'origine eucariote et procariote.
PCT/EP1997/002566 1996-05-24 1997-05-20 Phosphate cyclases a terminaison 3' d'arn de recombinaison et procedes de production WO1997045535A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30277/97A AU3027797A (en) 1996-05-24 1997-05-20 Recombinat rna 3'-terminal phosphate cyclases and production methods thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1833596P 1996-05-24 1996-05-24
US60/018,335 1996-05-24

Publications (1)

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WO1997045535A1 true WO1997045535A1 (fr) 1997-12-04

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005382A2 (fr) * 1998-07-23 2000-02-03 Genset Acide nucleique codant pour une geranylgeranyl pyrophosphate synthetase (ggpps) et marqueurs polymeres associes a un acide nucleique
WO2006056487A2 (fr) * 2004-11-24 2006-06-01 Theraptosis S.A. Nouveaux peptides utilises comme inhibiteurs doubles de la caspase-2/-6 et leurs applications biologiques
CN109971775A (zh) * 2017-12-27 2019-07-05 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法
EP4100416A4 (fr) * 2020-02-06 2024-03-20 Cornell University Compositions et procédés pour l'adénylation et le séquençage rapide d'arn

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001036A1 (fr) * 1987-07-23 1989-02-09 Celltech Limited Vecteurs d'expression a base d'adn recombinant
EP0543137A1 (fr) * 1991-11-18 1993-05-26 American Cyanamid Company Clonage et caractérisation d'une adenylyl-cyclase cardiaque

Patent Citations (2)

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WO1989001036A1 (fr) * 1987-07-23 1989-02-09 Celltech Limited Vecteurs d'expression a base d'adn recombinant
EP0543137A1 (fr) * 1991-11-18 1993-05-26 American Cyanamid Company Clonage et caractérisation d'une adenylyl-cyclase cardiaque

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GENSCHICK P. ET AL.: "The human RNA 3' -terminal phosphate cyclase is a member of a new family of proteins conserved in Eucarya, Bacteria and Archaea", EMBO JOURNAL, vol. 16, no. 10, 1997, EYNSHAM, OXFORD GB, pages 2955 - 2967, XP002044540 *
M. INNIS, D. GELFAND, SNINSKY, T. WHITE: "PCR Protocols. A guide to methods and applications", 1990, ACADEMIC PRESS INC., HARCOURT BRACE JOVANOVICH, PUBLISHERS, SAN DIEGO NEW YORK BOSTON LONDON SYDNEY TOKYO TORONTO, XP002044541 *
VICENTE O., FILIPOWICZ W.: "Purification of RNA 3'-terminal phosphate cyclase from HeLa cells. Covalent modification of the enzyme with different nucleotides", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 176, no. 2, 1988, BERLIN, DE, pages 431 - 440, XP002044539 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000005382A2 (fr) * 1998-07-23 2000-02-03 Genset Acide nucleique codant pour une geranylgeranyl pyrophosphate synthetase (ggpps) et marqueurs polymeres associes a un acide nucleique
WO2000005382A3 (fr) * 1998-07-23 2000-08-24 Genset Sa Acide nucleique codant pour une geranylgeranyl pyrophosphate synthetase (ggpps) et marqueurs polymeres associes a un acide nucleique
WO2006056487A2 (fr) * 2004-11-24 2006-06-01 Theraptosis S.A. Nouveaux peptides utilises comme inhibiteurs doubles de la caspase-2/-6 et leurs applications biologiques
WO2006056487A3 (fr) * 2004-11-24 2008-05-08 Theraptosis S A Nouveaux peptides utilises comme inhibiteurs doubles de la caspase-2/-6 et leurs applications biologiques
JP2008520620A (ja) * 2004-11-24 2008-06-19 テラプトシス エス アー カスパーゼ−２／−６二重阻害剤として有用な新規ペプチドおよびそれらの生物学的適用
US8324173B2 (en) 2004-11-24 2012-12-04 Chiesi Farmaceutici S.P.A. Peptides useful as dual caspase-2/-6 inhibitors and their biological applications
CN103059100A (zh) * 2004-11-24 2013-04-24 奇斯药制品公司 用作双重胱天蛋白酶-2/-6 抑制剂的新肽及其生物学应用
CN109971775A (zh) * 2017-12-27 2019-07-05 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法
CN109971775B (zh) * 2017-12-27 2021-12-28 康码（上海）生物科技有限公司 一种核酸构建物及其调控蛋白合成的方法
EP4100416A4 (fr) * 2020-02-06 2024-03-20 Cornell University Compositions et procédés pour l'adénylation et le séquençage rapide d'arn

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