JP2003523181A - DNA helicase, DNA molecule encoding human NHL - Google Patents

Abstract

  (57) [Summary] The present invention discloses an isolated nucleic acid molecule (polynucleotide) encoding a putative DNA helicase, NHL. The present invention relates to recombinant vectors and recombinant hosts containing DNA fragments encoding NHL, substantially purified forms of related NHL, related mutant proteins, and NHLs which are believed to be useful in the treatment of various neoplastic diseases. And methods related to identifying compounds that modulate. Disclosed are genomic clones containing the regulatory and intron sequences of human NHL, as well as exon structures and open reading frames.

Description

Detailed Description of the Invention

(Cross Reference to Related Applications) S. C. Claims the priority of US Provisional Application No. 60 / 169,970, filed December 9, 1999, under §119 (e).

[0002]     (Statement regarding federally sponsored R & D)   Unsuitable.

[0003]     (Refer to Microfish accessory)   Unsuitable.

FIELD OF THE INVENTION The present invention relates, in part, to an isolated nucleic acid molecule (polynucleotide) that encodes a putative DNA helicase, NHL. The present invention also provides that the gene is 20q13.3.
, Which is useful in the treatment of various neoplastic diseases, located on the right side and is directly adjacent to M68 / DcR3 involved in tumor growth.
Recombinant vectors and recombinant hosts comprising NA fragments, related NHLs in substantially purified form, related mutant proteins, and methods related to identifying compounds that modulate NHLs. In addition, additional genes (M68 / DcR3, SCLIP
, And ARP), and a human genome fragment corresponding to this part of the human genome is also included in the present invention.

Background of the Invention Naumovski et al. (1985, Mol. Cell Biol. 5: 17-
26; Reynolds et al. (1985 Nucleic Acid Res 1
3: 2357-2372) and Weber et al. (1990 EMBO J. 143).
7-1447) discloses members of the RAD3 / ERCC2 gene family of DNA helicases.

Actinomycin C1, daunorubicin and nogaramycin (Tuteja et al., 1997, Biochem. Biophys. Res. Comm. 236 (3
): 636-640), and the prostate cancer suppressor CI-958 (Lun et al., 1).
998, Cancer Chemother Pharmacol. 42 (6)
Several chemotherapeutic agents are known to inhibit helicase, including 447-453). Furthermore, some topoisomerases have been shown to have anti-tumor effects.

Despite the aforementioned identification of genes encoding helicase and chemotherapeutic agents, they encode additional genes residing within chromosomal regions associated with disease states such as cancer and the types of proteins thought to be associated with that disease. It would be beneficial to identify genes that do. The present invention addresses this need and meets that need by disclosing a DNA molecule that encodes a DNA helicase with a chromosomal location suggesting a link to cancer.

DISCLOSURE OF THE INVENTION The present invention provides an isolated or purified nucleic acid molecule encoding a novel mammalian DNA helicase (
Polynucleotide).

The present invention also relates to an isolated nucleic acid molecule (polynucleotide) encoding an mRNA expressing a novel human DNA helicase, NHL.

One preferred aspect of the present invention is isolated or purified DNA encoding human NHL.
The molecule relates to the nucleotide sequence as shown in FIGS. 1A-B and SEQ ID NO: 1.

The present invention also relates to a novel DNA helicase, a biologically active fragment or mutant of SEQ ID NO: 1 encoding an mRNA molecule expressing NHL. Such bioactive fragments and / or mutants are disclosed herein in FIG. 2 and represent either a protein or protein fragment that at least substantially mimics the biological properties of the human NHL protein set forth as SEQ ID NO: 2. To code. Such polynucleotides are useful in screening for agents and / or antagonists of NHL activity, such that the mutation encodes an mRNA expressing a functional NHL protein in a host cell. Included, but not necessarily limited to, substitutions, deletions, additions, amino terminal truncations and carboxy terminal truncations.

The present invention also relates to both prokaryotic and eukaryotic recombinant vectors and hosts that include the substantially purified nucleic acid molecules disclosed throughout this specification.

The invention also relates to a substantially purified form of the human NHL protein disclosed in FIG. 2 and comprising the amino acid sequence shown as SEQ ID NO: 2.

One preferred aspect of this part of the invention is the human NHL protein consisting of the amino acid sequence disclosed in FIG. 2 and shown as SEQ ID NO: 2. Another preferred aspect of the present invention is a substantially purified NHL protein obtained from a recombinant host cell comprising a DNA expression vector containing a nucleotide sequence as set forth in SEQ ID NO: 1 and expressing the respective NHL protein. , Preferably human NHL
Regarding proteins. It is especially preferred that the recombinant host cell is a eukaryotic host cell, such as a mammalian cell line.

The present invention also provides a selection wherein said mutation provides a protein or protein fragment for diagnostic, therapeutic or prophylactic use, including but not limited to agonists and / or antagonists for human NHL pharmacology. SEQ ID NO: 2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations, as useful for screening of modulators of interest.
A biologically active fragment and / or mutant of NHL protein containing the amino acid sequence as shown in.

One preferred aspect of the present invention is the respective amino acid sequence disclosed in FIG. 2 and set forth in SEQ ID NO: 2 and encoding human NHL. Characterization of one or more of these DNA helicase-like proteins enables screening methods to identify novel NHL modulators that may be useful in treating human neoplastic diseases. Modulators selected through such screening and selection protocols may be used alone or in combination with other cancer therapies. As mentioned above, heterologous expression of the NHL protein allows pharmacological analysis of compounds that modulate NHL activity and is therefore considered useful in various cancer therapies. To this end, heterologous cell lines expressing NHL proteins can be used to establish functional or binding assays to identify novel NHL modulators.

The invention also relates to polyclonal and monoclonal antibodies raised against either NHL or a biologically active fragment of NHL.

The present invention also relates to transgenic mice with altered genotype and phenotype with respect to NHL and its in vivo activity.

The present invention also relates to GFP (green fluorescent protein), MYC epitope and GST.
NHL fusion constructs including, but not limited to, fusion constructs expressing portions of the NHL protein linked to various markers including, but not limited to. Any such fusion construct is expressed in the cell line of interest,
It can be used to screen for NHL modulators.

Therefore, the present invention includes methods for expressing mammalian NHL, preferably human NHL, bioequivalents disclosed herein, assays using these gene products, DNA constructs expressing these proteins. Recombinant host cells and compounds identified through these assays that act as agonists or antagonists of NHL activity.

The present invention also relates to an isolated genomic sequence comprising SEQ ID NO: 1, a 115 kb genomic fragment shown here as SEQ ID NO: 3. As particularly preferred aspects of this part of the invention, the regulatory and coding regions of human NHL, and intervening sequences (introns)
There is a region of the genomic fragment of SEQ ID NO. This 115 kb fragment contains the coding regions of at least 4 genes, NHL, M68 / DcR3, SCLIP and ARP. As described herein, this region of chromosome 20 has been shown to be associated with tumor growth. Therefore, one aspect of the present invention also includes one or more of this 115 kb genomic sequence for identifying a compound that upregulates or downregulates the expression of one or more genes located within this 115 kb region. This up- or down-regulation results in interference with tumor growth, including the use of more regions. For example, the transcriptional element of one of these four genes is thought to be responsible for overexpression of M68 / DcR3 (and / or NHL) in tumors, and if M68 in tumors
Alternatively, if NHL overexpression plays an important role, it would be useful to block M68 / DcR3 or NHL overexpression in tumors by interfering with this transcription site.

An isolated nucleic acid fragment encoding a novel form of human NHL (eg, SEQ ID NO: 1), FIG.
And it is an object of the invention to provide a fragment, mutant or derivative of human NHL as shown in SEQ ID NO: 2. Such polynucleotides include nucleotide substitutions such that the mutation encodes an mRNA expressing a protein or protein fragment for diagnostic, therapeutic or prophylactic use and is useful for screening for selective modulators of human NHL activity. , Deletions, additions, amino-terminal truncations and carboxy-terminal truncations, but not necessarily limited thereto.

NHL in mammals, especially humans, encoded by the nucleic acid molecules referred to in the above section
It is a further object of the invention to provide proteins or protein fragments.

It is a further object of the invention to provide recombinant vectors and recombinant host cells comprising nucleic acid sequences encoding a mammalian, in particular human NHL protein and its bioequivalents.

It is one object of the present invention to provide a substantially purified form of human NHL, as shown in FIG. 2 and SEQ ID NO: 2.

Of a functional processed form, obtained from a recombinant host cell line transformed or transfected with a DNA expression vector containing the complete open reading frame as shown in SEQ ID NO: 1 and expressing appropriately. Produces NHL,
It is another aspect of the present invention to provide a substantially purified recombinant form of the NHL protein.
One purpose. It is especially preferred that the recombinant host cell is a eukaryotic host cell, such as a mammalian cell line.

SEQ ID NO: including, but not necessarily limited to, amino acid substitutions, deletions, additions, amino terminal truncations and carboxy terminal truncations, such that the mutation (s) provide a protein or protein fragment for diagnostic, therapeutic or prophylactic use. It is an object of the invention to provide bioactive fragments and / or mutants of mammalian, especially human, NHL as shown in 2.

It is also an object of the invention to use NHL proteins or bioequivalents to screen for modulators of human NHL activity, preferably selective modulators. Such compounds would be useful in screening and selecting compounds active against human neoplastic diseases.

As used herein, “substantially free of other nucleic acids” is at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9 of other nucleic acids. % Means not including. Thus, a preparation of human NHL DNA substantially free of other nucleic acids will yield a percentage of total nucleic acids of 10% or less,
Preferably 5% or less, more preferably 1% or less, even more preferably 0.1%
The following non-NHL nucleic acids will be included. Whether a given NHL DNA preparation is substantially free of other nucleic acids can be determined by conventional methods of assessing nucleic acid purity, such as agarose gel electrophoresis in combination with a suitable staining method, eg, ethidium bromide staining. It can be determined by a technique or by sequencing.

As used herein, “substantially free of other proteins” or “substantially purified” means at least 90%, preferably 95%, more preferably 99% of other proteins. %, And even more preferably 99.9%. Therefore, the NHL protein, which is substantially free of other proteins, can be prepared as a percentage of the whole protein by 10% or less, preferably 5% or less, more preferably 1% or less,
Even more preferably, it will contain 0.1% or less of non-NHL protein. Whether a given NHL protein preparation is substantially free of other proteins can be determined, for example, by appropriate detection methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with silver staining. It can be determined by conventional techniques for assessing protein purity or by immunoblotting. The phrase "isolated NHL protein" or "purified NHL protein" as used interchangeably herein with the phrase "substantially free of other proteins" or "substantially purified" also , Refers to NHL proteins isolated from natural sources. The use of the words "isolated" or "purified" refers to N
It shows that the HL protein is taken out of its normal cellular environment. Thus, the isolated NHL protein may be present in a cell-free solution or placed in a cellular environment different from that in which it naturally occurs. The term isolated does not mean that the isolated NHL protein is the only protein present, but that the isolated NHL protein does not naturally bind other NHL proteins in vivo to other proteins and non-amino acid substances such as nucleic acids. , Lipid,
Means that it is substantially free of carbohydrates. Therefore, an NHL protein that is recombinantly expressed in prokaryotic or eukaryotic cells and that is substantially purified from a host cell that naturally does not express this protein (ie, without intervention) is, of course, in any of the situations mentioned herein. It is also an “isolated NHL protein” below. As described above, the NHL protein, which is an isolated or purified NHL protein, may be prepared as a percentage of the whole protein in an amount of 10% or less, preferably 5% or less, more preferably 1% or less, and even more preferably 0.1% or less. % Of non-NHL proteins will be included.

“Functional equivalent” or “bioactive equivalent”, as used interchangeably herein, is meant strictly because of splicing changes, deletions, mutations, substitutions, or additions. By a protein that does not have the same amino acid sequence as naturally occurring NHL, but retains substantially the same biological activity as NHL. Such functional equivalents have significant amino acid sequence identity with the naturally occurring NHL, and the genes and cDNAs encoding such functional equivalents have low stringency with the DNA sequence encoding the naturally occurring NHL. It can be detected by gentency hybridization. For example, the naturally occurring NHL disclosed herein comprises the amino acid sequence set forth as SEQ ID NO: 2 and is encoded by SEQ ID NO: 1. A nucleic acid encoding a functional equivalent has at least about 50% identity to SEQ ID NO: 1 at the nucleotide level.

As used herein, a “conservative amino acid substitution” is another chemically similar substance.
Substitution of one amino acid residue by another. Examples of such conservative substitutions are the replacement of one hydrophobic residue (isoleucine, leucine, valine, or methionine) by another, one polar residue, another one of the same charge. Substitution with polar residues (eg arginine with lysine, glutamic acid with aspartic acid).

The term “mammal” as used herein refers to any mammal, including humans.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-B show a nucleotide sequence containing an open reading frame encoding human NHL, ie the nucleotide sequence shown as SEQ ID NO: 1. The start Met residue (ATG) and stop codon (TAG) are underlined. FIG. 2 shows the amino acid sequence of human NHL as shown in SEQ ID NO: 2. Figure 3 shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members. RepD (Dictyostelem discoid
eum); RAD3 (S. cerevisiae (brewer's yeast)); RAD1
5 (S. pombe) and XP_GroupD (Homo sapien (human)). FIG. 4 shows Northern blot analysis of NHL expression in multiple human tissues. 5A-B shows the genomic structure of the NHL gene (FIG. 5A), and NHL, M68 /.
The entire 115 kb genomic region containing the DcR3, SCLIP and ARP genes (Fig. 5
B) is shown.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an isolated or purified nucleic acid molecule encoding a novel mammalian DNA helicase (
Polynucleotide). A particularly preferred aspect of the present invention is novel human DNA
The present invention relates to an isolated nucleic acid molecule (polynucleotide) encoding an mRNA expressing helicase, NHL.

Gene M68 / DcR3 is a secreted TN that is overexpressed in many human tumors
A member of FR. M68 / DcR3 is located at 20q13.3, a known site associated with frequent gene amplification in cancer. M68 / DcR3 protein is FASL
And inhibit FAS-mediated apoptosis. Therefore, M68 / DcR3
Genes closely related to are considered to be co-regulated (eg co-overexpressed and / or co-amplified in tumors). In the process of cloning a genomic M68 / DcR3 fragment and identifying a gene that binds to M68 / DcR3 at 20q13.3, three genes were directly flanked, including one novel gene similar to the Rad3 / ERCC2 helicase family. It was identified in the (overlap) region (named NHL). NHL chromosomal location, human genetic disease and DNA
Given the frequent association of helicases (mutations in DNA helicases have been found to be associated with many diseases, including xeroderma pigmentosum, Cockayne syndrome, Bloom syndrome, and Werner syndrome), NHL Are candidate substances for their contribution to human neoplastic disease. For this, a genomic clone of this gene is disclosed and the complete sequence is determined. Transcripts were identified through exon prediction using GRAIL2 and sequence alignment to the adjacent 4.5 kilobase region of chromosome 4 (88% sequence identity). The complete exon structure of NHL is then converted to R
Confirmed by T-PCR analysis. A multiple sequence alignment of NHL to a known helicase showed that NHL contained seven critically important helicase domains.
BLAST analysis of the predicted 1,219 amino acid sequence showed that the DNA helicase R
It revealed about 26% sequence identity and 48% sequence similarity with the AD3 / ERCC2 gene family (Naumovski et al., 1985 Mol. Cell.
Biol. 5: 17-26; Reynolds et al., 1985 Nucleic.
Acids Res 13: 2357-72; Weber et al., 1990 EM.
BO J. 9: 1437-1447). The NHL mRNA expression pattern was also examined in a number of human tissues. Radiation hybrid chromosome mapping reconfirms that it binds to the M68 / DcR3 locus.

One preferred aspect of the present invention is isolated or purified DNA encoding human NHL.
Regarding the molecule, ie, the nucleotide sequence as shown in FIGS. 1A-B and SEQ ID NO: 1, as follows:

[0038]

[Chemical 3]

The isolated DNA molecules exemplified above, shown in FIGS. 1A-B and SEQ ID NO: 1, have an initiating Met at nucleotides 828-830 and a “T at nucleotides 4585-4587.
It contains 4946 nucleotides with the "AG" stop codon. Start Met and T
The AG stop codon is underlined.

The present invention also relates to a novel DNA helicase, a bioactive fragment or mutant of SEQ ID NO: 1 encoding an mRNA molecule expressing NHL. Such bioactive fragments and / or mutants are disclosed herein in FIG. 2 and represent either a protein or protein fragment that at least substantially mimics the biological properties of the human NHL protein set forth as SEQ ID NO: 2. Will code. Such a polynucleotide is N
An mRNA in which the mutation expresses a functional NHL protein in a host cell in order to be useful in screening for agents and / or antagonists of HL activity
, Including, but not necessarily limited to, nucleotide substitutions, deletions, additions, amino terminal truncations and carboxy terminal truncations.

The isolated nucleic acid molecule of the present invention may be single-stranded (coding strand or non-coding strand) or double-stranded, and deoxyribonucleic acid molecule (DNA) such as genomic DNA and complementary DNA (cDNA). , And synthetic DN such as synthetic single-stranded polynucleotides
A may be included. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).

The present invention also relates to both prokaryotic and eukaryotic recombinant vectors and recombinant hosts that include the substantially purified nucleic acid molecules disclosed throughout this specification.

The degeneracy of the genetic code is that for all but two amino acids, two or more codons code for one particular amino acid. This allows the construction of a synthetic DNA encoding the NHL protein, which nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequence of SEQ ID NO: 1, but still encodes the same NHL protein as SEQ ID NO: 2. Such synthetic DNA is intended to be within the scope of the present invention. Such synthetic DNA in a particular host cell or organism
Is desired to be expressed, the codon usage of such synthetic DNA is adjusted to reflect the codon usage of that particular host, thereby N
It is possible to lead to higher expression levels of the HL protein. In other words, this redundancy of the various codons coding for a particular amino acid is within the scope of the invention. Therefore,
The present invention is also directed to a DNA sequence encoding an RNA containing alternative codons encoding possible transcription of the same amino acids, as shown below: A = Ala = alanine: codons GCA, GCC, GCG, GCU C. = Cys = Cysteine: Codon UGC, UGU D = Asp = Aspartic acid: Codon GAC, GAU E = Glu = Glutamic acid: Codon GAA, GAG F = Phe = Phenylalanine: Codon UUC, UUU G = Gly = Glycine: Codon GGA, GGC , GGG, GGU H = His = histidine: codon CAC, CAU I = Ile = isoleucine: codon AUA, AUC, AUU K = Lys = lysine: codon AAA, AAG L = Leu = leucine: codon UUA, UUG, CUA, CUC , CUG, C
UU M = Met = methionine: codon AUG N = Asp = asparagine: codon AAC, AAU P = Pro = proline: codon CCA, CCC, CCG, CCU Q = Gln = glutamine: codon CAA, CAG R = Arg = arginine: codon AGA, AGG, CGA, CGC, CGG,
CGU S = Ser = serine: codons AGC, AGU, UCA, UCC, UCG, UC
UT = Thr = Threonine: Codons ACA, ACC, ACG, ACU V = Val = Valine: Codons GUA, GUC, GUG, GUU W = Trp = Tryptophan: Codon UGG Y = Tyr = Tyrosine: Codons UAC, UAU Therefore, The present invention discloses codon redundancy that can result in different DNA molecules expressing the same protein. For the purposes of this specification, a sequence bearing one or more substituted codons is defined as a degenerate mutation. Mutations in either the DNA sequence or the transcribed protein that do not substantially change the ultimate physical properties of the expressed protein are also included within the scope of the invention. For example, substitution of leucine with valine, lysine with arginine, and glutamine with asparagine would not cause a change in the functionality of the polypeptide.

It is known that the DNA sequence encoding a peptide can be altered to encode a peptide with properties that differ from those of the naturally occurring peptide. Methods of altering the DNA sequence include, but are not limited to, site-directed mutagenesis. Examples of altered properties include, but are not limited to, altered affinity of an enzyme for a substance or altered affinity of a receptor for a ligand.

The present invention also relates to both prokaryotic and eukaryotic recombinant vectors and hosts that include the substantially purified nucleic acid molecules disclosed throughout this specification. A "recombinant DNA molecule" encoding each NHL protein by binding the whole or part of the nucleic acid molecule of the present invention encoding the NHL protein to another DNA molecule, that is, a DNA molecule to which the NHL coding sequence does not naturally bind. Can be formed. The novel DNA sequences of this invention can be inserted into a vector containing a nucleic acid encoding NHL or a functional equivalent. These vectors can be DNA or RN
It may be composed of A; DNA vectors are preferred for most cloning purposes.
Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA capable of encoding NHL proteins. It is well within the level of ordinary skill in the art to determine the appropriate vector for transfer of a particular gene or other use.

DNA sequences that hybridize to SEQ ID NO: 1 under stringent conditions are included in the invention. By way of example and not limitation, the procedure using high stringency conditions is as follows: 6 × SSC, 5 × Denhardt's solution, and 100 μg /
DNA in a buffer consisting of denatured salmon sperm DNA at 65 ° C for 2 hours to overnight
Perform prehybridization of the filter containing 100 μg / ml
Filters are hybridized for 12 to 48 hours at 65 ° C. in a prehybridization mixture containing denatured salmon sperm DNA of 5 and 5-20 × 10 ⁶ cpm of ³² P-labeled probe. 1 at 37 ° C. in a solution containing 2 × SSC, 0.1% SDS
Wash the filter for a period of time. This is followed by 0.1 x SSC, 0.1% SD
After washing in S at 50 ° C. for 45 minutes, autoradiography is performed. Other procedures using high stringency conditions are 5x SSC, 5x Denhardt's solution, a hybridization step performed in 50% formaldehyde at 42 ° C for 12 to 48 hours, or 0.2x SSPE, 0.2% SDS. 30 to 60 at 65 ° C
It will include any of the wash steps performed for minutes.

The reagents referred to in the above procedure for performing high stringency hybridization are known in the art. Details of the composition of these reagents can be found, for example, in Sambrook et al., 1989, Molecular Cloning: Laboratory Manual (A Laboratory Man).
ual); Cold Spring Harbor Laboratory, C
It can be found in Old Spring Harbor, New York. In addition to the above, other conditions of high stringency that can be used are known in the art.

The present invention also discloses the amino acid sequence disclosed in FIG. 2 and shown as SEQ ID NO: 2 (1219
Individual amino acid residues) and a substantially purified form of the human NHL protein. A preferred aspect of this part of the invention is an NHL protein consisting of the amino acid sequence disclosed in FIG. 2 and shown as SEQ ID NO: 2 as follows:

[0049]

[Chemical 4]

The present invention also provides amino acid substitutions such that the mutations provide proteins or protein fragments for diagnostic, therapeutic or prophylactic use, and are useful for screening agents and / or antagonists of NHL function. , Deletions, additions, amino terminal truncations and carboxy terminal truncations, and biologically active fragments and / or mutants of the human NHL protein comprising an amino acid sequence as set forth in SEQ ID NO: 2.

Another preferred aspect of the invention is a substantially purified, obtained from recombinant host cell comprising a DNA expression vector comprising a nucleotide sequence as set forth in SEQ ID NO: 1 and expressing a human NHL protein, It relates to a fully processed NHL protein. It is particularly preferred that the recombinant host cell is a eukaryotic host cell such as a mammalian cell line.

As with many proteins, it is possible to modify many of the amino acids of the NHL protein and still retain substantially the same biological activity as the wild-type protein. Accordingly, the invention includes modified NHL polypeptides that comprise amino acid deletions, additions, or substitutions, yet still retain substantially the same biological activity as their respective NHLs. It is generally accepted that a single amino acid substitution does not normally alter the biological activity of a protein (eg, molecular biology of a gene (Molecular Bi
logic of the Gene), Watson et al., 1987, 4th edition,
The Benjamin / Cummings Publishing Co.
, Inc. p. 226; and Cunningham & Wells, 1989.
, Science 244: 1081-1085). Accordingly, the present invention includes a polypeptide with a single amino acid substitution in SEQ ID NO: 2, which still retains substantially the same biological activity as the corresponding NHL protein. The invention also has two or more amino acid substitutions in SEQ ID NO: 2,
It still includes polypeptides that retain substantially the same biological activity as the corresponding NHL protein. In particular, the invention includes embodiments in which the above substitutions are conservative substitutions.

Those skilled in the art will also follow polypeptides that are functional equivalents of NHL and that have changes from the NHL amino acid sequence that are small deletions or insertions of amino acids, according to the same guidelines (ie amino acids between NHL and related proteins). Minimize sequence differences: Small deletions or insertions are generally within the range of about 1 to 5 amino acids.
) Will recognize that it can be made. The effect of such small deletions or insertions on the biological activity of the modified NHL polypeptide is to make the polypeptide synthetically or to make the necessary changes to the NHL-encoding DNA and then recombinantly express the DNA. Then, it can be easily evaluated by assaying the protein produced by such recombinant expression.

The present invention also includes a truncated form of NHL, which comprises a region containing the active site of the enzyme. Such truncated proteins are useful in various assays described herein for crystallization studies and for structure-activity relationship studies.

The invention also relates to producing isolated nucleic acid molecules that are fusion constructs expressing fusion proteins useful in assays to identify compounds that modulate the activity of wild-type NHL, as well as antibodies to the NHL. One aspect of this part of the invention includes, but is not limited to, glutathione-S transferase (GST) -NHL fusion constructs. Recombinant GST-NHL fusion protein was isolated from Spo using a baculovirus expression vector (pAcG2T, Pharmingen).
doptera frugiperda (Sf21) insect cells (Invitro
gen) and various expression systems. Another aspect relates to NHL fusion constructs linked to various markers, including but not limited to GFP (Green Fluorescent Protein), MYC epitope and GST. Again, any such fusion construct is expressed in the cell line of interest,
It can be used to screen for modulators of one or more NHL proteins disclosed herein.

NHL can be cloned using any of a variety of procedures. These methods include (1) RACE PCR cloning method (Frohman et al., 1
988, Proc. Natl. Acad. Sci. USA 85: 8998-9.
002). 5'and / or 3'RACE can be performed to generate a full length cDNA sequence. This strategy involves the use of gene-specific oligonucleotide primers for PCR amplification of NHL cDNA. These gene-specific primers are designed through the identification of expression sequence tag (EST) nucleotide sequences identified by searching a number of publicly available nucleic acid and protein databases; (2) suitable expression vectors. Direct functional expression of NHL cDNA following construction of an NHL-containing cDNA library in a system; (3) NHL-containing cDNA constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide designed from the amino acid sequence of NHL protein Screen library; (4) NHL constructed in bacteriophage or plasmid shuttle vector with partial cDNA encoding NHL protein
Screen the containing cDNA library. This partial cDNA is NHL
Obtained by specific PCR amplification of NHL DNA fragments through the design of degenerate oligonucleotide primers from known amino acid sequences for other protein-related kinases; (5) partial cDNA with homology to mammalian NHL protein.
Alternatively, oligonucleotides are used to screen NHL-containing cDNA libraries constructed in bacteriophage or plasmid shuttle vectors. This strategy is also used for PC of NHL cDNA identified as EST as described above.
Can include using gene-specific oligonucleotide primers for R amplification; or (6) full-length cDNA can be generated by known RACE techniques, or part of the coding region by these same known RACE techniques. And a portion of the coding region used as a probe in screening one of many types of cDNA and / or genomic libraries to isolate the full-length NHL-encoding nucleotide sequence. SEQ ID NO: 1 is used as a template to design 5'and 3'gene-specific oligonucleotides so that they can be produced and isolated.

It will be appreciated by those skilled in the art that other types of libraries, as well as libraries constructed from other cell types or other species, may be useful for isolating NHL-encoding DNA or NHL homologues. It is easy to see. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells.

It will be readily apparent to those skilled in the art that suitable cDNA libraries can be prepared from cells or cell lines that have NHL activity. The choice of cell or cell line used in preparing a cDNA library to isolate a cDNA encoding NHL is first determined by assaying cell-associated NHL activity using any known assay that can be used for that purpose. Can be carried out by measuring

Preparation of a cDNA library can be performed by standard techniques known in the art. A known cDNA library construction method is, for example, Sambrook.
K. et al., 1989, Molecular Cloning: Laboratory Manual (Molecular
Cloning: A Laboratory Manual); Cold Sp
Ring Harbor Laboratory, Cold Spring H
It can be found in Arbor, New York. Complementary DNA libraries are also available from Clontech Laboratories, Inc. And Str
It is also available from a number of commercially available sources, including but not limited to atagene.

It will be readily apparent to one skilled in the art that the DNA encoding NHL may also be isolated from a suitable genomic DNA library. Construction of a genomic DNA library can be performed by standard techniques known in the art. Known methods for constructing genomic DNA libraries can be found in Sambrook et al., Supra. A genomic library can be prepared, especially in the P1 artificial chromosome vector, from which a genomic clone containing the NHL gene can be isolated using probes based on the NHL nucleotide sequences disclosed herein. Methods for preparing such libraries are known in the art (Ioannou et al., 1
994, Nature Genet. 6: 84-89).

In order to clone the NHL gene by one of the preferred methods, NHL
Alternatively, the amino acid sequence or DNA sequence of the homologous protein may be required. To achieve this, each NHL protein can be purified and the partial amino acid sequence determined by an automated sequence analyzer. It is not necessary to determine the entire amino acid sequence, but it is possible to determine the linear sequence of two regions of 6 to 8 amino acids for PCR amplification of a partial NHL DNA fragment. Once the appropriate amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Due to the degeneracy of the genetic code, more than one codon can be used to encode a particular amino acid, and thus the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Although only one member of the set is identical to the NHL sequence, the other members of the set will be able to hybridize to NHL DNA even in the presence of mismatched DNA oligonucleotides. The mismatched DNA oligonucleotides are still able to hybridize well to NHL DNA, allowing the identification and isolation of NHL-encoding DNA. Alternatively, the nucleotide sequence of a region of expressed sequences can be identified by searching one or more available genomic databases. Gene-specific primers can be used to perform PCR amplification of a cDNA of interest from a cDNA library or a population of cDNAs. As mentioned above,
A suitable nucleotide sequence for use in a PCR-based method is NHL
One or more cDNAs for the isolation of overlapping 5'and 3'RACE products for the production of a full length sequence encoding Alternatively, it can be obtained from SEQ ID NO: 1 to isolate a portion of the NHL-encoding nucleotide sequence for use as a probe to screen a genome-based library.

The present invention also provides a vector containing the NHL gene, a host cell containing the vector,
And introducing the NHL gene into a host cell and culturing the host cell under suitable conditions such that NHL is produced, thereby producing a substantially pure NHL protein. The NHL so produced can be harvested from the host cells by conventional methods. Therefore, the present invention also provides methods for expressing NHL proteins and bioequivalents disclosed herein, assays using these gene products, recombinant host cells containing DNA constructs expressing these proteins, and NHL. It relates to compounds identified through these assays that act as active agonists or antagonists.

The cloned NHL cDNA obtained through the method described above is an expression vector (pcDNA3.n) containing a suitable promoter and other suitable transcriptional regulatory elements.
eo, pcDNA3.1, pCR2.1, pBlueBacHis2 or pLI
Recombinant expression can be achieved by molecular cloning (such as TMUS28) and transfer into prokaryotic or eukaryotic host cells to produce recombinant NHL. Expression vectors are defined herein as the DNA sequences necessary for the transcription of cloned DNA and the translation of their mRNAs in a suitable host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, cyanobacteria, plant cells, insect cells and animal cells. Specially designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. A properly constructed expression vector should contain: an origin of replication for autonomous growth in the host cell, a selectable marker, a limited number of useful restriction enzyme sites, the 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 that triggers mRNA at a high frequency.
To determine the NHL cDNA sequence that yields optimal levels of NHL, cDNA molecules can be constructed, including but not limited to: a cDNA fragment containing the full length open reading frame for NHL and the protein of interest. Various constructs containing portions of the cDNA encoding only specific domains of or the rearranged domains of proteins. All constructs may be designed to contain none, all or part of the 5'and / or 3'untranslated regions of the NHL cDNA. NHL expression levels and activity can be determined after introduction of these constructs, alone and in combination, into appropriate host cells. After determining the NHL cDNA cassette that produced optimal expression in the transient assay, this N
The HL cDNA construct was used to bind mammalian cells, plant cells, insect cells, oocytes, bacteria,
And various expression vectors, including but not limited to yeast cells, including recombinant viruses. Techniques for such manipulations are described by Sambrook et al.
It has been mentioned above and is known and available to the person skilled in the art. Therefore, another aspect of the invention includes a host cell designed to contain and / or express a DNA sequence encoding an NHL protein. DN encoding an NHL-like protein
An expression vector containing A can be used for the expression of NHL in recombinant host cells. Such recombinant host cells can be cultured under suitable conditions to produce NHL or bioequivalent forms. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses. Commercially available mammalian expression vectors that are considered suitable for recombinant NHL expression are pcDNA3. neo (Invitrogen), pc
DNA3.1 (Invitrogen), pCI-neo (Promega),
pLITMUS28, pLITMUS29, pLITMUS38 and pLITM
US (New England Biolabs), pcDNAI, pcDN
AIamp (Invitrogen), pcDNA3 (Invitrogen)
, PMC1neo (Stratagene), pXT1 (Stratagene)
), PSG5 (Stratagene), EBO-pSV2-neo (ATCC
37593), pBPV-1 (8-2) (ATCC37110), pdBPV-.
MMTneo (342-12) (ATCC37224), pRSVgpt (AT
CC37199), pRSVneo (ATCC37198), pSV2-dhf
r (ATCC37146), pUCTag (ATCC37460), and IZD
35 (ATCC 37565), but is not limited thereto. A variety of bacterial expression vectors may also be used to express recombinant NHL in bacterial cells.
Commercially available bacterial expression vectors considered suitable for recombinant NHL expression are pCR2.
1 (Invitrogen), pET11a (Novagen), λgt11 (
Invitrogen), and pKK223-3 (Pharmacia), but are not limited thereto. In addition, various fungal cell expression vectors may also be used to express recombinant NHL in fungal cells. A commercially available fungal cell expression vector that is considered suitable for recombinant NHL expression is pYES2 (Invitrogen).
) And Pichia expression vector (Invitrogen). In addition, various insect cell expression vectors can also be used in recombinant N cells in insect cells.
It can be used to express HL. Commercially available insect cell expression vectors that are considered suitable for recombinant NHL expression are pBlueBacIII and pBlueBacHis.
2 (Invitrogen), and pAcG2T (Pharmingen), but are not limited thereto.

Recombinant host cells include bacterial cells such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines from bovine, porcine, simian and rodents, And prokaryotic or eukaryotic cells, including but not limited to insect cells, including but not limited to cell lines from Drosophila and silkworm. For example, one insect expression system is a baculovirus expression vector (pAcG2T, Pharminge
n) is used alongside Spodoptera frugiperda (Sf21) insect cells (Invitrogen). Also, considered appropriate, mammalian species that are commercially available, L cells ^{L-M (TK -) (} ATCC CCL 1.3), L cells L-M (ATCC CCL1.2), Saos-2 (ATCC HTB -85
), 293 (ATCC CRL1573), Raji (ATCC CCL86)
, CV-1 (ATCC CCL70), COS-1 (ATCC CRL 165).
0), COS-7 (ATCC CRL1651), CHO-K1 (ATCC C
CL61), 3T3 (ATCC CCL92), NIH / 3T3 (ATCC C
RL1658), HeLa (ATCC CCL2), C127I (ATCC C
RL1616), BSC-1 (ATCC CCL26), MRC-5 (ATCC
CCL171) and CPAE (ATCC CCL209), but are not limited thereto.

As disclosed in Chapter 1 of the Examples, a 115 kb BAC clone (Genom
e Systems) and subjected to restriction and sequence analysis. M68 / DcR3, NHL, SCLIP and A at chromosomal location 20q13.3
Four genes were identified, including RP (Figure 5A). This BAC clone, hbm1
The nucleotide sequence of 68 is shown below:

[0066]

[Chemical 5]

The present invention also relates to the 5 ′ regulatory region, exons, introns and human NHL of the present invention.
It relates to the part of SEQ ID NO: 3 with the 3'untranslated region containing the gene. Such regulatory sequences can be found within various regions of this 115 kb fragment. The 5'portion of SEQ ID NO: 1 begins at nucleotide 47095 of SEQ ID NO: 3; the starting ATG of human NHL is nucleotides 46887-48689 of SEQ ID NO: 3 and ends at "
The "TAG" codon is at nucleotides 84855-84857, while the 3'end of SEQ ID NO: 1 (GCAGTGCCC) as disclosed herein is nucleotide 85.
Corresponding to 308-85316. To this end, one preferred aspect of the invention is
An isolated genomic fragment (one or more) comprising about nucleotide 47000 to about nucleotide 85500 of SEQ ID NO: 3, said fragment comprising a portion of a genomic clone encoding an mRNA transcript responsible for human NHL. Including (Fig. 5A
-See B). The genomic sequence encodes an NHL containing 35 exons.
A particularly preferred aspect of the invention is a human genomic fragment (one or more) comprising from about nucleotide 47095 to about nucleotide 85316 of SEQ ID NO: 3. As mentioned with respect to SEQ ID NO: 1, the invention also relates to a DNA vector and a recombinant host comprising at least part of SEQ ID NO: 3. Portions of the 115 kb genomic fragment can be accommodated in multiple vectors / hosts to optimize handling of the DNA sequence within SEQ ID NO: 3. Therefore, the invention provides an isolated genomic sequence set forth as SEQ ID NO: 3, a region of SEQ ID NO: 3 that includes coding and non-coding regions of human NHL, and NH.
The cis sequence in SEQ ID NO: 3 that affects the regulation of transcription of one or more genes located within this 115 kb human genomic fragment, including regulatory regions that affect the levels of L, M68 / DcR3, SCLIP and ARP. Acting sequence. As mentioned above, this region of chromosome 20 (20q13.3) is associated with tumor growth. Therefore, one aspect of the present invention also includes, as an example, this 115 kb genomic sequence as a target to antagonize the action of transcription factors that upregulate the expression of genes that normally have an important role in tumor growth. The use of one or more regulatory regions of Alternatively, one can select compounds that interact with specific cis-acting sequences that upregulate genes in this region if upregulation results in reduced tumor growth.

The present invention is also directed to methods of screening for compounds that modulate the expression of DNA or RNA encoding the NHL protein. Compounds that modulate these effects can be DNA, RNA, peptides, proteins, or non-proteinaceous organic substances. Compounds may be modulated by enhancing or attenuating the expression of NHL-encoding DNA or RNA or the function of NHL-based proteins. Compounds that modulate the expression of the DNA or RNA encoding NHL or its biological function can be detected by various assays. The assay can be a simple "yes / no" assay to determine if there is a change in expression or function. The assay can be made quantitative by comparing the expression or function of the test sample to the level of expression or function in a standard sample. NHL, antibody to NHL, or modified NHL
A kit containing the can be prepared by known methods for such use.

Using the DNA molecules, RNA molecules, recombinant proteins and antibodies of the present invention, NHL
Can be screened and levels can be measured. Recombinant proteins, DNA molecules, RNA molecules and antibodies are compatible with kit formats suitable for NHL detection and typing. Such a kit would include a compartmentalized carrier suitable for holding at least one container in a strictly localized condition. The carrier will further include reagents such as recombinant NHL or anti-NHL antibodies suitable for detecting NHL. The carrier may also include means for detection such as labeled antigen or enzyme substrates and the like.

The assay described above can be performed on cells transiently or stably transfected with NHL. Expression vectors can be introduced into host cells through any of a number of techniques, including but not limited to transformation, transfection, protoplast fusion, and electroporation. Transfection is intended to include any method known in the art for introducing NHL into a test cell. For example, transfection is transfection via calcium phosphate or calcium chloride,
Includes lipofection, infection with retroviral constructs containing NHL, and electroporation. Cells containing expression vectors are analyzed individually to determine if they produce the NHL protein. Identification of NHL-expressing cells was performed using anti-NH
It may be carried out by several means, including but not limited to immunoreactivity with L antibodies, labeled ligand binding, the presence of host cell associated NHL activity.

The specificity of binding of compounds showing affinity for NHL is demonstrated by measuring the affinity of the compounds for recombinant cells expressing NHL. Screening of human NHL expression and compounds that bind to NHL or bind to known radiolabeled ligands of NHL provides an effective method for rapid selection of compounds with high affinity for NHL. . Such ligands do not necessarily have to be radiolabeled and may be non-isotopic compounds that can be used to replace bound radiolabeled compounds or can be used as activators in functional assays.
The compounds identified by the above methods are likely to be NHL agonists or antagonists and may be peptides, proteins, or non-proteinaceous organic molecules.

Accordingly, the present invention is directed to methods for screening for compounds that modulate the expression of DNA or RNA encoding the NHL protein as well as compounds that affect the function of the NHL protein. Methods for identifying other receptor agonists and antagonists are known in the art and can be adapted to identify NHL agonists and antagonists. For example, Cascieri et al. (1992, Mo
lec. Pharmacol. 41: 1096-1099) describe a method for identifying substances which inhibit an agent binding to the rat neurokinin receptor, and thus are potential agonists or antagonists of the neurokinin receptor. Such a method involves the use of an expression vector containing the rat neurokinin receptor in COS.
Transfecting cells, allowing the transfected cells to grow for a period of time sufficient for expression of the neurokinin receptor, collecting the transfected cells, and known radiolabeling of the neurokinin receptor in the presence or absence of the substance. Suspending the cells in an assay buffer containing the agent and then measuring the binding of the known radiolabeled agent of the neurokinin receptor to the neurokinin receptor. A substance is a potential agonist or antagonist of the neurokinin receptor if the amount of binding of the known agent is less in the presence of the substance than in the absence of the substance. When measuring the binding of a substance such as an agonist or an antagonist, such binding can be measured by using a labeling substance or a labeling agent. The substance or agent may be labeled by any convenient method known in the art, eg radioactively, fluorescently or enzymatically.

The invention therefore includes an assay by which a modulator of NHL is identified. As mentioned above, methods for identifying agonists and antagonists are known in the art and can be adapted to identify compounds that affect in vivo levels of NHL. Accordingly, the invention includes a method for determining whether a substance is a potential modulator of mammalian NHL, which method comprises: (a) transfecting the cell with an expression vector that directs the expression of NHL in the cell. Thereby providing a test cell, (b) contacting the test cell with the substance, (c) measuring the amount of the substance bound to NHL, and (d) the amount of the substance bound to the NHL in the test cell calculated as NHL. Or comparing the amount of the substance bound to control cells not transfected at that portion, and if the amount of substance bound is greater in the test cell compared to the control cells, the substance binds to NHL be able to.

The conditions under which step (b) of such a method is carried out are those typically used in the art for testing protein-ligand interactions, eg physiological pH, generally such as PBS. Salt conditions, such as those typified by the buffers used or in tissue culture medium, temperatures of about 4 ° C to about 55 ° C.

The assay described above can be performed on cells transiently or stably transfected with NHL. Transfection is intended to include any method known in the art for introducing NHL into a test cell. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with retroviral constructs containing NHL, and electroporation.

When measuring the binding of a substance or agent to NHL, such binding can be measured by using a labeling substance or labeling agent. The substance or agent may be labeled by any convenient method known in the art, eg radioactively, fluorescently or enzymatically.

The specificity of binding of compounds having an affinity for NHL is therefore demonstrated by measuring the affinity of the compounds for recombinant cells expressing cloned receptors or membranes from these cells. . Screening for compounds that bind to cloned receptor expression and NHL binding or known radiolabeled ligands of NHL to these cells is effective for rapid selection of compounds with high affinity for NHL. To provide a simple method. Such ligands do not necessarily have to be radiolabeled and may be non-isotopic compounds that can be used to replace bound radiolabeled compounds or can be used as activators in functional assays.
It is also possible to construct assays in which compounds are tested for their ability to modulate helicase activity in in vitro or in vivo based assays. The compounds identified by the above methods are likely to be NHL agonists or antagonists and may be peptides, proteins, or non-proteinaceous organic molecules. As mentioned elsewhere in this specification, compounds modulate by enhancing or attenuating the expression of NHL-encoding DNA or RNA, or by acting as agonists or antagonists of NHL receptor proteins. sell. Again, these compounds that modulate the expression of DNA or RNA encoding NHL or its biological function can be detected by various assays. The assay can be a simple "yes / no" assay to determine if there is a change in expression or function. The assay can be made quantitative by comparing the expression or function of the test sample to the level of expression or function in a standard sample.

Expression of NHL DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNAs can be efficiently translated in cell-based systems including, but not limited to, microinjection into frog oocytes, including but not limited to wheat germ extract and reticulocyte extract. It is efficiently translated even in various cell-free systems, and microinjection into frog oocytes is preferable.

After expression of NHL in the host cell, the NHL protein is recovered to produce the active form of NHL.
L protein can be provided. Several NHL protein purification procedures are available and suitable for use. Recombinant NHL proteins may be purified from cell lysates and extracts by various combinations or individual applications of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography and hydrophobic interaction chromatography. In addition, recombinant NHL proteins can be separated from other cellular proteins by the use of immunoaffinity columns made of monoclonal or polyclonal antibodies specific for full-length NHL protein or polypeptide fragments of NHL protein.

Polyclonal or antibody monoclonals may be raised against NHL or against synthetic peptides (usually about 9 to about 25 amino acids in length) from the portion of NHL disclosed in SEQ ID NO: 2. Monospecific antibodies to NHL have been purified from mammalian antisera containing antibodies reactive to NHL, or Kohler and Mi.
Is prepared as a monoclonal antibody reactive with NHL using the procedure of Istein (1975, Nature 256: 495-497). Monospecific antibody as used herein is defined as a single antibody species or a multi-antibody species with uniform binding properties for NHL. Homogenous binding, as used herein, refers to the ability of the antibody species to bind a specific antigen or epitope, such as those associated with NHL as described above. Human NHL-specific antibodies are available in mouse, rat, guinea pig, rabbit,
It is elicited by immunizing animals such as goats, horses and the like with appropriate concentrations of NHL protein or synthetic peptides made up of portions of NHL with or without an immunoadjuvant.

Pre-immune serum is collected prior to the first immunization. Each animal receives from about 0.1 mg to about 1000 mg of NHL protein with an acceptable immunoadjuvant.
Such an acceptable adjuvant is Corynebacterium pa
Includes, but is not limited to, Freund's complete, incomplete, Freund's alum precipitate, including rvum and tRNA, water-in-oil emulsion. Initial immunity is subcutaneous (SC
), NHL protein or a peptide fragment thereof in a multi-site, preferably Freund's complete adjuvant by the intraperitoneal (IP) or both routes. Blood is drawn from each animal at regular intervals, preferably once a week, to determine antibody titers. After the initial immunization, animals may or may not be given booster injections. Animals receiving booster injections are generally given an equivalent amount of NHL in Freund's incomplete adjuvant by the same route. Booster injections are given at approximately 3 week intervals until maximal titers are obtained. Approximately 7 days after each booster immunization, or approximately weekly after each immunization, animals are bled to collect serum and aliquots are stored at approximately -20 ° C.

An inbred mouse, preferably Balb / c, is immunized with NHL protein to produce a monoclonal antibody (mAb) reactive with NHL. Mice were dosed by the IP or SP route to approximately 0.5 m incorporated into an acceptable equal volume of adjuvant as described above.
Immunize with about 1 mg to about 100 mg, preferably about 10 mg of NHL protein in 1 buffer or saline. Freund's complete adjuvant is preferred. Mice are given an initial immunization on day 0 and rested for about 3 to about 30 weeks. Immunized mice are administered by intravenous (IV) route from about 1 to about 1 in a buffer such as phosphate buffered saline.
One or more booster immunizations of 00 mg NHL are performed. Spleens are excised from immunized mice by standard procedures known in the art to obtain lymphocytes, preferably splenic lymphocytes, from antibody positive mice. The splenic lymphocytes are mixed with a suitable fusion partner, preferably myeloma cells, under conditions that allow the formation of stable hybridomas to produce hybridoma cells. Fusion partners include, but are not limited to, mouse myeloma, P3 / NS1 / Ag4-1; MPC-11; S-194 and Sp2 / 0, with Sp2 / 0 being preferred. Antibody-producing cells and myeloma cells,
Fuse in polyethylene glycol of about 1000 molecular weight at a concentration of about 30% to about 50%. The fused hybridoma cells are prepared by procedures known in the art.
Selection by growth in Dulbecco's Modified Eagle Medium (DMEM) supplemented with hypoxanthine, thymidine and aminopterin. Supernatants were collected from growth positive wells on days 14, 18 and 21 and solid phase radioimmunoassay using NHL as the antigen (S
Screening for antibody production by immunoassays such as PIRA). The cultures are also tested in the Octaronie precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody-positive wells were transferred to MacPherson, 197.
3, "Soft Agar Technologies", Tiss
From ue Culture Methods and Applications, it is cloned by a method such as Kruse and Paterson's editing, Academic Press's soft agar method.

Balb / c mice primed with pristane were injected with the monoclonal antibody in vivo by injection of about 0.5 ml / mouse containing about 2 × 10 ⁶ to about 6 × 10 ⁶ hybridoma cells about 4 days after the initial immunization. To make. About 8-1 of cell introduction
Ascites fluid is collected 2 days later and the monoclonal antibody is purified by techniques known in the art.

In vitro generation of anti-NHL mAbs is performed by growing hybridomas in DMEM with about 2% fetal bovine serum to obtain sufficient quantities of specific mAbs. The mAb is purified by techniques known in the art.

Antibody titers in ascites or hybridoma cultures can be assayed for various serological conditions including, but not limited to, precipitation reactions, passive agglutination reactions, enzyme-linked immunosorbent antibody (ELISA) techniques and radioimmunoassay (RIA) techniques. Alternatively, it is measured by an immunological assay. The agitation assay is used to detect the presence of NHL in body fluids or tissues and in cell extracts.

It will be readily apparent to those skilled in the art that the methods described above for making monospecific antibodies can be used to make antibodies specific for the NHL peptide fragment or the respective full length NHL. .

For example, N may be used so that the antibody forms a covalent bond with the agarose gel bead support.
-Hydroxysuccinimide ester pre-activated gel carrier, Affigel
An antibody is added to -10 (Biorad) to prepare an NHL antibody affinity column. The antibody is then attached to the gel through an amide bond with the spacer arm. The remaining activated ester is then quenched with 1M ethanolamine HCl (pH 8). The column is washed with water and then 0.23 M glycine HCl (pH 2.6) to remove unconjugated antibody or foreign protein. Next, phosphate buffered saline (p
The column is equilibrated in H7.3) and the cell culture supernatant or cell extract containing full length NHL or NHL protein fragment is slowly passed through the column. Thereafter the optical density (A _{28 0)} is washed column until reduced to background with phosphate buffered saline,
The protein is eluted with 0.23M glycine HCl (pH 2.6). Then purified NHL
The protein is dialyzed against phosphate buffered saline.

Pharmaceutically useful compositions containing modulators of NHL may be formulated according to known methods, such as by mixing pharmaceutically acceptable carriers. Examples of such carriers and methods of formulation may be found in "Remington's Pharm Chemistry.
"Medical Sciences)". In order to form a suitable pharmaceutically acceptable composition for effective administration, such composition comprises a protein, DNA, RNA, a modified NHL, or an NHL effect including a tyrosine kinase activator or inhibitor. It will include an effective amount of the substance or antagonist.

The therapeutic or diagnostic composition of the present invention is administered to an individual in an amount sufficient to treat or diagnose a disease. The effective amount can vary depending on various factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.

The pharmaceutical composition may be provided to the individual by a variety of routes including subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” refers to a molecule that contains additional chemical moieties not normally part of the base molecule. Such components may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively, the component may diminish the adverse side effects of the base molecule or reduce the toxicity of the base molecule. Examples of such ingredients are described in various texts such as "Remington's Pharmaceutical Chemistry."

The compounds identified according to the methods disclosed herein can be used alone in appropriate administration. Alternatively, simultaneous or sequential administration of other agents may be desirable.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel therapeutic methods of the present invention. The compositions containing a compound identified according to the present invention as an active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, oral dosage forms such as tablets, capsules (including time-controlled and sustained release formulations, respectively), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, Alternatively, the compound can be administered by injection. Similarly, intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular forms can all be administered, all of which are well known to those of ordinary skill in the pharmaceutical arts. use.

Conveniently, the compounds of the invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times daily. In addition, the compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using the forms of transdermal patches well known to those of ordinary skill in that art. For administration in the form of transdermal delivery systems, it will be appreciated that administration will be continuous rather than intermittent throughout the dosing regimen.

For combination treatment with two or more active ingredients in which the active ingredients are in separate administration formulations, the active ingredients can be administered simultaneously or each can be administered separately with a time difference.

Dosage regimens using the compounds of the invention include patient type, species, age, weight, sex and medical condition; severity of the condition being treated; route of administration; patient renal, hepatic and cardiac function; and Selection will depend on a variety of factors, including the particular compound used. A physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal details in achieving drug concentrations within the range that yields efficacy without toxicity require a regimen based on the kinetics of the drug's availability to target sites. This includes consideration of drug distribution, balance and excretion.

The present invention is also useful in examining the ability of various compounds to act as modulators of NHL or alternative functional NHLs in vivo or in vitro by providing cells for culture. Regarding transgenic animals. With respect to the transgenic animals of the present invention, reference is made to transgenes and genes. As used herein, a transgene is a genetic construct that includes a gene. The transgene is integrated into one or more chromosomes within the cell of the animal by methods known in the art. Once integrated, the transgene is transported to at least one position within the chromosome of the transgenic animal. Of course, a gene is a nucleotide sequence that encodes a protein, such as one or a combination of the cDNA clones described herein. Genes and / or transgenes may also include genetic regulatory and / or structural elements known in the art. One type of target cell for transgene transfer is embryonic stem cells (ES
). ES cells are obtained from preimplantation embryos that have been cultured in vitro and fused with the embryo (Evans et al., 1981, Nature 292: 154-156; Bra.
dley et al., 1984, Nature 309: 255-258; Gossle.
r et al., 1986, Proc. Natl. Acad. Sci. USA 83:90
65-9069; and Robertson et al., 1986 Nature 322.
: 445-448). The transgene can be efficiently introduced by various standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resulting transformed ES cells can then be combined with undifferentiated embryo cells from non-human animals. The ES cells introduced thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaenisch, 1988, Science 240: 1468-147).
4). Also in the wild-type background and in one of the NHL subunits.
One or more C. deficient transgenic or knockout invertebrates expressing the NHL transgene in elegans mutants (eg C
． It will be within the skill of the artisan to produce elegans). These organisms will further be useful in determining the predominant negative effects of NHL, as well as in selecting from compounds that modulate this effect.

The present invention also relates to non-human transgenic animals that are heterozygous for the functional NHL gene native to that animal. As used herein, functional is used to describe a gene or protein that, when present in a cell or in vitro system, functions normally as if it were in its native or native state or environment. An animal of this aspect of the invention is the N in an animal that has only one functional copy of the gene.
It is useful for examining retinal-specific expression or activity of HL. Such animals can also be grown in vivo, or in vitro by providing cells for culture.
It is useful to investigate the ability of various compounds to act as modulators of HL activity. Again, as used herein, a modulator is a compound that causes a change in NHL expression or activity, or a change in the effect of the interaction of NHL with its ligand or other protein. In one embodiment of this aspect, such animals are used in a method for the production of additional animals lacking a functional native NHL gene. In another embodiment, animals of this aspect are treated with natural N
Use in a method for preparing an animal expressing a non-natural NHL gene without expression of the HL gene. In a particular embodiment, the non-human animal is a mouse. In a further embodiment, the non-natural NHL is a wild-type human NHL disclosed herein, or some other bioequivalent form of the human NHL gene as also disclosed herein.

With respect to the transgenic animals of the present invention, reference is made to transgenes and genes. As used herein, a transgene is a genetic construct that includes a gene. The transgene can be expressed in the cells of an animal by methods known in the art.
It is integrated into one or more chromosomes. Once integrated, the transgene is transported to at least one position within the chromosome of the transgenic animal. Of course, a gene is a nucleotide sequence that encodes a protein, such as human or mouse NHL. Genes and / or transgenes may also include genetic regulatory and / or structural elements known in the art.

Another aspect of the invention is a non-human animal embryo lacking native NHL expression.
This embryo is useful in studying the effects of NHL deficiency on developing animals. In a particular embodiment, the animal is a mouse. Animal embryos are also useful as a source of cells lacking a functional native NHL gene. Such cells are useful in in vitro culture studies in the absence of NHL.

One aspect of the invention is a method for obtaining an animal whose cells lack the functional gene NHL that is native to the animal. Such methods include providing the gene in the form of a transgene with an altered form of the NHL gene native to the animal and targeting such transgene to the animal's chromosome at the location of the native NHL gene.
Transgenes can be introduced into embryonic stem cells by a variety of methods known in the art, including electroporation, microinjection, and lipofection. Next, the cells carrying the transgene are injected into undifferentiated embryo cells, which are then transplanted into pseudopregnant animals. In an alternative embodiment, transgene-targeted embryonic stem cells can be co-incubated with fertilized eggs or morula and then transferred into females. After pregnancy, the animals obtained are chimeric fo
Under) transgenic animals. The founder animal, in a further embodiment, has been bred with a wild-type animal and is heterozygous for the altered NHL gene.
It can be used to produce animals. In a further embodiment, these heterozygous animals are interbreeded to obtain non-viable transgenic embryos that are homozygous for the somatic and germline altered NHL gene and thus lack a functional NHL gene. You can In other embodiments, heterozygous animals can be used to generate cell lines. In a preferred embodiment, the animal is a mouse.

A further aspect of the invention is a transgenic non-human animal expressing non-natural NHL in a background free of natural NHL. In certain embodiments, the absent background is created by producing animals with altered native NHL genes that are non-functional or knockout. The animal is heterozygous (ie, has a different allelic expression on each pair of chromosomes of the diploid genome) or homozygous (ie has the same expression on each of a pair of chromosomes of the diploid genome) for the altered NHL gene. ), And may be hemizygous (ie, a gene expressed on only one of a pair of chromosomes in the diploid genome) or homozygous for the non-natural NHL gene. In a preferred embodiment, the animal is a mouse. In particular embodiments, the non-natural NHL gene can be a mutant allele, including mutant alleles associated with wild type or disease. In a further embodiment, the non-natural NHL is human NHL. In a further embodiment, the non-natural NHL is operably linked to a promoter. As used herein, operably linked is used to describe a functional connection between two elements whose orientation with respect to each other may change. It will be apparent in the art that in this particular case, the promoter may be positioned at various distances from the coding sequence within the genetic construct and operably linked to the coding sequence of the gene which directs the expression of the coding sequence.

One aspect of the invention is a method of producing a transgenic animal that comprises a transgene that includes a non-natural NHL gene in a background free of natural NHL. Such methods include providing a transgenic animal of the invention, the cells of which are heterozygous for a natural gene encoding a functional NHL protein and an altered natural NHL gene. These animals are crossed with transgenic animals of the invention that are hemizygous for transgenes, including non-natural NHL genes, and are heterozygous for altered natural NHL genes and hemizygous for non-natural NHL genes. Get the animals. The latter animals are crossbred to obtain animals that are homozygous or hemizygous for the non-natural NHL and homozygous for the altered natural NHL gene. In a particular embodiment, the cell line is generated from any of the animals produced in such method steps.

The transgenic animals and cells of the invention are useful in determining the in vivo function of non-native NHL in the animal's central nervous system and other tissues. Such animals are also useful in examining tissue and transient specific expression patterns of non-natural NHL throughout the animal. Animals are also useful in determining the ability of various forms of wild-type and mutant alleles of non-natural NHL to rescue a natural NHL-absence defect. Animals may also be used to identify and study the ability of various compounds to act as modulators of non-natural NHL expression or activity for in vitro studies by providing cells for in vivo or culture. It is useful.

As used herein, a “target gene” or “knockout” (KO) is DNA introduced into the germline of a non-human animal by human intervention, including, but not limited to, the methods described herein. It is an array. The target gene of the present invention comprises a nucleic acid sequence designed to specifically alter a cognate endogenous allele. Changed N
The HL gene should not completely encode the same NHL as is native to the host animal, and its expression product may be slightly or significantly altered, or even absent. If it is useful to express the non-natural NHL gene in transgenic animals in the absence of the natural NHL gene, then the altered N
It is preferred that the HL gene does not introduce a lethal knockout phenotype into the animal. However, more mildly modified NHL genes may be useful and are within the scope of the invention.

The target cell type for transgene transfer is embryonic stem cells (ES). ES
Cells are obtained from preimplantation embryos that have been cultured in vitro and fused with the embryo (Evans).
Et al., 1981, Nature 292: 154-156; Bradley et al., 1
984, Nature 309: 255-258; Gossler et al., 1986.
, Proc. Natl. Acad. Sci. USA 83: 9065-9069.
And Robertson et al., 1986 Nature 322: 445-44.
8). The transgene can be efficiently introduced by various standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resulting transformed ES cells can then be combined with undifferentiated embryo cells from non-human animals. The ES cells introduced thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaeni).
sch, 1988, Science 240: 1468-1474).

Methods for assessing targeted recombination events as well as for generating knockout mice are readily available and known in the art. Such a method is
DNA (Southern) hybridization, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) to detect target alleles
And Western blots for detecting DNA, RNA and proteins, but are not limited thereto.

The following examples are presented to illustrate, but not limit, the invention.

Example 1 Characterization of a DNA molecule encoding NHL Identification of M68 / DcR3-Human osteoprotegerin (OPG) sequence (Acc.
# U94332) was used to search Genbank with the TBLASTN and TFASTX3 programs to identify members of a novel gene family. OP
Two EST sequences (GenBa) showing sequence similarity with the cysteine repeats of the G sequence.
nk Acc. # AA155701 and AA025672) were identified. These EST sequences were then used to identify additional EST sequences to form one EST cluster (GenBank Acc. #S aa577703, aa60.
3704, aa613366, aa158406, w67560, aa3258
43, aa155646, aa025673, aa514270, m91489.
). Colon and germ cell tumor libraries (Research Geneti)
cs, Inc. 2) was further characterized. DNA sequence analysis revealed two alternative spliced forms of the 5'-end UTR of M68 / DcR3. M68 / DcR3 open reading frame,
Confirmed by sequence analysis of clones obtained by PCR cloning from a normal human cDNA library (Clontech).

Identification and Sequencing of M68 / DcR3 BAC-To further elucidate the gene structure of M68 / DcR3, human "Down to the Well" ^TM genomic bacterial artificial chromosome (BAC) library (Genome Systems)
, Inc. ) Was used according to the manufacturer's protocol to obtain genomic DNA.
Two sets of PCR primers, C68.36F: 5'-CACAGGTTC
AGCATGTTTGTGCGTC-3 '(SEQ ID NO: 4) and C68.275R:
5'-CACAGTCCCTGCTGGCCTCTGTCTA-3 '(SEQ ID NO: 5), and E68.715F: 5'-CAGGACCATCTCCATCAAGA.
GGCTGC-3 ′ (SEQ ID NO: 6) and E68.972R: 5′-AATAAGA
PCR using GGGGGGCCAGGATCAGTGC-3 ′ (SEQ ID NO: 7)
Reactions were performed to identify positive wells containing the full length M68 / DcR3 gene. The PCR conditions used were 94 ° C for 9 minutes (94 ° C, 30 seconds, 68 ° C, 3 minutes).
35 cycles of, followed by 72 ° C. for 10 minutes. Two positive BAC clones were identified and characterized by restriction digest and BAC end sequence analysis, among which hbm
168 was selected for shotgun sequencing.

A shotgun library for BAC hbm168 was constructed using conventional strategies. Briefly, two 150 ml bacterial cultures were combined and purified using a modified protocol of the Plasmid-Maxi kit (QIAGEN),
It was then purified on a CsCl gradient. After butanol extraction and isopropanol precipitation, BAC DNA was sprayed at 10 psi for 60 seconds to generate randomly sheared fragments. After the ethanol precipitation reaction, the fragment was end-repaired using T4 polymerase (Promega), and the BstXI adapter (Invitrogen) was ligated overnight. Excess unbound adapters were removed and size selection was performed using a cDNA size selection column (Life Technologies, Inc.) to generate genomic fragments within the size range of 1500 to 3000 bp. The adapter-ligated fragment was modified with pBlueScript SK ⁺ vector (Strata
gene) and XL2-Blue ultracompetent cells (St
(Ratagene). Isolated about 1000 clones,
The plasmid was purified using the Turbo miniprep kit (QIAGEN), and both ends of the plasmid were added to the BigDye terminator kit (Perkin-E).
lmer). Directed sequence determination strategy (directed sequence)
to fill the single-stranded and gap regions using Ph.
Sequence data was assembled using red / Phrap / Consed.

[0112]   Identification and sequencing of NHL-obtaining a genomic clone for the NHL gene,
Sequenced. For GRAIL2 and adjacent 4.5 kilobase region of chromosome 4
Identifies transcripts through exon prediction using sequence alignments (88% sequence identity).
Decided The complete exon structure of NHL was then confirmed by RT-PCR analysis.
It was The exon structure is based on the human colorectal adenocarcinoma cell line, SW480 (Clontech).
) Was confirmed by RT-PCR using poly A RNA from In exon
Primers were designed based on the expected genomic sequence. SW480 poly
For A RNA, 94 ° C for 12 minutes, (94 ° C, 30 seconds, 60 ° C, 30 seconds
, And 68 cycles of 68 ° C. for 2-6 minutes, followed by Taq at 68 ° C. for 7 minutes
The RT-PCR reaction was performed using standard conditions by Gold Enzyme.
. Most of the sequence confirmation was performed by RT-PCR, but between the exons 1 and 2
The first bond is by 5'RACE and the bond between exons 26-29 is RC.
Confirmed by CA. The primers used were as follows:   Exon binding      Confirmation by primer   H01 / H02 hdkw (5'RACE)   H02 / H03 hdii, hdiz   H03-H09 hdja, jdi, hdja, hdjb   H09-H13 hdja, hdie   H13-H18 hdje, hdjf   H18-H23 hdjg, hdjh   H23-H26 hdji, hdjj   H26-H29 hdkv, r543 (RCCA)   H29-H31 hdij, hdmu, hdnd, hdne   H31 / H32 hdij, hdmu   H32 / H34 hdip, hdil, hdmv, hdik, hdli   H34 / H35 hdng, hdnh   HDID-5'- GTGAATGGCATCCTGGAGAG-3 '(SEQ ID NO: 8)   HDIE-5'- GTCTCCAGGCAGCTCAACAG-3 '(SEQ ID NO: 9)   HDIJ-5'- ACCCTGTCCCTCCTGTCTGA-3 '(SEQ ID NO: 10)   HDYY-5'- AGACCCTAAGATGTTCGGAG-3 '(SEQ ID NO: 11)   HDIZ-5'-GATGACCTGTGTGAGTTGCG-3 '(SEQ ID NO: 12)   HDJA-5'-CGCAACTCACACAGGTCATC-3 '(SEQ ID NO: 13)   HDJB-5'- GGAGTCAGGTCAAAGGATGC-3 '(SEQ ID NO: 14)   HDJC-5'-GCATCCTTTGACCTGACTCC-3 '(SEQ ID NO: 15)   HDJD-5'- GGTCTGAAACGTGATCTGGG-3 '(SEQ ID NO: 16)   HDJE-5'- CCCAGATCACGTTTCAGACC-3 '(SEQ ID NO: 17)   HDJF-5'-CGATGATGTGTGGGTTCTCC-3 '(SEQ ID NO: 18)   HDJG-5'- GGAGAACCCACACATCATCG-3 '(SEQ ID NO: 19)   HDJH-5'-CGTGTCTGAGAAGTCCAGCC-3 '(SEQ ID NO: 20)   HDJI-5'-CGCTGGACTTCTCAGACACG-3 '(SEQ ID NO: 21)   HDJJ-5'- ACAGCATCTTCTCCACGCAC-3 '(SEQ ID NO: 22)   HDMU-5'- AGTCCTCTGGCTTTGCAGTG-3 '(SEQ ID NO: 23)   HDKV-5'-TGTGCGTGGAGAAGATGCTG-3 '(SEQ ID NO: 24)   HDKW-5'- GGCTGGAAAGGGAAGTCTAC-3 '(SEQ ID NO: 25)   HDND-5'-TGGTTCAGGTGCTCTTGGGG-3 '(SEQ ID NO: 26)   HDNE-5'-CGTGAAGCAGGAGTTGAGCC-3 '(SEQ ID NO: 27)   HDIK-5'- ATCTTGCTCTGGGTCTTCCC-3 '(SEQ ID NO: 28)   HDIL-5'-CACTGCAAAGCCAGAGGACT-3 '(SEQ ID NO: 29)   HDIP-5'-ATAAGCAAGACGACGACCTC-3 '(SEQ ID NO: 30)   HDLI-5'- CTATTCTGTTGGGTGGGTTC-3 '(SEQ ID NO: 31)   HDMV-5'- CGTGCCTCCTGTGCTTACCC-3 '(SEQ ID NO: 32)   HDNG-5'- CAGACCCCAAGGTAGCTCAG-3 '(SEQ ID NO: 33)   HDNH-5'- GGAAGACCCAGAGCAAGATC-3 '(SEQ ID NO: 34).

Amplification products were either purified from agarose gel and then subjected to direct sequencing, or for sequencing, TOPO PCR cloning vector (Invitroge).
n). A multiple sequence alignment to the known helicases showed that NHL contained all seven important helicase domains. BLAST analysis of the predicted 1,219 amino acid sequence (see FIG. 2, SEQ ID NO: 2) shows the DNA helicase RA
It shows about 26% sequence identity and 48% sequence similarity with the D3 / ERCC2 gene family (see Figure 3). Examining this sequence data, two partial human DNA
It shows that the clones (Acc. No. a108127 and ab029011) have been deposited. No. a108127 covers exons 25-35 and ab
029011 covers exons 9-35. Ab029011 differs not only at amino acid 240 of the full-length human NHL protein disclosed herein, but also at exon 35, and appears to be a fusion transcript with M68. This cd
NA has been isolated from brain tissue and is known to express rare transcripts.

Example 2 Northern Analysis of Human NHL Expression Messenger RNA (mRNA) was obtained from human brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes. Poly A ⁺ R
2 μg NA was applied to a denaturing formaldehyde 1% agarose gel in each lane and transferred to a charge modified nylon membrane. NHL cDNA 1174-1907
A probe was prepared using the 733 bp fragment derived from ³² P of this fragment
Labeled through the dCTP random priming method (Ambion). Exp
Hybridization was performed according to the manufacturer's protocol, except for the final wash at 55 ° C. in resHyb (Clontech).
The membrane was exposed to X-ray film using an intensifying screen at −80 ° C. overnight. Northern data are shown in FIG. Hybridization of the NHL probe to the approximately 4.4 kb transcript is observed. The 7.5 kb transcript is believed to suggest alternative splicing of NHL RNA.

Example 3 Chromosome Orientation To map the position of M68 / NHL in the human genome, primers MIT 6868F and C68.36F were used to map MIT GeneBrid.
A PCR reaction was performed on 93 clones of ge 4 panel (Research Genetics) and the results were submitted to MIT for analysis. M68 / D
cR3 was 28qR from 20q13.3, D20S173, and was mapped with rod score 13 to the most advanced telomere on chromosome 20. Stanford G
A similar procedure was performed for 83 clones of the 3-radiation hybrid panel and the PCR results were analyzed by the Stanford Genome Center for analysis.
Submitted to. Analysis with another pair of PCR primers specific for NHL yielded the same results. A normal human male fibroblast cell line, L136 (Coriell Cell Repository, Ca) for fluorescence in situ (FISH) analysis.
Mden, NJ) was arrested for mitosis with colcemid (10 μg / ml). Human chromosome 20 α-satellite probe (Vysis,
Downers Grove, IL) to Spectrum Orange dU
It was directly labeled with TP and used to identify chromosome 20. M68 BAC clones were nick translated into Spectrum Green d
Directly labeled with UTP (Vysis). The slide glass was counterstained with DAPI stain, and O was obtained using narrow blue and DAPI / TRITC filters.
It was observed under a lympus microscope. Fifty metaphase cells were counted, and it was confirmed that the M68 probe was located on the same chromosome as the human chromosome 20 probe. Radiation hybrid chromosome mapping has reconfirmed that it binds to the M68 locus at 20q13.3.

[Brief description of drawings]

1A shows a nucleotide sequence comprising an open reading frame encoding human NHL, ie the nucleotide sequence shown as SEQ ID NO: 1.

FIG. 1B shows a nucleotide sequence containing an open reading frame encoding human NHL, ie the nucleotide sequence shown as SEQ ID NO: 1.

[Fig. 2]   3 shows the amino acid sequence of human NHL as shown in SEQ ID NO: 2.

FIG. 3A shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members.

FIG. 3B shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members.

FIG. 3C shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members.

FIG. 3D shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members.

FIG. 3E shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members.

FIG. 3F shows an alignment of the amino acid sequences of human NHL to ERCC2 / RAD3 gene family members.

[Figure 4]   4 shows Northern blot analysis of NHL expression in multiple human tissues.

FIG. 5A   The genomic structure of the NHL gene is shown.

FIG. 5B shows the entire 115 kb genomic region containing the NHL, M68 / DcR3, SCLIP and ARP genes.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/00 C12N 5/00 A (72) Inventor Bai, Jiang New Jersey, 07065-0907, Lowway, East Lincarn Avenyu-126 (72) Inventor Metzker, Michael El United States, New Jersey 07065-0907, Lowway, East-Lincoln Avenyu 126 F term (reference) 4B024 AA01 BA07 CA04 GA11 HA01 4B050 CC01 CC04 DD11 EE10 LL01 4B065 AA26X AA72X AA90X AA93Y AB01 AC14 BA02 CA27 CA44

Claims (26)

[Claims] 1. A purified DNA molecule encoding a mammalian NHL protein. 2. Amino acid sequence: A purified DNA molecule according to claim 1, which encodes a human NHL protein, comprising: 3. An expression vector for expressing an NHL protein in a recombinant host cell, the expression vector comprising the DNA molecule according to claim 2. 4. A host cell expressing a recombinant NHL protein, which comprises:
A host cell containing the expression vector according to 1. 5. The host of step (a) under conditions that (a) transfect the expression vector of claim 3 into a suitable host cell and (b) express the NHL protein from the expression vector. A method of expressing an NHL protein in a recombinant host cell, comprising culturing the cell. 6. Amino acid sequence: A purified DNA molecule encoding the human NHL protein consisting of: 7. An expression vector for expressing an NHL protein in a recombinant host cell, the expression vector comprising the DNA molecule according to claim 6. 8. A host cell expressing a recombinant NHL protein, which comprises:
A host cell containing the expression vector according to 1. 9. A method for expressing an NHL protein in a recombinant host cell, comprising: (a) introducing the expression vector of claim 7 into a suitable host cell, and (b) expressing the NHL protein from the expression vector. A method comprising culturing the host cell of step (a) under conditions that allow it to be expressed. 10. A purified DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1. 11. An expression vector for expressing an NHL protein in a recombinant host cell, the expression vector comprising the DNA molecule of claim 10. 12. A host cell expressing a recombinant NHL protein, the host cell comprising the expression vector of claim 11. 13. A purified DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1. 14. An expression vector for expressing an NHL protein in a recombinant host cell, the expression vector comprising the DNA molecule of claim 13. 15. A host cell expressing a recombinant NHL protein, the host cell comprising the expression vector of claim 14. 16. The purified DNA of claim 13, which consists of the nucleotide sequence shown in SEQ ID NO: 1 from about nucleotide 828 to about nucleotide 4587.
molecule. 17. An expression vector for expressing an NHL protein in a recombinant host cell, the expression vector comprising the DNA molecule of claim 16. 18. A host cell expressing a recombinant NHL protein, the host cell comprising the expression vector of claim 17. 19. A substantially purified NHL protein comprising the amino acid sequence set forth in SEQ ID NO: 2. 20. A substantially purified NHL protein consisting of the amino acid sequence shown in SEQ ID NO: 2. 21. A substantially purified NHL protein comprising the amino acid sequence set forth in SEQ ID NO: 2, which comprises SEQ ID NO: 1 and is contained within a recombinant host cell.
The NHL protein, which is a product of an NA expression vector. 22. A method of identifying a modulator of NHL activity, comprising: (a) combining a test compound with an NHL protein comprising the amino acid sequence set forth in SEQ ID NO: 2, and (b) adding the test compound to the NHL protein. A method comprising measuring an effect. 23. An isolated DNA molecule comprising the nucleotide sequence set forth in SEQ ID NO: 3. 24. The isolated DNA molecule of claim 20, comprising from about nucleotide 47000 to about nucleotide 85500 of SEQ ID NO: 3. 25. The isolated DNA molecule of claim 23, which comprises from about nucleotide 47095 to about nucleotide 85316 of SEQ ID NO: 3. 26. The substantially purified NHL protein of claim 21, comprising about nucleotide 828 to about nucleotide 4587 set forth in SEQ ID NO: 1 and contained within a recombinant host cell. The NHL protein, which is a product of a DNA expression vector.