WO2002040718A2 - Method to identify genes associated with chronic myelogenous leukemia - Google Patents
Method to identify genes associated with chronic myelogenous leukemia Download PDFInfo
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- WO2002040718A2 WO2002040718A2 PCT/US2001/043781 US0143781W WO0240718A2 WO 2002040718 A2 WO2002040718 A2 WO 2002040718A2 US 0143781 W US0143781 W US 0143781W WO 0240718 A2 WO0240718 A2 WO 0240718A2
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Definitions
- Chronic myelogenous leukemia is a lethal disease of hematopoietic stem cells, characterized by a specific chromosomal translocation between human chromosome 9 and human chromosome 22.
- the chromosome resulting from this translocation is commonly referred to as the Philadelphia chromosome (Darnell et al., 1990).
- the c-abl gene ABL
- BCR c-bcr gene
- the translocation places the promoter distal three exons of ABL, including those elements which encode the tyrosine kinase domain, downstream of either the first or second exon of BCR (Chung and Wong, 1995).
- the product of the translocation between human chromosome 9 and human chromosome 22 is a chimeric gene, BCR- ABL, which encodes a fusion protein, often referred to as pl 85 BCR - ABL Qr p21 Q BCR - AB ⁇ depending upon the inclusion of the second exon of BCR (Bartram et al, 1983).
- pi 85 BCR_ABL causes acute leukemia, typically lymphoblastic; p210 BCR"AB usually causes CML, but can occasionally also cause acute leukemia.
- Ph + cells are able to differentiate and mature along the various myeloid lineages while retaining the capacity to function as their normal, unaffected counterparts.
- CML is characterized in its initial chronic phase by the circulation of malignant progenitors in the peripheral blood (Kantarjian et al., 1985). After three to five years, the disease transforms into a blast crisis, in which presumed additional genetic abnormalities prevent an early myeloid or lymphoid progenitor from differentiating (Clarkson and Strife, 1993; Daley and Ben Neriah, 1991; Deisseroth and Arlinghaus, 1991; Sawyers et al., 1991). In the chronic phase, the pool of malignant progenitors and precursors is also massively expanded (Kantarjian et al., 1985).
- ⁇ 210 BCR"AB Compared with the native pl45 ABL protein, which is found mainly in the cell nucleus, ⁇ 210 BCR"AB is located exclusively in the cytoplasm (VerfaiUie et al., 1997; VerfaiUie, 1998).
- the tyrosine kinase function and F-actin-binding function of p210 BCR"ABL is significantly elevated compared with that of the native p210 BCR protein (Konopka et al., 1989; VerfaiUie et al., 1997; VerfaiUie, 1998).
- the increased kinase activity and cytoplasmic location of the BCR- ABL gene product are essential elements of its transforming abilities (McWhirter et al., 1991).
- BCR- ABL Several in vitro and in vivo studies demonstrate that the presence of BCR- ABL is necessary and sufficient for transformation (Daley et al., 1990).
- the introduction of BCR- ABL cDNA into hematopoietic cell lines causes growth factor-independent growth in vitro and tumorigenicity in vivo.
- transplantation of murine stem cells transduced with BCR-ABL cDNA causes a CML-like phenotype, and transgenic expression of BCR-ABL causes a syndrome with myeloproliferative or acute leukemia-type characteristics.
- the invention provides a method for the differential isolation of nucleic acid sequences that are present in one nucleic acid population and not in another.
- the method is based upon employing a first population of cells which express a gene product that is associated with a particular phenotype or disease, and another (second) population of cells which does not express that gene product.
- the method comprises contacting nucleic acid from a first sample with nucleic acid from a second sample under hybridization conditions so as to form binary complexes.
- the first sample comprises nucleic acid from cells which express a protein that is associated with malignancy, e.g., hematopoietic stem or progenitor cell malignancy
- the second sample comprises nucleic acid from cells which do not express the protein. Then a nucleic acid molecule is identified which is present in the first sample which is not present in the second sample.
- the identified nucleic acid molecule is isolated and characterized.
- the first population of cells comprises a vector that encodes a first gene product such as a chimeric protein, e.g., p210 BCR"ABL , l ⁇ S 3 ⁇ , p230 BCR/ABL , TEL-ABL, PDGF-ABL, AML-ETO, PML-RAR ⁇ , MDS/EV1 and the like.
- a first gene product such as a chimeric protein, e.g., p210 BCR"ABL , l ⁇ S 3 ⁇ , p230 BCR/ABL , TEL-ABL, PDGF-ABL, AML-ETO, PML-RAR ⁇ , MDS/EV1 and the like.
- the vector also comprises a marker gene which encodes a second gene product that is detectable.
- the second population of cells preferably comprise a vector comprising the marker gene.
- Cells from each population that express the second gene product are selected or detected, e.g., by sorting in a FACS, and nucleic acid molecules are identified that are preferentially expressed in the first population.
- the identified nucleic acid molecules e.g., isolated RNAs or cDNAs, are then characterized, e.g., by sequencing.
- the method of the invention can be applied to identify genes associated with disease.
- the nucleic acid molecules of the invention are not limited to those identified by any particular method.
- CD34 + cells were transduced with an MS CV-retro virus vector containing either eGFP alone (eGFP) or BCR/ABL cDNA-LRES-eGFP (p210-eGFP).
- GFP + cells were FACS selected (> 90% purity), and subtractive hybridization performed between the two populations using the Clontech PCR-SelectTM System.
- cDNA clones that were expressed in p210-eGFP but not eGFP transduced CD34 + cells were sequenced and analyzed. Forty-one of the sequences did not encode a characterized human protein. Of these, at least 15 were closely homologous to expressed sequence tags (ESTs), at least 10 had homology to genomic clones for which no mRNA transcripts or proteins had been identified, while 1 sequence had no match in available genomic, RNA or protein databases. In addition, at least 22 sequences had significant homologies to known genes. These genes are involved in protein degradation, signal transduction, cell cycle regulation, and in RNA splicing.
- ESTs expressed sequence tags
- genes hypothesized to be downstream molecules for p210 BCR" ⁇ BL i.e., c-myb, c-KIT, c-rav and c-myc, were not identified by the method of the invention.
- SRPK1, Sty, Gu, SNRNP-G differentially expressed genes
- CML CD34 + cells and cell lines express a number of alternatively spliced proteins, including Pyk2, beta r B integrin, CSCP, and MPP1 (Verfaille et al., unpublished results; Deminger et al., 2000).
- the invention also provides isolated nucleic acid molecules comprising nucleic acid segments encoding polypeptides that are expressed in cells that express a protein, e.g., p210 BCR"ABL , that is associated with disease.
- the invention includes isolated nucleic acid molecules comprising an open reading frame comprising any one of SEQ ID NOs:l-79, or the complement thereof, or nucleic acid molecules which hybridize thereto, e.g., under moderate and/or stringent hybridization conditions.
- Preferred nucleic acid molecules comprise.
- an open reading frame comprising any one of SEQ ID NOs: 1, 3-4, 11, 14-17, 19, 22, 25, 27, 29, 32, 35, 37-39, 41, 44-47, 52-54, 57-58, 60-62, 64, 67- 79, the complement thereof, or nucleic acid molecules which hybridize thereto, e.g., under moderate and/or stringent hybridization conditions.
- Moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. (1989).
- stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50°C, or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% FicoU/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
- SSC sodium lauryl sulfate
- a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% FicoU/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
- Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.
- sequences of the nucleic acid molecules are useful as probes, to obtain full length sequences, i.e., a sequence that comprises an open reading frame that encodes a full length polypeptide, and in expression cassettes, as well as to prepare primers (oligonucleotides) for amplification or non-amplification- based methods to detect expression of the corresponding genes, e.g., using RT- PCR or linear amplification, in cells, such as in primary CML cells or CML lines, relative to normal cells.
- the invention also provides probes and primers comprising at least a portion of the nucleic acid molecules of the invention.
- the probes or primers of the invention are preferably detectably labeled or have a binding site for a detectable label.
- the probes or primers of the invention are at least about 7, more preferably at least about 15, but less than about 200, more preferably less than about 50, contiguous nucleotides bases having at least about 80% identity, more preferably at least about 90% identity, to the isolated nucleic acid molecules of the invention.
- Such probes or primers are useful to detect, quantify, isolate and/or amplify DNA strands that are related to the nucleic acid molecules of the invention.
- an expression cassette comprising an open reading frame comprising any one of the nucleic acid molecule of the invention operably linked to a promoter functional in a host cell, as well as a host cell, the genome of which is augmented with the expression cassette.
- Preferred host cells are vertebrate cells, e.g., mammalian cells.
- isolated polypeptides encoded by the nucleic acid molecules of the invention are provided.
- partial or full length sequences corresponding to the nucleic acid molecules of the invention in sense or antisense orientation may be employed to further characterize the role of the encoded gene(s) product in diseased and/or in normal cells.
- overexpression or aberrant expression e.g., expression which is spatially or temporally different, of one or more of the identified genes, may result in identifying the mechanism associated with transformation, e.g., p210 BCR" ⁇ B transformation, and identifying other molecular target(s) for therapy.
- agents that interact with that target including RNA or polypeptides, are employed to inhibit or prevent disease.
- antisense sequences or ribozymes specific for the RNA target, or agents that bind to the encoded polypeptide and inhibit its activity may be used to inhibit or prevent disease.
- a PCR-based subtractive hybridization method is employed to identify genes, the expression of which is altered in cells that express a gene associated with hematopoietic stem cell malignancy.
- Those identified genes are useful as probes or primers to detect expression of those genes in malignant and normal cells, as well as therapeutic targets, e.g., via antisense expression or agents that alter the activity or amount of the encoded gene product.
- the method comprises contracting a cell or cell extract thereof with the agent, wherein the cell expresses a nucleic acid molecule comprising any one of SEQ ID Nos: 1-79. Then an agent is identified that inhibits or reduces expression of the nucleic acid molecule or the polypeptide encoded thereby.
- the agent may a ribozyme, DNAzyme, antibody, e.g., a polyclonal, monoclonal, humanized or ScFv antibody, or antisense molecule.
- the agent inhibits or reduces cell migration, cell proliferation, cell death or genetic instability, or increases cell adhesion.
- Figure 1 shows a schematic of a method to identify a gene, the expression of which is altered in p210 BCR"ABL cells.
- Figure 2 depicts the sequence identifier number associated with each sequence identified by the method of the invention.
- Figure 3 shows the nucleic acid sequences identified by the method of the invention.
- Marker genes are genes that impart a distinct phenotype to cells expressing that gene and thus allow such cells to be distinguished from cells that do not have the marker gene. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can 'select' for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a "reporter” trait that one can identify through observation or testing, i.e., by 'screening'.
- a selective agent e.g., a herbicide, antibiotic, or the like
- reporter simply a "reporter” trait that one can identify through observation or testing, i.e., by 'screening'.
- suitable marker genes are known to the art and can be employed in the practice of the invention.
- Screenable markers that may be employed include, but are not limited to, a ⁇ -glucuronidase or uidA gene (GUS), a ⁇ -lactamase gene, a ⁇ -galactosidase gene, a luciferase (luc) gene, an aequorin gene, a green fluorescent protein gene (GFP) gene, a blue, red or yellow fluorescent protein gene, a chloramphenicol acetyltransferase gene (CAT), horseradish peroxidase gene (HRP), an alkaline phosphatase gene (AP) and others.
- GUS ⁇ -glucuronidase or uidA gene
- GUS ⁇ -lactamase gene
- a ⁇ -galactosidase gene a luciferase (luc) gene
- an aequorin gene a green fluorescent protein gene (GFP) gene
- GFP green fluorescent protein gene
- CAT chloramphenicol ace
- Means of detecting labels are well known to those of skill in the art.
- means for detection include a scintillation counter or photographic film as in autoradiography.
- the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
- CCDs charge coupled devices
- enzymatic labels may be detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product.
- simple colorimetric labels are often detected simply by observing the color associated with the label.
- an isolated nucleic acid molecule of the invention is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that hybridize to the RNA or DNA corresponding to any one of SEQ ID Nos. 1-79, or the complement thereof, and remain stably bound under moderate or stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al. (1989).
- a nucleic acid molecule which "hybridizes" to a reference nucleic acid sequence duplexes or binds to that nucleic acid.
- a nucleic acid molecule which "hybridizes" to a reference sequence can include sequences which are shorter or longer than the reference sequence.
- the DNA:DNA hybridization is done in a Southern blot protocol using a 0.2X SSC, 0.1% SDS, 65°C wash.
- SSC refers to a citrate-saline solution of 0.15 M sodium chloride and 20 mM sodium citrate. Solutions are often expressed as multiples or fractions of this concentration.
- 6X SSC refers to a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate.
- 0.2X SSC refers to a solution 0.2 times the SSC concentration or 0.03 M sodium chloride and 4 mM sodium citrate.
- Accepted means for conducting hybridization assays are known and general overviews of the technology can be had from a review of: Hames and Higgins, 1985; Meinkoth and Wahl, 1984; Sambrook et al., 1989; and Innis et al., 1990. "Moderate” and “stringent” hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. (1989).
- stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50°C, or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
- SSC sodium lauryl sulfate
- a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
- Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 42 ° C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.
- Denaturation refers to the process by which a double-stranded nucleic acid is converted into its constituent single strands. Denaturation can be achieved, for example, by the use of high temperature, low ionic strength, acidic or alkaline pH, and/or certain organic solvents. Methods for denaturing nucleic acids are well-known in the art.
- Annealing refers to tlie process by which complementary single-stranded nucleic acids form a double-stranded structure, or duplex, mediated by hydrogen-bonding between complementary bases in the two strands.
- Annealing conditions are those values of, for example, temperature, ionic strength, pH and solvent which will allow annealing to occur. Many different combinations of the above-mentioned variables will be conducive to annealing. Appropriate conditions for annealing are well-known in the art, and will generally include an ionic strength of 50 mM or higher monovalent and/or divalent cation at neutral or near-neutral pH.
- An annealing mixture is a composition containing single-stranded nucleic acid at the appropriate temperature, pH and ionic strength to allow annealing to occur between molecules sharing regions of complementary sequence.
- a “duplex” refers to a double-stranded polynucleotide.
- “Amplification” is the process by which additional copies of a nucleic acid sequence or collection of nucleic acid sequences are generated. Amplification is generally achieved enzymatically, using a DNA polymerase enzyme. Current techniques allow exponential amplification of any sequence flanked by binding sites for a pair of oligonucleotide primers, through reiterative application of denaturation, primer annealing and polymerase extension steps, commonly known as a polymerase chain reaction.
- U.S. Patent No. 4,683,202 Saiki et al., 1988; Innis et al., 1990; Ehrlich, 1989.
- the rate of polymerization is approximately 1,000-2,000 nucleotides per minute. Accordingly, the maximum length of amplifiable sequence will be limited by the reaction conditions (for example, the duration of the extension step).
- the ability to control the extent of elongation in a polymerase chain reaction can be used to advantage to generate lower-complexity subsets of amplified fragments from an initial fragment collection of high complexity.
- Nucleic acid molecules of interest in the present invention may be cloned or amplified, e.g., by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Q ⁇ replicase amplification system (QB).
- PCR polymerase chain reaction
- LCR ligase chain reaction
- TAS transcription-based amplification system
- 3SR self-sustained sequence replication system
- QB Q ⁇ replicase amplification system
- nucleic acid segment is a polymer of nucleotides, and the terms are meant to encompass both RNA and DNA, as well as single-stranded and double-stranded polynucleotides, as well as molecules containing modifications of the base, sugar or phosphate groups as are known in the art.
- a "population" of polynucleotides, or nucleic acid molecules, sequences or segment is any collection comprised of different nucleotide sequences.
- polynucleotide populations include, but are not limited to, those which represent the genome of a normal cell, the genome of an infected cell, the genome of a neoplastic cell, the genome of a cell existing in a pathological state, the DNA that is characteristic of a particular cell, multicellular structure, organism, state of differentiation, pathological or non-pathological state, the total RNA population of a cell, the polyadenylated RNA population of a cell, or a cDNA population representative of the mRNA population of a particular cell, multicellular structure, organism, state of differentiation, pathological or non- pathological state.
- a sample population of polynucleotides comprising more than one different polynucleotide sequence is compared to a sample from a control population of polynucleotides to identify nucleic acid molecules that are unique to the sample population.
- a “primer” is an oligonucleotide capable of base-pairing with a polynucleotide and serving as a site from which polymerization can be initiated.
- An "oligonucleotide” is a short nucleic acid, generally DNA and generally single-stranded. Generally, an oligonucleotide will be shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, even more particularly, 50 nucleotides or shorter, but greater than 7 nucleotides, and preferably greater than 10 nucleotides, in length.
- “Genomic” DNA is DNA obtained from a cell representing all or part of the genome of that cell.
- cDNA or “complementary DNA” is DNA obtained from copying RNA by reverse transcription. It most often represents the population of mRNA molecules found in a particular cell, cell type, state of development or pathological state.
- a "variant" polypeptide of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous amino acid sequence identity to a polypeptide having an amino acid sequence encoded by an open reading frame comprising any one of SEQ TD NOs:l-79, or a fragment thereof.
- a preferred variant polypeptide includes a variant polypeptide or fragment thereof having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%, the activity of the polypeptide having the amino acid sequence encoded by DNA comprising any one of SEQ ID NOs:l-79.
- a "variant" nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, but less than 100%, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising any one of SEQ ID NOs:l-79, or a fragment thereof.
- the amino acid and/or nucleic acid similarity (or homology) of two sequences may be determined manually or using algorithms well known to the art.
- sequence homology or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences.
- sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
- percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
- percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
- the identical nucleic acid base e.g., A, T, C, G, U, or I
- substantially identical denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
- Gaps are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
- the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%).
- Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
- two protein sequences or polypeptide sequences derived from them of at least 30 amino acids in length
- reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
- two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may fiirther comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
- a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
- default parameters are employed.
- nucleic Acid Molecules of the Invention and the Polypeptides Encoded Thereby The ability to identify nucleic acid sequences which are expressed in one nucleic acid sample and not in another is of intense interest in the field of molecular biology. The identification of differentially expressed nucleic acid sequences can provide valuable clues as to genetic bases for disease, inherited dominant and recessive traits, genetic alterations which give rise to diseases such as cancer, determining species similarities and differences, genotyping, and taxonomic classification.
- the present invention relates to isolated nucleic acid molecules that are specifically or differentially expressed in cells which express a protein associated with disease.
- the expression of the nucleic acid molecules of the invention is induced by the protein associated with disease or are otherwise downstream of the expression of the disease-associated protein in a pathway that links the two.
- Exemplary nucleic acid molecules which are expressed in p210 BCR_ABL - expressing cells are described in Figure 3. These sequences are useful as nucleic acid probes for hybridization assays and as primers, ' e.g., for use in amplification reactions.
- the present invention also relates to isolated polypeptides, as well as to methods for obtaining isolated polypeptides, e.g., by producing recombinant polypeptides, encoded by the nucleic acid molecules of the invention.
- the present invention also relates to antibodies which specifically bind to the encoded gene products, and methods of therapy for diseases, e.g., for ALL and CML and other types of cancer.
- RNA of which is expressed in p210 BCR"AB expressing cells.
- exemplary nucleic acid molecules include RNA or DNA corresponding to any one of SEQ ID NOs:l-79, the complement thereof, or a variant thereof.
- this embodiment of the invention includes nucleic acid molecules comprising: a) the nucleotide sequence of any one of SEQ ID NOs:l- 79 or a portion thereof; b) DNA, genomic or cDNA, comprising an open reading frame comprising any one of SEQ ID NOs:l-79, or the complement thereof, e.g., one which encodes a full length polypeptide; or c) DNA which hybridizes to any one of SEQ TD NOs:l-79, the complement thereof, or a portion thereof, e.g., under moderate hybridization conditions.
- the nucleic acid molecules can be obtained from sources in which it occurs in nature (e.g., tissue or cell samples), from a DNA library, by means of recombinant technology or amplification procedures, or by synthetic techniques.
- the nucleic acid source is mammalian, preferably primate, and more preferably human, DNA.
- the nucleic acid molecules of the present invention can be antisense nucleic acid molecules.
- Antisense nucleic acid is complementary, in whole or in part, to a sense strand and can hybridize with the sense strand.
- the target can be DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart).
- antisense nucleic acid can inhibit the expression of the gene encoded by the sense strand.
- Antisense nucleic acids can be produced by standard techniques.
- the nucleic acid molecule is wholly or partially complementary to and hybridizes with a nucleic acid having a sequence comprising any one of SEQ ID NOs: 1-79.
- nucleic acids referred to herein as “isolated” are nucleic acids separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing.
- isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated.
- Nucleic acids referred to as "recombinant" are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as PCR and/or cloning into a vector using restriction enzymes.
- a nucleic acid molecule of the invention can be operably linked to one or more expression control elements (an expression cassette) which is incorporated into a vector, e.g., a viral vector, and the resulting construct introduced into host cells, which are maintained under conditions suitable for expression of the encoded polypeptide.
- the construct can be introduced into cells by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, or infection).
- the encoded polypeptide can be isolated from the host cells or medium.
- the nucleic acid molecules can be also used as probes to detect and/or isolate (e.g., by hybridization with RNA or DNA) variants or homologs (i.e., in other species) thereof, and/or to identify sequences corresponding to full length open reading frames. Moreover, the presence or frequency of the RNA corresponding to a nucleic acid molecule of the invention may be indicative of a disease.
- nucleic acid molecules of the invention are also useful for therapeutic purposes.
- sense or anti-sense DNA fragments can be introduced into expression cassette which are subsequently cells in which expression of a particular nucleic acid sequence is to be reduced.
- the sense or anti-sense DNA fragments are expressed so as to result in reduced expression of the corresponding gene.
- compositions which comprise nucleic acid molecules of the present invention and a suitable carrier (e.g., a buffer) are also the subject of this invention.
- a suitable carrier e.g., a buffer
- the compositions can include additional components, such as stabilizers.
- the present invention also relates to polypeptides encoded by the nucleic acid molecules of the invention, including a variant polypeptide which has at least 80% or more contiguous amino acid sequence identity to the polypeptide encoded by an open reading frame comprising a nucleic acid molecule of the invention, and which variant polypeptide has at least 1%, preferably 10% or more, of the activity of the non- variant (wild type) polypeptide, which can be obtained (isolated) from sources (e.g., cells) in which they occur in nature, produced using recombinant or genetic engineering methods or synthesized chemically. It also relates to pharmaceutical compositions which comprise the polypeptide and an appropriate carrier, such as a buffer. It may also comprise other components, such as stabilizers and other drags.
- Polypeptides referred to herein as “isolated” are polypeptides purified to a state beyond that in which they exist in cells in which they are produced. "Isolated” polypeptides include polypeptides obtained by methods described herein, similar methods or other suitable methods, including essentially pure proteins or polypeptides isolated from the source in which they occur, polypeptides produced by chemical synthesis (e.g., synthetic peptides), or by combinations of biological and chemical methods, and recombinant polypeptides which are isolated.
- Polypeptides referred to herein as "recombinant” or “recombinantly produced” are polypeptides produced by the expression of nucleic acids encoding the polypeptides in a host cell which is modified to contain the nucleic acids encoding the polypeptide (e.g., by transfection with exogenous DNA which encodes the polypeptide) or is modified to express a gene which induces the expression of the nucleic acid molecules of the invention.
- Another aspect of the invention relates to a method of producing polypeptide encoded by a nucleic acid molecule of the invention, a variant, or a fragment thereof.
- Recombinant polypeptide can be obtained, for example, by the expression of a recombinant DNA molecule encoding the polypeptide, a variant or a fragment thereof in a suitable host cell.
- recombinantly produced polypeptide is expressed in a suitable host cell by turning on or enhancing expression of a gene corresponding to the nucleic acid molecule of the invention present in (endogenous to) the host cell.
- Constructs suitable for the expression of the polypeptide or a variant can be introduced into a suitable host cell. Cells which express a recombinantly produced polypeptide or variant thereof, can be maintained in culture.
- Suitable host cells can be prokaryotic, including bacterial cells such as E. coli, B. subtilis and other suitable bacteria (e.g., Streptococci) or eukaryotic, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus species,
- a nucleic acid (e.g., DNA) encoding the polypeptide is inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, cosmid, phage, virus or other suitable replicon for expression.
- the resulting vector is introduced into a host cell, using known methods, and the host cell is maintained under conditions appropriate for growth of the host cell and expression of the endogenous DNA.
- a nucleic acid encoding the polypeptide or a variant thereof can be incorporated into a vector, operably linked to one or more expression control elements, and the construct can be introduced into host cells, which are maintained under conditions suitable for expression of the polypeptide.
- the construct can be introduced into cells by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, or infection).
- the encoded polypeptide can be isolated from the host cells or medium.
- Suitable expression vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, terminator), and/or one or more translation signals; a signal sequence or leader sequence for membrane targeting or secretion (of mammalian origin or from a heterologous mammal or non-mammalian species).
- a transcriptional control element e.g., a promoter, an enhancer, terminator
- translation signals e.g., a signal sequence or leader sequence for membrane targeting or secretion (of mammalian origin or from a heterologous mammal or non-mammalian species).
- the present invention also relates to antibodies, both polyclonal and monoclonal, which bind the polypeptides of the invention in vitro and/or in vivo and, optionally, inhibit an activity or function characteristic of the polypeptide.
- the present invention further relates to pharmaceutical compositions which comprise anti-polypeptide antibodies and a suitable carrier, such as a buffer; they can also include further components, such as stabilizers.
- a suitable carrier such as a buffer
- the antibodies of the present invention are useful in a variety of applications, including separation techniques, research, diagnostic and therapeutic applications.
- antibodies of the present invention can be used to detect and/or measure the level of the polypeptide in a sample (e.g., tissue or body fluid) obtained from an individual (e.g., a human).
- a sample e.g., tissue and/or fluid
- a suitable immunological method can be used to detect and/or measure polypeptide levels.
- antibodies which bind the polypeptide are used to analyze tissues or cells in mammals for reactivity and/or expression (e.g., immunohistologically).
- the antibodies of the present invention are useful in immunological diagnostic methods of assessing expression of the polypeptide in normal tissues or cells and cancerous tissues or cells.
- Anti-polypeptide antibodies also have therapeutic uses.
- An anti- polypeptide antibody can be administered in an amount effective to inhibit the activity of the polypeptide.
- an effective amount is sufficient to achieve the desired therapeutic and/or prophylactic effect.
- the antibody can be administered in a single dose or multiple doses.
- the dosage can be determined by methods known in the art and is dependent, for example, upon the individual's age, sensitivity, tolerance and overall well-being. Suitable dosages for antibodies can be from 0.1-1.0 mg/kg body weight per treatment.
- an antibody can be administered to an individual (e.g., a human) alone or in conjunction with another agent, which is administered before, along with or subsequent to administration of the antibody.
- Compositions of the present invention can be administered by a variety of routes, including, but not limited to, parenteral (e.g., injection, including but not limited to, intravenous, intraarterial, intramuscular, subcutaneous; inhalation, including but not limited to, intrabronchial, intranasal or oral inhalation, intranasal drops; topical) and non-parenteral (e.g., oral, including but not limited to, dietary; rectal).
- parenteral e.g., injection, including but not limited to, intravenous, intraarterial, intramuscular, subcutaneous
- inhalation including but not limited to, intrabronchial, intranasal or oral inhalation, intranasal drops; topical
- non-parenteral e.g., oral, including but not limited to,
- compositions comprising the nucleic acids, proteins or antibodies to be administered can be prepared in a physiologically acceptable vehicle or carrier.
- suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
- Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers.
- the compound can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
- a suitable dispenser for administration e.g., an atomizer, nebulizer or pressurized aerosol dispenser.
- Nucleic acids, proteins and antibodies can be administered individually, together or in combination with other drugs or agents (e.g., other chemotherapeutic agents, immune system enhancers).
- the invention in its broadest sense provides a method to identify genes associated with a particular phenotype, e.g., disease or malignancy, by identifying genes that are differentially expressed and so are associated with the phenotype.
- Methods for identifying differentially expressed transcripts, transcripts which differ in abundance between samples being compared, are known to the art.
- subtractive hybridization An enhancement of differential screening, called subtractive hybridization, enriches samples for differentially expressed transcripts prior to differential screening. Exemplary subtractive hybridization methods are described hereinbelow, although any other methods which identify differentially expressed genes may be employed to identify the nucleic acid molecules of the invention (e.g., see U.S. Patent Nos. 5,726,022, 5,827,658, 5,958,738 and 6,066,457 and Matz et al., 1998).
- DD differential screening
- subsets cDNA fragments
- fingerprints the analogous pools obtained from the compared RNA samples are resolved side by side on a polyacrylamide gel (the produced band patterns are called fingerprints)
- fragments defined by length that are present in only one sample (or much more abundant in one than in the other) are excised from the gel and investigated
- DD by changing the parameters of the pool generation protocol, other pools are produced and studied in a similar way to compare more mRNAs.
- the advantage of such an approach is that the abundance of several dozen randomly picked cDNA fragments can be checked simultaneously.
- Another important feature of DD is the ability to compare more than two RNA samples at once.
- the methods of the invention may be employed with any source of nucleic acid in which one is interested in comparing for differences.
- the sources may be eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non- mammalian, plant and others.
- the source is a vertebrate source such as a mammalian source, e.g., from canine, bovine, murine, ovine, caprine, equine, and primates such as humans.
- RNA may be isolated by any known means as a subset of the genomic nucleic acid and subsequent synthesis of cDNA. It is also desirable to use cDNA as the first nucleic acid sample in assays in which cDNA or RNA is used as the second nucleic acid sample to prevent the isolation of products that are derived from intronic genomic sequences.
- One subtractive hybridization method employs first and second nucleic acid samples that are separately subjected to at least one restriction endonuclease.
- the restriction endonuclease may provide for blunt ends or staggered (sticky) ends, usually staggered ends. It is preferred that both first and second nucleic acid samples are subjected to the same restriction endonuclease and that such endonuclease is one which recognizes and cuts at a four base site. For the subsequent steps it is further preferred that such restriction endonuclease be one which recognizes a four base sequence found within a longer six base or eight base sequence recognized by a restriction endonuclease. Almost 1500 restriction endonucleases are now known and at least 150 are commercially available. Complete lists plus details of restriction sites and reaction conditions are published, for example in Brown, 1991.
- double- stranded oligonucleotide adaptors are ligated onto the ends of each of the strands of the fragments.
- the adaptor will usually be staggered at both ends, with one strand being longer than the other.
- the adaptors will generally serve to provide the sequence complementary to a primer to be used when a subsequent amplification step is employed.
- typically one end of the adaptor will be double-stranded and have one end complementary to the ends of the double- stranded nucleic acid fragments from the digestion, sometimes referred to herein as the proximal end of the adaptor.
- Each adaptor will preferably further contain a restriction site located distal to the proximal end.
- the restriction site in the adaptor is preferably one which has a six or eight base consensus sequence, and most preferably is such a one that further contains a 3 ' sequence that ends in a four base consensus sequence that has ends that are complementary to the same ends that are created by the six or eight base cutter that is adjacent and external to it.
- restriction endonucleases include, but are not limited to, DpnJI ('GATC); BgUI (A'GATCT); BamHJ (G'GATCC); Tsp509J ( ⁇ ATT), EcoRI (G'AATTC) and Pad (TT'AATTAA).
- the adaptors used for the first nucleic acid sample fragments contain a restriction site which is different than the one used in the adaptors for the second nucleic acid sample fragments.
- the adaptor may further optionally contain a ligand binding end.
- a ligand binding end is particularly important if the fragments will not be amplified.
- a ligand or ligand binding end is a molecule which binds to another molecule and so permits the physical or chemical separation of molecules which are physically linked to the ligand or ligand binding end, e.g., biotin binds to avidin or streptavidin and digoxigenin binds to anti-digoxigenin antibodies.
- the adaptor have one strand longer than the other to serve as a complement to primers if the fragments are to be amplified.
- the first nucleic acid sample fragments will contain adaptors having a restriction site.
- the second nucleic acid sample fragments do not necessarily need to have adaptors or a primer used for amplification with a restriction site. If this embodiment is employed, the adaptors and/or primers for the second nucleic acid sample fragments will have a ligand binding moiety to enable capture of the second nucleic acid sample fragments.
- nucleic acid sample and the second nucleic acid sample it is possible to ligate the same adaptors onto nucleic acid of the first nucleic acid sample and the second nucleic acid sample if different primers are subsequently used to amplify the two sample populations so long as a restriction endonuclease site is encoded within the primers used to amplify the first nucleic acid sample fragments.
- the adaptor ligated onto the first nucleic acid sample fragments may have "non-nested" restriction endonuclease sites; e.g. 5 'EcoRI— GATC3 ', where the EcoRI site is external to an initial DpnII digestion site.
- This protocol is less preferred, however, because when the EcoRI site is subsequently targeted by the restriction endonuclease in order to release the homoduplex from its biotinylated adaptors, approximately 1/16 of the cDNA molecules may contain an internal EcoRI site.
- the first and second nucleic acid fragments may be separately amplified to enhance the assay, preferably by PCR or other methods, using primers containing a sequence complementary to the respective adaptors and a ligand binding end.
- the second nucleic acid sample fragments and the first nucleic acid sample fragments may be amplified separately by adding appropriate primers complementary to the adaptors using PCR, typically for about 10-35 cycles, more typically about 20 cycles, depending upon the initial concentration of second or first nucleic acid sample fragments being amplified.
- PCR for a general overview of PCR, see Innis et al.,1990; and U.S. Patent Nos. 4,683,195 and 4,683,202.
- the adaptors do not need to be removed.
- the amplified first and second nucleic acid sample fragments are combined under hybridization conditions such that the fragments hybridize together creating essentially several possible complexes: first nucleic acid/second nucleic acid matches, second nucleic acid/second nucleic acid matches, and first nucleic acid/first nucleic acid matches. It is preferred that the second nucleic acid fragments are present in excess of the first nucleic acid sample fragments to increase the probability that the first nucleic acid/first nucleic acid complexes are representative of nucleic acid not found in the second nucleic acid sample.
- second nucleic acid sample fragments are combined with the adaptor-ligated first nucleic acid fragments, with the second nucleic acid sample fragments present in excess, usually at least 5-fold excess and less than 500-fold excess, preferably about 100-fold excess for the first cycle of hybridization.
- Hybridization will be allowed to proceed at high stringency temperatures, usually about 60°-70° C.
- Various buffers and salt concentrations may be used to adjust for the desired stringency as will be appreciated by those in the art.
- the first nucleic acid/first nucleic acid complexes present in the combined first and second nucleic acid solution can be readily separated from the other complexes depending upon the ligand used. Most conveniently, all of the combined fragments will be subjected to a restriction enzyme which recognizes the site in the first nucleic acid sample adaptors which will effectively remove the ligand binding end from the first nucleic acid/first nucleic acid molecules and not from the others. Thus, by capture technology which will attract the ligand binding end of the second nucleic acid/first nucleic acid complexes, one can readily separate out the first nucleic acid/first nucleic acid complexes.
- the first nucleic acid/first nucleic acid complexes may be further amplified and isolated, by, for example, ligating new adaptors onto the ends of the molecules and amplifying by PCR. It may be of interest to carry out the process more than once, where different restriction endonucleases are used. Different fragments may be obtained and result in additional information.
- any resulting unique first nucleic acid sequences can be used as probes to identify sites in the first nucleic acid sample which differ from the second nucleic acid source.
- they may be labeled in a variety of ways.
- the first nucleic acid sample sequences may be cloned by inserting into an appropriate cloning vector for cloning in a prokaryotic host.
- the cloned DNA may be sequenced to determine the nature of the target DNA.
- the cloned DNA may be labeled and used as probes to identify fragments in libraries carrying the target DNA.
- the target DNA may be used to identify the differences which may be present between the two sources of nucleic acid.
- the resulting target DNA will be greatly enriched. It may be used as a probe to identify sites on the first nucleic acid sample sequences which differ from the second nucleic acid.
- the target nucleic acid may be sequenced directly by PCR or it may be cloned by inserting it in a cloning vector for cloning into a host cell.
- the cloned DNA can be sequenced to determine the nature of the target DNA through the use of dot blotting or other procedure. It may also be labeled and used as probes to identify fragments in libraries carrying the target DNA. Sequences can be identified and cloned for sequencing. Comparative searches with sequences described in accessible libraries such as Genbank (National Center for Biotechnology Information, Natl.
- the genes are isolated from tumorigenic tissue or cells having a malignant or neoplastic phenotype, as well as from normal cells.
- the method entails substantive hybridization of two sets of cDNA, one provided from the tissue of interest and the other from normal tissue.
- primers for first strand synthesis can include polyT, "anchored" polyT, primers with restriction enzyme recognition sequences build-in, primers to complement a polyN tail created by the terminal transferase, where N stands for any one of the four nucleotides to be used.
- the polymerases can be any of the available and known polymerases. Either set of cDNAs could be maintained and amplified by cloning into a plasmid. A set of cDNAs from the normal tissue can serve as subtractor cDNA for annealing to cDNA from samples from multiple tissues of interest. Further variations and options would be readily apparent to one skilled in the art.
- oligo is attached to the ends of the cDNA set prepared from the tissue of interest.
- the oligo set is composed of two, at least partially complementary synthetic oligos. They are attached to the cDNA by a DNA ligase. The attachment can be preceded by creation of protruding ends on the cDNA by cleavage with a restriction endonuclease for which a recognition side was built- in on the cDNA ends by the choice of oligos used to create the cDNA set.
- the oligo set to be attached is designed to create, after self annealing, complementary ends to the cDNA.
- the cDNA is made blunt-ended by enzymatic reaction, Klenow fragment by example. Then the oligo set would be ligated to the cDNA by a blunt-ended ligation.
- the cDNA ends are made blunt-ended as described above and ligated to an adapter set which is blunt ended at least at one end.
- the cDNA derived from the normal tissue is biotinylated. Again, this requirement can be achieved by any of a number of methods readily apparent to one skilled in the art.
- the biotin label can be incorporated into the cDNA starting with the synthesis of a second strand or can result from PCR amplification of a pre-made cDNA set.
- the label can also be introduced by PCR amplification or by "nick- translation" of a cDNA set or by photobiotinylation.
- the invention also includes a mixing of the two cDNA sets, derived from the tissue of interest and from the normal tissues, followed by denaturation and annealing.
- An excess of subtracter cDNA will increase the efficiency of annealing (and eventual removal, see below) of the sequences that are common to the two cDNA sets and are not present in the tissue of interest.
- the melting and annealing conditions are standard for such experiments and known to one skilled in the art.
- the annealing results in populations of hybrid cDNAs. Magnetic spheres make the j ob of removal of biotin containing DNA easier. Thus, little biotin labeled DNA should escape untrapped, reducing the background levels of cDNA recovered from the subtractive hybridization.
- Streptavidin coated beads are available commercially. They are used by other to remove biotin labeled DNA, unlike the current disclosure that employs them within in a subtractive hybridization protocol.
- the subtractive hybridization results in a cDNA fraction, hereafter called flow-through, enriched in cDNAs representing genes expressed in tumorigenic tissue or cells having a malignant to neoplastic phenotype, but not free of all other cDNAs.
- Initial analysis is sometimes facilitated by cloning the cDNAs of the flow-through.
- the cloning step itself is facilitated by first carrying out a PCR amplification of the flow-through cDNAs. Both of these steps can be accomplished by use of the previously described adapter set that can a) contain a restriction enzyme recognition site and b) one of the oligos in the set can be used as PCR primer.
- the chosen cloned cDNA(s) described above would be hybridized to equivalent amounts of nucleic acids, DNA and/or RNA, from both the tissue of interest and the normal tissue.
- the relative intensity of the bands would be compared spectrophotometrically and result in a estimate of copy number.
- variations and shortcuts will be readily apparent. For example, but not limited to this examples, one could use dot blots rather than gels and blotting, or one can incorporate a control hybridization with a probe not expected to hybridize to the gene, to standardize the amount of nucleic acids from the two tissues used.
- CB Cord blood
- CD34 + cells (Zhao et al., 1999) were transduced with a MS CV-retro virus vector containing either eGFP alone (eGFP) or BCR/ABL cDNA-IRES-eGFP (p210-eGFP).
- GFP + cells were FACS selected (> 90% purity), and subtractive hybridization performed between the two populations using the Clontech PCR-SelectTM System, according to the manufacturer' s recommendations .
- PCR-Select method requires only one round of subtractive hybridization to subtract and equalize cDNAs, combined with suppression PCR for efficient amplification of target molecules. This dramatically increases the probability of obtaining differentially expressed, rare transcripts.
- traditional subtractive hybridization methods require a large amount of poly A + RNA and the tedious separation of ss and ds cDNA fractions.
- cDNA is synthesized from only 0.5-2.0 ⁇ g of poly A + RNA prepared from the two types of tissue or cells under comparison. A total of 79 subtracted cDNA clones were expressed in p210-eGFP + but not eGFP + transduced CD34 + cells. The cDNAs were between 400-900 bp in length.
- cDNA sequences represent uncharacterized human proteins. Of these, at least 15 were closely homologous to expressed sequence tags (ESTs), at least 10 had homology to genomic clones for which no mRNA transcripts or proteins had been identified, while 1 sequence had no match in available genomic, RNA or protein databases. In addition, at least 22 sequences were identified that have significant homologies to known genes, which are involved in protein degradation, signal transduction, cell cycle regulation, and in RNA splicing.
- ESTs expressed sequence tags
- RNA splicing may provide an explanation for the finding that CML CD34 + cells and cell lines express a number of alternatively spliced proteins, including Pyk2, beta r B integrin, CSCP
- chondroitin sulfate core protein chondroitin sulfate core protein
- MPP1 human palmitoylated erythrocyte membrane protein
- the identified sequences and/or their full length genes are used as probes to detect expression patterns in normal and CML cells, and/or cloned into expression vectors.
- the identified sequences and/or their full length equivalent may also be used to prepare primers for RT-PCR analysis of the expression of these sequences in primary CML cells.
- gene expression analyses will lead to important new insights in the molecular mechanisms underlying CML and may identify critical targets for novel therapies for this disease.
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AU2002225702A AU2002225702A1 (en) | 2000-11-14 | 2001-11-14 | Method to identify genes associated with chronic myelogenous leukemia |
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US7449548B2 (en) | 2001-12-07 | 2008-11-11 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
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US7449548B2 (en) | 2001-12-07 | 2008-11-11 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
US7615379B2 (en) | 2001-12-07 | 2009-11-10 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
US7659377B2 (en) | 2001-12-07 | 2010-02-09 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
US7732584B2 (en) | 2001-12-07 | 2010-06-08 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
US7968099B2 (en) | 2001-12-07 | 2011-06-28 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
US8188228B2 (en) | 2001-12-07 | 2012-05-29 | Agensys, Inc. | Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer |
WO2005014853A1 (en) * | 2003-07-28 | 2005-02-17 | Universität Bremen | Comparative expression analysis for the identification of genes responsible for phenotypic differences |
Also Published As
Publication number | Publication date |
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EP1399586A2 (en) | 2004-03-24 |
AU2002225702A1 (en) | 2002-05-27 |
WO2002040718A3 (en) | 2003-12-31 |
CA2428716A1 (en) | 2002-05-23 |
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