EP1240311A1 - Molecules d'adn codant la nhl humaine, une helicase d'adn - Google Patents

Molecules d'adn codant la nhl humaine, une helicase d'adn

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Publication number
EP1240311A1
EP1240311A1 EP00983952A EP00983952A EP1240311A1 EP 1240311 A1 EP1240311 A1 EP 1240311A1 EP 00983952 A EP00983952 A EP 00983952A EP 00983952 A EP00983952 A EP 00983952A EP 1240311 A1 EP1240311 A1 EP 1240311A1
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EP
European Patent Office
Prior art keywords
nhl
protein
seq
host cell
expression vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00983952A
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German (de)
English (en)
Inventor
Xiaomei Liu
Chang Bai
Michael L. Metzker
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Merck and Co Inc
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Merck and Co Inc
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Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1240311A1 publication Critical patent/EP1240311A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/90—Isomerases (5.)

Definitions

  • the present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode NHL, a putative DNA helicase.
  • the present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding NHL, substantially purified forms of associated NHL, associated mutant proteins, and methods associated with identifying compounds which modulate NHL, which will be useful in the treatment of various neoplastic disorders, given that this gene is located at 20ql3.3 and immediately adjacent to M68/DcR3, which is involved in tumor growth.
  • a human genomic fragment representing this portion of the human genome, along with three additional genes (M68/DcR3, SCLIP, and ARP).
  • chemotherapeutic agents inhibit helicases, including actinomycin Cl, daunorubicin and nogalamycin (Tuteja, et al., 1997, Biochem. Biophys. Res. Comm. 236(3):636-640), and a prostate cancer drug, CI-958 (Lun, et al.,1998, Cancer Chemother. Pharmacol. 42(6):447-453).
  • actinomycin Cl daunorubicin and nogalamycin
  • CI-958 a prostate cancer drug
  • some topoisomerases have been shown to have anti-cancer activity.
  • helicase-encoding genes and chemotherapeutic agents it would be advantageous to identify additional genes which reside within chromosomal regions associated with a disease state such as cancer as well as a gene which encodes a type of protein which may be associated with that disease.
  • the present invention addresses and meets this need by disclosing a DNA molecule encoding a DNA helicase with a chromosomal location suggestive of association with cancer.
  • the present invention relates to an isolated or purified nucleic acid molecule (polynucleotide) which encodes a novel mammalian DNA helicase.
  • the present invention also relates to an isolated nucleic acid molecule (polynucleotide) which encodes mRNA which expresses a novel human DNA helicase, NHL.
  • a preferred aspect of the present invention relates to an isolated or purified DNA molecule which encodes human NHL, the nucleotide sequence as set forth in Figure 1A-B and SEQ ID NO: l.
  • the present invention also relates to biologically active fragments or mutants of SEQ ID NO: 1 which encode a mRNA molecule expressing a novel DNA helicase, NHL.
  • Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the biological properties of the human NHL protein disclosed herein in Figure 2 and as set forth as SEQ ID NO:2.
  • Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy- terminal truncations such that these mutations encode mRNA which express a functional NHL protein in a host cell, so as to be useful for screening for agonists and/or antagonists of NHL activity.
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • the present invention also relates to a substantially purified form of a human NHL protein which comprises the amino acid sequence disclosed in Figure 2 and set forth as SEQ ID NO:2.
  • a preferred aspect of this portion of the present invention is a NHL protein which consists of the amino acid sequence disclosed in Figure 2 and set forth as SEQ ID NO:2.
  • a substantially purified NHL protein preferably a human NHL protein
  • obtained from a recombinant host cell containing a DNA expression vector comprises a nucleotide sequence as set forth in SEQ ID NO: 1 and expresses the respective NHL protein.
  • the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.
  • the present invention also relates to biologically active fragments and/or mutants of a NHL protein comprising the amino acid sequence as set forth in SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators, including but not limited to agonists and/or antagonists for human NHL pharmacology.
  • a preferred aspect of the present invention is disclosed in Figure 2 and is set forth as SEQ ID NO:2, a respective amino acid sequence which encodes human NHL. Characterization of one or more of these DNA helicase-like proteins allows for screening methods to identify novel NHL modulators that may be useful in the treatment of human neoplastic disorders. The modulators selected through such screening and selection protocols may be used alone or in conjunction with other cancer therapies. As noted above, heterologous expression of a NHL protein will allow the pharmacological analysis of compounds which modulate NHL activity and hence may be useful in various cancer therapies. To this end, heterologous cell lines expressing a NHL protein can be used to establish functional or binding assays to identify novel NHL modulators.
  • the present invention also relates to polyclonal and monoclonal antibodies raised in response to either the NHL or a biologically active fragment of NHL.
  • the present invention relates to transgenic mice comprising altered genotypes and phenotypes in relation to NHL and its in vivo activity.
  • the present invention also relates to NHL fusion constructs, including but not limited to fusion constructs which express a portion of the NHL protein linked to various markers, including but in no way limited to GFP (Green fluorescent protein), the MYC epitope, and GST. Any such fusion constructs may be expressed in the cell line of interest and used to screen for NHL modulators.
  • GFP Green fluorescent protein
  • MYC epitope MYC epitope
  • GST GST
  • the present invention relates to methods of expressing mammalian NHL, and preferably human NHL, biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these proteins, and compounds identified through these assays which act as agonists or antagonists of NHL activity.
  • the present invention also relates to the isolated genomic sequence which comprises SEQ ID NO:l, a 115 kb genomic fragment set forth herein as SEQ ID NO:3.
  • SEQ ID NO:3 As especially preferred aspect of this portion of the invention is the region of the genomic fragment of SEQ ID NO:3 which comprises the regulatory and coding regions of human NHL, as well as intervening sequences (introns).
  • This 115 kb fragment contains at least the coding region of four genes, NHL, M68/DcR3, SCLIP and ARP. As discussed herein, it has been shown that this region of chromosome 20 is associated with tumor growth.
  • an aspect of this invention also comprises the use of one or more regions of this 115 kb genomic sequence to identify compounds which up or downregulate expression of one or more of the genes localized within this 115 kb region, wherein this up or down regulation results in an interference of tumor growth.
  • a transcription element of one of these four genes may be responsible for M68/DcR3 ( and/or NHL) overexpression in tumors, and if M68 or NHL overexpression in tumors has a caustic role, blockage of M68/DcR3 or NHL overexpression in tumors by interfering with this transcription site will be useful.
  • It is an object of the present invention to provide an isolated nucleic acid molecule (e.g., SEQ ID NO:l) which encodes novel form of human NHL, or fragments, mutants or derivatives of human NHL as set forth in Figure 2 and SEQ ID NO:2.
  • Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for selective modulators of human NHL activity.
  • the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.
  • NHL proteins or biological equivalent to screen for modulators, preferably selective modulators, of human NHL activity. Any such compound may be useful in screening for and selecting compounds active against human neoplastic disorders.
  • substantially free from other nucleic acids means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids.
  • a human NHL DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-NHL nucleic acids.
  • Whether a given NHL DNA preparation is substantially free from other nucleic acids can be determined by such conventional techniques of assessing nucleic acid purity as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.
  • substantially free from other proteins or “substantially purified” means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins.
  • a NHL protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-NHL proteins.
  • Whether a given NHL protein preparation is substantially free from other proteins can be determined by such conventional techniques of assessing protein purity as, e.g., sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.
  • SDS-PAGE sodium dodecyl sulfate polyacryl amide gel electrophoresis
  • detection methods e.g., silver staining or immunoblotting.
  • the terms “isolated NHL protein” or “purified NHL protein” also refer to NHL protein that has been isolated from a natural source. Use of the term “isolated” or “purified” indicates that NHL protein has been removed from its normal cellular environment.
  • an isolated NHL protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally.
  • isolated does not imply that an isolated NHL protein is the only protein present, but instead means that an isolated NHL protein is substantially free of other proteins and non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the NHL protein in vivo.
  • non-amino acid material e.g., nucleic acids, lipids, carbohydrates
  • a NHL protein preparation that is an isolated or purified NHL protein will be substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of non-NHL proteins.
  • a functional equivalent or “biologically active equivalent” means a protein which does not have exactly the same amino acid sequence as naturally occurring NHL, due to alternative splicing, deletions, mutations, substitutions, or additions, but retains substantially the same biological activity as NHL.
  • Such functional equivalents will have significant amino acid sequence identity with naturally occurring NHL and genes and cDNA encoding such functional equivalents can be detected by reduced stringency hybridization with a DNA sequence encoding naturally occurring NHL.
  • a naturally occurring NHL disclosed herein comprises the amino acid sequence shown as SEQ ID NO:2 and is encoded by SEQ ID NO:l.
  • a nucleic acid encoding a functional equivalent has at least about 50% identity at the nucleotide level to SEQ ID NO: 1.
  • a conservative amino acid substitution refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
  • substitution of one hydrophobic residue isoleucine, leucine, valine, or methionine
  • substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid).
  • the term “mammalian” will refer to any mammal, including a human being.
  • Figure 1A-B shows the nucleotide sequence which comprises the open reading frame which encodes human NHL, the nucleotide sequence set forth as SEQ ID NO:l.
  • the initiating Met residue (ATG) and the stop codon (TAG) are underlined.
  • Figure 2 shows the amino acid sequence of human NHL as set forth in SEQ ID NO:2.
  • Figure 3 shows the alignment of amino acid sequences of human NHL to ERCC2/RAD3 gene family members.
  • Rep D Dermatyosteliem discoideum
  • RAD 3 S. cerevisiae
  • RAD 15 S. pombe
  • XP_GroupD Homo sapien
  • Figure 4 shows Northern analysis of NHL expression in multi-human tissues.
  • Figure 5A-B show the genomic structure of the NHL gene (Figure 5A) and the entire 115 kb genomic region ( Figure 5B) containing the NHL, M68/DcR3, SCLIP and ARP genes.
  • the present invention relates to an isolated or purified nucleic acid molecule (polynucleotide) which encodes a novel mammalian DNA helicase.
  • An especially preferred aspect of this invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes mRNA which expresses a novel human DNA helicase, NHL.
  • M68/DcR3 is a secreted TNFR member that is overexpressed in a number of human tumors.
  • M68/DcR3 is located at 20ql3.3, a known site that is associated with frequent gene amplification in cancer.
  • M68 DcR3 protein binds to FASL and inhibit FAS mediated apoptosis.
  • genes tightly linked to M68/DcR3 may be coregulated (e.g. co overexpressed and/or amplified in tumors).
  • NHL neoplastic disorders
  • the transcript was identified through exon prediction using GRAIL2 and sequence alignment to a contiguous 4.5 kilobase region of chromosome 4 (88% sequence identity). The complete exon structure of NHL was subsequently confirmed by RT-PCR analysis. Multiple sequence alignment of NHL to known helicases showed that NHL contains all the seven critical helicase domains.
  • BLAST analysis of the predicted 1,219 amino acid sequence revealed an approximately 26% sequence identity and 48% sequence similarity to the RAD3/ERCC2 gene family of DNA helicases (Naumovski et al., 1985 Mol. Cell Biol. 5:17-26; Reynolds et al., 1985 Nucleic Acid Res 13:2357-72; Weber et al., 1990 EMBO J. 9:1437-1447).
  • a preferred aspect of the present invention relates to an isolated or purified DNA molecule which encodes human NHL, the nucleotide sequence as set forth in Figure 1A-B and SEQ ID NO:l, which is as follows:
  • the above-exemplified isolated DNA molecule shown in Figure 1A-B and SEQ ID NO:l comprise 4946 nucleotides, with an initiating Met at nucleotides 828-
  • TAG termination codon are underlined.
  • the present invention also relates to biologically active fragments or mutants of SEQ ID NO:l which encode a mRNA molecule expressing a novel DNA helicase, NHL.
  • Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the biological properties of the human NHL protein disclosed herein in Figure 2 and as set forth as
  • any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy- terminal truncations such that these mutations encode mRNA which express a functional NHL protein in a host cell, so as to be useful for screening for agonists and/or antagonists of NHL activity.
  • the isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
  • RNA ribonucleic acid molecule
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • the degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon encodes a particular amino acid. This allows for the construction of synthetic DNA that encodes the NHL protein where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequence of SEQ ED NO: 1 but still encodes the same NHL protein as SEQ ID NO: 1
  • Such synthetic DNAs are intended to be within the scope of the present invention. If it is desired to express such synthetic DNAs in a particular host cell or organism, the codon usage of such synthetic DNAs can be adjusted to reflect the codon usage of that particular host, thus leading to higher levels of expression of the NHL protein in the host. In other words, this redundancy in the various codons which code for specific amino acids is within the scope of the present invention. Therefore, this invention is also directed to those DNA sequences which encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:
  • the present invention discloses codon redundancy which may result in differing DNA molecules expressing an identical protein.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
  • DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide.
  • Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • the nucleic acid molecules of the present invention encoding a NHL protein in whole or in part, can be linked with other DNA molecules, i.e, DNA molecules to which the NHL coding sequence are not naturally linked, to form "recombinant DNA molecules" which encode a respective NHL protein.
  • the novel DNA sequences of the present invention can be inserted into vectors which comprise nucleic acids encoding NHL or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred.
  • Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode a NHL protein. It is well within the purview of the skilled artisan to determine an appropriate vector for a particular gene transfer or other use.
  • DNA sequences that hybridize to SEQ ID NO:l under stringent conditions include DNA sequences that hybridize to SEQ ID NO:l under stringent conditions.
  • a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hours to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe.
  • Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
  • Other procedures using conditions of high stringency would include either a hybridization step carried out in 5XSSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.
  • the present invention also relates to a substantially purified form of a human NHL protein which comprises the amino acid sequence (1219 amino acid residues) disclosed in Figure 2 and set forth as SEQ ID NO:2.
  • a preferred aspect of this portion of the present invention is a NHL protein which consists of the amino acid sequence disclosed in Figure 2 and set forth as SEQ ID NO:2, as follows:
  • GPSQSSGPPH GPAASEWGL* (SEQ ID NO: 2).
  • the present invention also relates to biologically active fragments and/or mutants of the human NHL protein comprising the amino acid sequence as set forth in SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagon i sts of NHL f uncti on .
  • Another preferred aspect of the present invention relates to a substantially purified, fully processed NHL protein obtained from a recombinant host cell containing a DNA expression vector which comprises a nucleotide sequence as set forth in SEQ ID NO:l and expresses the human NHL protein. It is especially preferred is that the recombinant host cell be a eukaryotic host cell, such as a mammalian cell line.
  • this invention includes modified NHL polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as a respective, corresponding NHL. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology ofthe Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085).
  • the present invention includes a polypeptide where one amino acid substitution has been made in SEQ ID NO:2 wherein the polypeptide still retains substantially the same biological activity as a corresponding NHL protein.
  • the present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ ID NO:2 wherein the polypeptide still retains substantially the same biological activity as a corresponding NHL protein.
  • the present invention includes embodiments where the above-described substitutions are conservative substitutions.
  • polypeptides that are functional equivalents of NHL and have changes from the NHL amino acid sequence that are small deletions or insertions of amino acids could also be produced by following the same guidelines, (i.e, minimizing the differences in amino acid sequence between NHL and related proteins. Small deletions or insertions are generally in the range of about 1 to 5 amino acids).
  • the effect of such small deletions or insertions on the biological activity of the modified NHL polypeptide can easily be assayed by producing the polypeptide synthetically or by making the required changes in DNA encoding NHL and then expressing the DNA recombinantly and assaying the protein produced by such recombinant expression.
  • the present invention also includes truncated forms of NHL which contain the region comprising the active site of the enzyme.
  • truncated proteins are useful in various assays described herein, for crystallization studies, and for structure-activity- relationship studies.
  • the present invention also relates to isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which modulate wild-type NHL activity, as well as generating antibodies against NHL.
  • One aspect of this portion of the invention includes, but is not limited to, glutathione S-transferase (GST)-NHL fusion constructs.
  • GST-NHL fusion proteins may be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (pAcG2T, Pharmingen).
  • Another aspect involves NHL fusion constructs linked to various markers, including but not limited to GFP (Green fluorescent protein), the MYC epitope, and GST.
  • GFP Green fluorescent protein
  • any such fusion constructs may be expressed in the cell line of interest and used to screen for modulators of one or more of the NHL proteins disclosed herein.
  • Any of a variety of procedures may be used to clone NHL. These methods include, but are not limited to, (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998-9002). 5' and/or 3' RACE may be performed to generate a full-length cDNA sequence. This strategy involves using gene-specific oligonucleotide primers for PCR amplification of NHL cDNA.
  • These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases; (2) direct functional expression of the NHL cDNA following the construction of a NHL-containing cDNA library in an appropriate expression vector system; (3) screening a NHL-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of the NHL protein; (4) screening a NHL-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the NHL protein.
  • EST expressed sequence tag
  • This partial cDNA is obtained by the specific PCR amplification of NHL DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other kinases which are related to the NHL protein; (5) screening a NHL- containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA or oligonucleotide with homology to a mammalian NHL protein.
  • This strategy may also involve using gene-specific oligonucleotide primers for PCR amplification of NHL cDNA identified as an EST as described above; or (6) designing 5' and 3' gene specific oligonucleotides using SEQ ID NO: 1 as a template so that either the full-length cDNA may be generated by known RACE techniques, or a portion of the coding region may be generated by these same known RACE techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full-length version of the nucleotide sequence encoding NHL.
  • libraries as well as libraries constructed from other cell types-or species types, may be useful for isolating a NHL-encoding DNA or a NHL homologue.
  • Other types of libraries include, but are not limited to, cDNA libraries derived from other cells.
  • suitable cDNA libraries may be prepared from cells or cell lines which have NHL activity.
  • the selection of cells or cell lines for use in preparing a cDNA library to isolate a cDNA encoding NHL may be done by first measuring cell-associated NHL activity using any known assay available for such a purpose.
  • cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Complementary DNA libraries may also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc. and Stratagene.
  • DNA encoding NHL may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Sambrook, et al., supra. One may prepare genomic libraries, especially in PI artificial chromosome vectors, from which genomic clones containing the NHL gene can be isolated, using probes based upon the NHL nucleotide sequences disclosed herein. Methods of preparing such libraries are known in the art (Ioannou et al., 1994, Nature Genet. 6:84-89).
  • the amino acid sequence or DNA sequence of a NHL or a homologous protein may be necessary.
  • a respective NHL protein may be purified and the partial amino acid sequence determined by automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids can be determined for the PCR amplification of a partial NHL DNA fragment.
  • the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides.
  • the nucleotide sequence of a region of an expressed sequence may be identified by searching one or more available genomic databases.
  • Gene-specific primers may be used to perform PCR amplification of a cDNA of interest from either a cDNA library or a population of cDNAs.
  • the appropriate nucleotide sequence for use in a PCR-based method may be obtained from SEQ ID NO:l either for the purpose of isolating overlapping 5' and 3' RACE products for generation of a full-length sequence coding for NHL, or to isolate a portion of the nucleotide sequence coding for NHL for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding NHL or NHL-like proteins.
  • This invention also includes vectors containing a NHL gene, host cells containing the vectors, and methods of making substantially pure NHL protein comprising the steps of introducing the NHL gene into a host cell, and cultivating the host cell under appropriate conditions such that NHL is produced.
  • the NHL so produced may be harvested from the host cells in conventional ways. Therefore, the present invention also relates to methods of expressing the NHL protein and biological equivalents disclosed herein, assays employing these gene products, recombinant host cells which comprise DNA constructs which express these proteins, and compounds identified through these assays which act as agonists or antagonists of NHL activity.
  • the cloned NHL cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.neo, pcDNA3.1, pCR2.1, pBlueBacHis2 or pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant NHL.
  • Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells.
  • RNA-yeast or bacteria-animal cells Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells.
  • An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
  • a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full- length open reading frame for NHL as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a NHL cDNA. The expression levels and activity of NHL can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
  • this NHL cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells. Techniques for such manipulations can be found described in Sambrook, et al., supra, are well known and available to the artisan of ordinary skill in the art. Therefore, another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding the NHL protein. An expression vector containing DNA encoding a NHL-like protein may be used for expression of NHL in a recombinant host cell.
  • Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • Commercially available mammalian expression vectors which may be suitable for recombinant NHL expression include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLlTMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pS V2-neo (ATCC 37593) pBPV- 1 (8-2)
  • bacterial expression vectors may be used to express recombinant NHL in bacterial cells.
  • Commercially available bacterial expression vectors which may be suitable for recombinant NHL expression include, but are not limited to pCR2.1 (Invitrogen), pETl la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia).
  • a variety of fungal cell expression vectors may be used to express recombinant NHL in fungal cells.
  • Commercially available fungal cell expression vectors which may be suitable for recombinant NHL expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
  • a variety of insect cell expression vectors may be used to express recombinant protein in insect cells.
  • Commercially available insect cell expression vectors which may be suitable for recombinant expression of NHL include but are not limited to pBlueBacUI and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
  • Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of bovine, porcine, monkey and rodent origin; and insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • bacteria such as E. coli
  • fungal cells such as yeast
  • mammalian cells including, but not limited to, cell lines of bovine, porcine, monkey and rodent origin
  • insect cells including but not limited to Drosophila and silkworm derived cell lines.
  • one insect expression system utilizes Spodoptera frugiperda (Sf21) insect cells (Invitrogen) in tandem with a baculovirus expression vector (pAcG2T, Pharmingen).
  • pAcG2T baculovirus expression vector
  • mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS- C-l (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).
  • L cells L-M(TK') ATCC CCL 1.3
  • L cells L-M ATCC CCL 1.2
  • Saos-2 ATCC HTB-85
  • CTAAGCACAC CCAGGCAGGT GTCCTGGCAG ATGAGGACCA CATGCAGAGC CTCGGCCAGC 120
  • ATCTGACTAG TGTGATCTCG CAAGGAACAT TCCAGACACA GTGGAGCTAG AAGGTTCTTC 840 TCCAAACAAG GAATCCCCAG GGGATCAAAT TGTTTTGCAT CGGCCAGACA TGGTGGCTCA 900
  • CAGTCCCAGC ACTGTACTAA AAATCTACAC GGGGCCGGGC ATGGTGGCAC ATGCCTGTAG 1080
  • GACGTGCCAT AACCAAGAAG CCCCAGCCAC ACCCAGACCC GATGTGGCCA CAAGGGGTGA 1620
  • CTTTCCTCTG CAACTGTGGG CTTACGGGGC AAAGAAGTCC AGGCCTCCAG GTGGAGGATC 1860
  • CAAGACACCC ACAGAGGAGA GCTCTAAGCC ACAACTGTGT ACGAAGACAA CTGTGCAGGA 2640 TTTTATTACT ACAACATTTT TGTTTTCTTT TTTTTTTTTT TTTGAGACTG AGTCTCGCTC 2700
  • CACATTTCAA ATGGGTAACT CCAGTGTCCT TGATGCTCCT GCGACATGTT CGTGAGACTT 3060
  • ATGGTTCACA CTCCTTACCC TGCCGCTTTG TCTTGTATCC AATAAATAGC GCAACCTGGC 4080 ATTCGGGGCC GCTACCAGTC TCCGCGTCTT GGTGGTAGTG GTCCCCCAGG CCCAGCTGTC 4140
  • AAATGGCAAA TTAGACACAC ACATGTGGGC CGGGTACAGT GGCTCGCGCC TGTAATTCCA 5340
  • CCCTGCCTAC AGGACCCTGA GAGCTAGGGG AAGGCGTTAT CCTGAACTGT GTCCCCCGTA 6000
  • GAGATAATTT AAATGAGGTC ATATAAGTTG
  • GCCCTCATCC AGTAAGACTT TGACCTTCTG 6120 GTGGTTTTTT TTTTTTTGGA
  • GACTGGGTCT CACTCTATCA CTCAGGTTGG AGTACAGTGG 6180
  • ATCTGTCTCC CTCGGCCTCC TGCAGTGCTG GAATTACAGG TATGAGCCAC CGCGCCTGGC 6420 CGACCGTGAC CTTCTAAGAA GTGAAAGAGA AAGATCTTTC TCTCTCCCTC CCTCTCCATC 6480
  • GGCAGCCGCGCG GCCACGGTGT CAGGGCTCAG GTGAGGAGAG TTGGATATGG GACTGGGCCT 6960 ACCCCGAGGC TGCTTCCACC CAGACGCCTG GGTGGGTGAC ACGAAAGCTG GGCTCAGTTG 7020
  • CACGGCGTCC CAAGGGAGGG ACTTGGGCAC TGCCTCTCTG GGCAAGAGTG GGGAGGTGTG 7260 GGGTGGGAGA TGTCTGGAAA CATCATGGAC AC TGCCGGG AAAACACGGA AGCTGTGCAC 7320
  • GCCTCCACCA CCCTGACATG CAGGAGGGAG GTCAAAGCCT CGGGTCCAAC AACAGGCTCC 7560 ACAGCAAGGG AAGAAAGGCA GGAAGGAACT CAGGGCCAGG TCCTCCCAGG CAGCAGCTGC 7620
  • CTGCACGCTG TCCACCAAGG GAGGTCTGAC CTACACCGCA CAGGGGTTGG CAGTCTAGAG 7680
  • GGCCAACACA GTGAAATCCT GTCTTGACTA AAAATACTAA AAATTAGCCA GGCATGGTGG 10680
  • TTCTTGCTAA ATCTTACTCA ACCGACATTT TCCAGCATGG GAACATTTTT CTGAATGTCT 11640 TAGGGAGAGG AAGTCCGCAA GAGAACAAAA GGTCCTCAGG CCACCCTAGC TTCTTTTCCT 11700
  • AAAAATACAA AAATTAGCCG GGCGCGGTGG CAGGTGCCTG TAATCTCAGC TACTTGGGAG 12840 GCTGAGGCAG GAGAATCGCT TGAACCTGGG CAGCAGAGGT TGCAGTGAGC CAAGATCATG 12900
  • CTACTGATCT CCCGTGCTGA CTTCGGGG TTTAACTCTC ACTGAGGAGA CGCTGCTTTC 13680 ATAAGGGTAA GCTCAGCAGG GGCAACTAAA GTCATTTAAG CAGAGAGCTG CAAAGAGGCA 13740
  • CCGTGTTAGC CAGGCTGCTC TCAAACTCCT GACCTCATGA TCCGCCCACG TCGGGCTCCC 15000
  • GGCACTCACC CCGATCGCAT AGCATAGCTG ATACCCCGAT CCCACCCCAG TCCCATAGCC 17100
  • CTTCCTTCTA AAATATTTAT CATTTTTGTT TTGGGGATTT TTTTGGTTTG GTTTTTTTTG 19140
  • AACACGGTGA AACCCCGTCT CTACTAAAAA TACAAAAAAT TAGCCGGGCG TGGTAGCGGG 19440
  • AAAATACAAA AAAATTAGCC AGGCATGGTG ACGGGCGCCT GTAATCTCAG CTACTTGGGA 20640
  • TGGTGGCTCA CACCTGTAAT CCCAGCTACG TGGCAGGCTG AGGCAGGAGA ATCGCTTGAA 21240 CCTGGGAGGC GGAGGTTGTA GGGAGCTGAG ATCGCACCAC TGCACTCCAG CCTGGGCAAC 21300
  • ATCTCTACTA AAAATACAAA AGTTAGCTGG GTGTGTACAT GTAGTCTCAG CTACTTGGGA 21960
  • CAGCCACAGT CAATACCTCG CTTCTGCAGG GACGGTGGCT GCCAGAGTGG GAGGCTTTGG 22260
  • CTTTGCCCGA CACGAGTGCA CAGCAGGCTG TGGGGGAGCA ACTGGTTGAG TCAGGCCTCC 24780 ACTTGTGCCG TATCCCCACC TGCTTTGCTG GACACCCCTG TTTGGGGGGC ACCCACTGCT 24840
  • TAATCCCAGC TACTTGGGAG ACTGAGGCAG GAGAATCGCT TATAACCTGG GAGGTGGAGG 25620 TTGCAGTGAG CTGAGATCAC ACCGCTACAC TCTAGCTTGG GCAACAAGAG TGAAACTCCG 25680
  • GCAGATCTTC ACTCCCAGAC AGGGAGCCCG CAGCTGCCCC CGACCCCACA GGTGCAGGAC 28860 ACACACAGAC AGTTCAACCA TGTCTTAAAC ACACAGGTGT TTATTTAATT GTTCATTTGA 28920
  • CGCCATCCTC 29400 CAGCTGACCG TCCTCCAAGG CCAGCACTGG GCGTCCAAGG GAAAGAAGGA ACTCAGCCCA 29460
  • TCCTGCGCAC CTCGGCCGCGCG TGCAGCTCCT GCAGGACAGG GGGCGGGAGG GCCTGAGGGC 30600 GGGGGTGGCT TGGGGCGACT CCGGGAACCC CCAGGCGC AGGCCGTGGC GCCCTGGCAC 30660
  • ATTTAGCCAT CTATTACTGC GGCTAGTTAC TGTCCCGCCA GGACCAGACT CTGGACCTGC 30900 CTCGTGCGCT GCTGGGGACG CCCAGTAAAC ACGGGAGGAG CCCCCGACCC CCACCCCAGC 30960
  • CTTCTCCTCC GCCTGGCGGC TGAAGTTGTT ATTCTCCTCC AGCGCCTTGT GCAGCACCTC 31620
  • GGGCCTGCCC ACCTGGGCCC CCGTTTTCCC TCCCCATGGC TGCCTCTATC ATGTCTCTGT 33780 GAGACACGGA GCTGCCCAGC ACGCTCTCTT GTGTGTCTCC ACACCGCCGG CCCCTTCGTC 33840
  • CTGTCTTTCT CCCTGAGTGC ATCTTTCTGT GATTCCTTGT CACTGTGTGT CTTTCTGACT 34500
  • GATAGTCTCG ATCTCCTGAC CTCGTGATCC GCCCGCCTCG ACCTCCCAAA GTGCTGGGAT 36000
  • CTCCAGGCCC CTACCCTTCA GCTCATCCTT CCTTATCACA CATCCAAAAC TCTGAATGTG 37740
  • ATCTCGGCTC ACGGCAAGCT CCGCCTCCCG GATTCACGCC ATTCTCCTGC CTCAGCCTCC 38940
  • GGCTCACTGC AACCTCTGCC TCCCCAGTTC AAGTGATTCT CCTGCCTCAG CCTCCCAAGT 39240
  • TGAGGTTTCA CCATGTTGGC CAGGCTGGTC TTGAACTCCT GACCTCCGGT GATCTGCCCA 39360 CCTCAGCCTC CCAAAGTGCT GGGATGACAG GCGTGAGCCC CCGCGCCTGG CCCCCCGCAG 39420
  • TTCTCGCAGT GAGCTGGGCT TGTTTTGTCT CCCTGCTTCT CTTTGTACTA AACATTAGAT 39600 ACCGAGGAAA TGCGGATTGG CCTTTGGATG ATTCATGAGC AGGAGTCAGA AAAAGGCACC 39660
  • GGAGAGGTGC CAGCACCGTC ATCTCTACCC AGATAAGGAG ACCCAGGTCC TGAGAGGTTA 41040
  • CTCCCAGCTC AGCCCCAGGA ACCGAGCCCA TGGGGAGGGA CCGTCAGGGA AAGGCTGTCA 41220
  • TGACTCAGAC TTCAGCTCAG TCCACAGGAC AGCCTTTTCT GGCCACTGCT CTCAGGAGAT 46020 GAGATGTGTG GCTGCAAAAG GTAAACTCCT GGCTCCTGAG CAGGCTCTGG GCAATCTGCT 46080
  • AAATATAAAA AATTAGCCGG GCTTAGTGGT GCACACCTGT AATCCCAGCT ACTTGAGAGG 46320 CTGAGGCAGG AGAATCACTT GAACCCAGGA GGTGGAGGTT GCAGTGAGCC AAGATTGTGC 46380
  • TAGTCCCAGT TCCCGGGCGG GATTGAGGCT TAGAGAAGTT GAGTGATTTG CTGAGGGCTG 48060 CACGGGTTGG CATCCCGGCA TGCTCTTTCG CTACTTTGGC TGCATCTGGT TGCCCACCCG 48120
  • CTCTTCAGCC CTCACGCTCTCT TGTGGAAGTC GCGGAATTAC TGCAGGCGGA ACTTGCAGCA 48300
  • CTGTGGGCGT CTTTTCCAGA GAAGGACGGA GTTGTGGGGC GGGAGGATAA GGCAAGGCCC 48360 AGCCACTTCG CATCTTCGCC CCGCCAGCTC CTCGAGATGG GATATACCAG GGTTGCTCTC 48420
  • CTTCAGCTGC GCACTCTGCC CTTCCTCCCA CAGATCCACT TGTGCCGTAA GAAGGTGGCA 52140 AGTCGCTCCT GTCATTTCTA CAACAACGTA GAAGGTACAA GCAGCTGGGT GGGACCAGGG 52200
  • GCACACACCA CCACCCCCTG CTAATTTTTG TATTTTTAGT AGAGACGGGG TTTTACCATG 61200 TTGGCCAGGC TGGTCTTGAA CTCCTGACCT CGTGATCCGC CCGCCTCGGC CTCCCAAAGT 61260
  • CTTCCTGACT GCGGTGGCCG GGGGCTCCCA GGGCATCGTG GCCGTCTGTC TTGCTGAGCG 61440
  • GGTATTGTTC AGTAGTTCTG GTATTTTCCA AAGACCTATG TCTTCTCCCA GCCAGTATCA 63540 ACTTGGCCTC TACTGTGTAA AACTGGAAAA CTCTACTTTG TGAAGCTGAG TTGGGAGCAT 63600
  • AAAAATTAGC CAGGTGTGGT GGTGTGCTCC TGTGGTCCAA GCTTTTCTGG AGGCCGAAGT 63720
  • AAGTTGGCAT TTGTTTAGTA CAGAAGTTAT CAGGTGTTCT GGCTTTAGAA TCCCTTTATA 64620
  • CTCCTCTCCA CAGTCCCCCA ACCCCACCTC TCTAACGGGG TGGACGGCCG CCTCTTTCCA 64920
  • AACTTCAGTT TTCATCCCTA TCTGTTCCCC CACCCCTTTG GAGATGGGGT CTCACTCTGT 65220
  • CTATAGTCCC AGCTAGTCGG GAGACAGACA CGAGAATTGC TTGAACCTGG GACATGGAGG 66660
  • CTCTTCCTTT CCATGTTGGT GTCCTTTTTT CCATGCCAGG AATCCTGGTT CTCAAGGGCG 67200
  • CTGGTCCAGT CCGTCATTTG AGCACAGGTG CCTGTTAGAA CGAGACCTTC TTGTTAGGAC 68160 GATGAGTGTC CCAGCCACCA CCTCTTTTGG ACTCCGGGAG GCCTGGAACG TTCTGAACGC 68220

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Abstract

La présente invention concerne des molécules d'acide nucléique isolé (oligonucléotides) qui codent la NHL que l'on suppose être une hélicase d'ADN. L'invention concerne plus particulièrement des vecteurs de recombinaison et des hôtes de recombinaison contenant un fragment d'ADN codant la NHL, des formes sensiblement purifiées de la NHL associée, des protéines mutantes associées, et des procédés associés visant à identifier des composés qui modulent la NHL et qui devraient convenir au traitement de divers troubles néoplasiques. L'invention concerne également, d'une part un clone génomique contenant des séquences régulatrices et d'intron, et d'autre part la structure d'exon et un cadre de lecture ouvert de la NHL humaine.
EP00983952A 1999-12-09 2000-12-07 Molecules d'adn codant la nhl humaine, une helicase d'adn Withdrawn EP1240311A1 (fr)

Applications Claiming Priority (3)

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US16997099P 1999-12-09 1999-12-09
US169970P 1999-12-09
PCT/US2000/033065 WO2001042434A1 (fr) 1999-12-09 2000-12-07 Molecules d'adn codant la nhl humaine, une helicase d'adn

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CN1247567A (zh) 1997-01-14 2000-03-15 人体基因组科学有限公司 肿瘤坏死因子受体6α和6β
US7285267B2 (en) 1997-01-14 2007-10-23 Human Genome Sciences, Inc. Tumor necrosis factor receptors 6α & 6β

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US5466576A (en) * 1993-07-02 1995-11-14 Fred Hutchinson Cancer Research Center Modulation of PIF-1-type helicases
US5843737A (en) * 1994-12-30 1998-12-01 Chen; Lan Bo Cancer associated gene protein expressed therefrom and uses thereof
US5888792A (en) * 1997-07-11 1999-03-30 Incyte Pharmaceuticals, Inc. ATP-dependent RNA helicase protein

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