EP1181357A2 - Molecules for disease detection and treatment - Google Patents

Molecules for disease detection and treatment

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Publication number
EP1181357A2
EP1181357A2 EP00938108A EP00938108A EP1181357A2 EP 1181357 A2 EP1181357 A2 EP 1181357A2 EP 00938108 A EP00938108 A EP 00938108A EP 00938108 A EP00938108 A EP 00938108A EP 1181357 A2 EP1181357 A2 EP 1181357A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
mddt
sequence
sequences
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00938108A
Other languages
German (de)
French (fr)
Inventor
David M. Hodgson
Stephen E. Lincoln
Frank D. Russo
Peter A. Spiro
Steven C. Banville
Shawn R. Bratcher
Gerard E. Dufour
Howard J. Cohen
Bruce H. Rosen
Michael S. Chalup
Jennifer L. Hillman
Anissa Lee Jones
Jimmy Y. Yu
Lila B. Greenawalt
Scott R. Panzer
Ann M. Roseberry
Rachel J. Wright
Susan E. Daniels
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Genomics Inc
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Filing date
Publication date
Application filed by Incyte Genomics Inc filed Critical Incyte Genomics Inc
Publication of EP1181357A2 publication Critical patent/EP1181357A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to molecules for disease detection and treatment and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases associated with disease detection and treatment molecules
  • the human genome is comprised of thousands of genes, many encoding gene products that function in the maintenance and growth of the various cells and tissues in the body Aberrant expression or mutations in these genes and their products is the cause of, or is associated with, a variety of human diseases such as cancer and other cell proliferative disorders
  • the identification of these genes and their products is the basis of an ever-expanding effort to find markers for early detection of diseases, and targets for their prevention and treatment
  • cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body
  • a wide variety of molecules, either aberrantly expressed or mutated can be the cause of, or involved with, various cancers because tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis Cell proliferation must be regulated to maintain both the number of cells and their spatial organization This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals such as growth factors and other mitogens, and intracellular cues such as DNA damage or nutrient starvation Molecules
  • DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes
  • a genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes
  • the interactions may be expected, such as when the genes are part of the same signaling pathway
  • the interactions may be totally unexpected Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes
  • the present invention relates to human polynucleotides encoding molecules for disease detection and treatment (mddt) as presented in the Sequence Listing Some of the mddt uniquely identify genes encoding structural, functional, and regulatory molecules for disease detection and treatment
  • the invention provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d)
  • the polynucleotide composes a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14
  • the polynucleotide comprises at least 60 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting
  • the invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide compnsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d)
  • the method comprises a) hybridizing the sample with a probe compnsing at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby
  • the invention further provides a recombinant polynucleotide comprising a promoter sequence operably linked to an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 - 14, b) a naturally occurnng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d)
  • the invention provides a cell transformed with the recombinant polynucleotide
  • the invention provides a transgenic organism comprising the recombinant polynucleotide
  • the invention provides a method for producing a disease
  • the invention also provides a purified disease detection and treatment molecule polypeptide (MDDT) encoded by at least one polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14 Additionally, the invention provides an isolated antibody which specifically binds to the disease detection and treatment molecule polypeptide
  • the invention further provides a method of identifying a test compound which specifically binds to the disease detection and treatment molecule polypeptide, the method comprising the steps of a) providing a test compound, b) combining the disease detection and treatment molecule polypeptide with the test compound for a sufficient time and under suitable conditions for binding, and c) detecting binding of the disease detection and treatment molecule polypeptide to the test compound, thereby identifying the test compound which specifically binds the disease detection and treatment molecule polypeptide
  • the invention further provides a microarray wherein at least one element of the microarray is an isolated polynucleotide comprising at least 60 contiguous nucleotides of
  • the invention provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide compnses a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d)
  • the method compnses a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide
  • the invention further provides a method for detecting a target polynucleo
  • Table 1 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with their GenBank hits (GI Numbers), probability scores, and functional annotations corresponding to the GenBank hits
  • Table 2 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated “start” and “stop” nucleotide positions The reading frames of the polynucleotide segments and the Pfam hits, Pfam descnptions, and E-values corresponding to the polypeptide domains encoded by the polynucleotide segments are indicated
  • Table 3 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated “start” and “stop” nucleotide positions The reading frames of the polynucleotide segments are shown, and the polypeptides encoded by the polynucleotide segments constitute either signal peptide (SP) or transmembrane (TM) domains, as indicated
  • Table 4 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with component sequence identification numbers (component IDs) corresponding to each template
  • SEQ ID NO s sequence identification numbers
  • template IDs template identification numbers
  • component IDs component sequence identification numbers
  • Table 5 summarizes the bioinformatics tools which are useful for analysis of the polynucleotides of the present invention
  • the first column of Table 5 lists analytical tools, programs, and algorithms, the second column provides brief descriptions thereof, the third column presents approp ⁇ ate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences)
  • mddt refers to a nucleic acid sequence
  • MDDT refers to an amino acid sequence encoded by mddt
  • a “full-length” mddt refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in human tissue
  • adjuvants are materials such as Freund's adjuvant, mineral gels (aluminum hydroxide), and surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol) which may be administered to increase a host's immunological response
  • Alleles result from a "mutation," a change or an alternative reading of the genetic code Any given gene may have none, one, or many allelic forms Mutations which give rise to alleles include deletions, additions, or substitutions of nucleotides Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence
  • allelic mddt “Amino acid sequence” refers to a peptide, a polypeptide, or a protein of either natural or synthetic origin The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a fragment, epitope, va ⁇ ant, or derivative of a protein expressed by a nucleic acid sequence.
  • Amplification refers to the production of additional copies of a sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art
  • Antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic determinant
  • Antibodies that bind MDDT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen
  • the polypeptide or peptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used earners that are chemically coupled to peptides include bovine serum albumin, thyroglobulm, and keyhole limpet hemocyanin (KLH) The coupled peptide is then used to immunize the animal
  • Antisense sequence refers to a sequence capable of specifically hyb ⁇ dizing to a target sequence
  • the antisense sequence may include DNA, RNA, or any nucleic acid mimic or analog such as peptide nucleic acid (PNA), oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyurac ⁇ l, or 7-deaza-2'-deoxyguanosme.
  • Antisense sequence refers to a sequence capable of specifically hybridizing to a target sequence
  • the antisense sequence can be DNA, RNA, or any nucleic acid mimic or analog
  • Antisense technology refers to any technology which relies on the specific hybridization of an antisense sequence to a target sequence
  • a "bin” is a portion of computer memory space used by a computer program for storage of data, and bounded in such a manner that data stored in a bin may be retrieved by the program
  • Bioly active refers to an amino acid sequence having a structural, regulatory, or biochemical function of a naturally occurnng amino acid sequence
  • “Clone joining” is a process for combining gene bins based upon the bins' containing sequence information from the same clone.
  • the sequences may assemble into a primary gene transcript as well as one or more splice variants
  • “Complementary” descnbes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing (5'-A-G-T-3 T pairs with its complement 3'-T-C-A-5')
  • a “component sequence” is a nucleic acid sequence selected by a computer program such as PHRED and used to assemble a consensus or template sequence from one or more component sequences
  • a "consensus sequence” or “template sequence” is a nucleic acid sequence which has been assembled from overlapping sequences, using a computer program for fragment assembly such as the GELVIEW fragment assembly system (Genetics Computer Group (GCG), Madison WI) or using a relational database management system (RDMS)
  • Constant amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, 1 e , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions
  • Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain
  • “Deletion” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or am o acid residue, respectively, is absent
  • Derivative refers to the chemical modification of a nucleic acid sequence, such as by replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group
  • element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray
  • E-value refers to the statistical probability that a match between two sequences occurred by chance
  • a "fragment" is a unique portion of mddt or MDDT which is identical in sequence to but shorter in length than the parent sequence
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/ amino acid residue
  • a fragment may comprise from 10 to 1000 contiguous amino acid residues or nucleotides
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues or nucleotides in length
  • Fragments may be preferentially selected from certain regions of a molecule
  • a polypeptide fragment may compnse a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing and the figures, may be encompass
  • a “full length” nucleotide sequence is one containing at least a start site for translation to a protein sequence, followed by an open reading frame and a stop site, and encoding a "full length” polypeptide
  • “Hit” refers to a sequence whose annotation will be used to descnbe a given template Criteria for selecting the top hit are as follows if the template has one or more exact nucleic acid matches, the top hit is the exact match with highest percent identity If the template has no exact matches but has significant protein hits, the top hit is the protein hit with the lowest E-value If the template has no significant protein hits, but does have significant non-exact nucleotide hits, the top hit is the nucleotide hit with the lowest E-value "Homology” refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of an mddt or between a reference amino acid sequence and a fragment of an MDDT
  • Hybndization refers to the process by which a strand of nucleotides anneals with a complementary strand through base pairing Specific hybndization is an indication that two nucleic acid sequences share a high degree of identity Specific hybndization complexes form under defined annealing conditions, and remain hybndized after the "washing" step
  • the defined hybridization conditions include the annealing conditions and the washing step(s), the latter of which is particularly important in determining the stnngency of the hybndization process, with more stnngent conditions allowing less non-specific binding, I e , binding between pairs of nucleic acid probes that are not perfectly matched
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency
  • stnngency of hybridization is expressed with reference to the temperature under which the wash step is earned out Generally, such wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe
  • High stnngency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0 2 x SSC and about 0 1 % SDS, for 1 hour Alternatively, temperatures of about 65°C, 60°C, or 55°C may be used SSC concentration may be varied from about 0 2 to 2 x SSC, with SDS being present at about 0 1 %
  • blocking reagents are used to block non-specific hybndization
  • Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200 ⁇ g/ml
  • Hybndization particularly under high stringency conditions, may be suggestive of evolutionary similanty between the nucleotides Such similarity is strongly indicative of a similar role for the nucleotides and their resultant proteins
  • Other parameters such as temperature, salt concentration, and detergent concentration may be varied to achieve the desired stnngency
  • Denaturants such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as RNA DNA hybridizations
  • Appropnate hybndization conditions are routinely determinable by one of ordinary skill in the art "lmmunogenic" descnbes the potential for a natural, recombinant, or synthetic peptide, epitope, polypeptide, or protein to induce antibody production appro
  • “Insertion” or “addition” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or residue, respectively, is added to the sequence
  • Labeling refers to the covalent or noncovalent joining of a polynucleotide, polypeptide, or antibody with a reporter molecule capable of producing a detectable or measurable signal
  • “Microarray” is any a ⁇ angement of nucleic acids, amino acids, antibodies, etc , on a substrate
  • the substrate may be a solid support such as beads, glass, paper, nitrocellulose, nylon, or an appropriate membrane
  • Linkers are short stretches of nucleotide sequence which may be added to a vector or an mddt to create restnction endonuclease sites to facilitate cloning
  • Polylinkers are engineered to incorporate multiple restriction enzyme sites and to provide for the use of enzymes which leave 5' or 3' overhangs (e g , BamHI, EcoRI, and Hindlll) and those which provide blunt ends (e g , EcoRV, SnaBI, and Stul)
  • Naturally occurring refers to an endogenous polynucleotide or polypeptide that may be isolated from viruses or prokaryotic or eukaryotic cells
  • Nucleic acid sequence refers to the specific order of nucleotides joined by phosphodiester bonds in a linear, polymenc arrangement Depending on the number of nucleotides, the nucleic acid sequence can be considered an oligomer, oligonucleotide, or polynucleotide
  • the nucleic acid can be DNA, RNA, or any nucleic acid analog, such as PNA, may be of genomic or synthetic origin, may be either double-stranded or single-stranded, and can represent either the sense or antisense (complementary) strand
  • Oligomers refers to a nucleic acid sequence of at least about 6 nucleotides and as many as about 60 nucleotides, preferably about 15 to 40 nucleotides, and most preferably between about 20 and 30 nucleotides, that may be used in hybridization or amplification technologies Oligomers may be used as, e g , pnmers for PCR, and are usually chemically synthesized
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence
  • operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame
  • PNA protein nucleic acid
  • PNA refers to a DNA mimic in which nucleotide bases are attached to a pseudopeptide backbone to increase stability PNAs, also designated antigene agents, can prevent gene expression by targeting complementary messenger RNA
  • percent identity and % identity as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algonthm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI) CLUSTAL V is described in Higgins, D.G. and Sharp, P.M ( 1989) CABIOS 5: 151-153 and in Higgins, D.G. et al. ( 1992)
  • CABIOS 8 189-191.
  • the "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequence pairs Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S F. et al. ( 1990) J Mol. Biol.
  • BLAST 215 403-410 which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http7/www. ncbi.nlm.nih.gov/BLAST/
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to determine alignment between a known polynucleotide sequence and other sequences on a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www ncbi.nlm nih.gov/gorf/bl2/.
  • BLAST 2 Sequences can be used for both blastn and blastp (discussed below) BLAST programs are commonly used with gap and other parameters set to default settings For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences” tool Version 2.0.9 (May-07-1999) set at default parameters
  • Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm
  • Some alignment methods take into account conservative amino acid substitutions Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity of the substituted residue, thus preserving the structure (and therefore function) of the folded polypeptide
  • NCBI BLAST software suite may be used for example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2 0 9 (May-07-1999) with blastp set at default parameters
  • Such default parameters may be, for example
  • Filter on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured
  • Post-translational modification of an MDDT may involve hpidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art These processes may occur synthetically or biochemically Biochemical modifications will vary by cell type depending on the enzymatic milieu and the MDDT
  • Probe refers to mddt or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes
  • Pnmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing The primer may then be extended along the target DNA strand by a DNA polymerase enzyme Pnmer pairs can be used for amplification (and identification) of a nucleic acid sequence, e g , by the polymerase chain reaction (PCR)
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence In order to enhance specificity, longer probes and primers may also be employed, such as probes and primer
  • PCR pnmer pairs can be denved from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0 5, 1991 , Whitehead Institute for Biomedical Research, Cambndge MA) Oligonucleotides for use as pnmers are selected using software known in the art for such purpose For example, OLIGO 4 06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases Similar primer selection programs have incorporated additional features for expanded capabilities For example, the P ⁇ mOU pnmer selection program (available to the public from the Genome Center at University of Texas
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e g , by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell
  • such recombinant nucleic acids may be part of a viral vector, e g , based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal
  • Regulatory element refers to a nucleic acid sequence from nontranslated regions of a gene, and includes enhancers, promoters, introns, and 3' untranslated regions, which interact with host proteins to carry out or regulate transcription or translation
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, an amino acid, or an antibody They include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cof actors, inhibitors, magnetic particles, and other moieties known in the art
  • RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymme are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxy ⁇ bose
  • Samples may contain nucleic or amino acids, antibodies, or other materials, and may be derived from any source (e g , bodily fluids including, but not limited to, saliva, blood, and urine, chromosome(s), organelles, or membranes isolated from a cell, genomic DNA, RNA, or cDNA in solution or bound to a substrate, and cleared cells or tissues or blots or imprints from such cells or tissues)
  • source e g , bodily fluids including, but not limited to, saliva, blood, and urine, chromosome(s), organelles, or membranes isolated from a cell, genomic DNA, RNA, or cDNA in solution or bound to a substrate, and cleared cells or tissues or blots or imprints from such cells or tissues
  • Specific binding or “specifically binding” refers to the interaction between a protein or peptide and its agonist, antibody, antagonist, or other binding partner The interaction is dependent upon the presence of a particular structure of the protein, e g , the antigenic determinant or epitope, recognized by the binding molecule For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody
  • substitution refers to the replacement of at least one nucleotide or ammo acid by a different nucleotide or amino acid
  • Substrate refers to any suitable rigid or semi-rigid support including, e g , membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles or capillaries
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound
  • a "transcript image” refers to the collective pattern of gene expression by a particular tissue or cell type under given conditions at a given time
  • Transformation refers to a process by which exogenous DNA enters a recipient cell Transformation may occur under natural or artificial conditions using vanous methods well known in the art Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell The method is selected based on the host cell being transformed
  • Transformants include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as cells which transiently express inserted DNA or RNA
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et
  • a “variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 25% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters
  • Such a pair of nucleic acids may show, for example, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even at least 98% or greater sequence identity over a certain defined length
  • the variant may result in "conservative" amino acid changes which do not affect structural and/or chemical properties
  • a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymorphic” variant
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing
  • variants of the polynucleotides of the present invention may be generated through recombinant methods
  • One possible method is a DNA shuffling technique such as MOLECULARBREEDING (Maxygen Inc , Santa Clara CA, described in U S Patent Number 5,837,458, Chang, C -C et al ( 1999) Nat Biotechnol 17 793-797, Chnstians, F C et al (1999) Nat Biotechnol 17 259-264, and Crame ⁇ , A et al ( 1996) Nat.
  • MOLECULARBREEDING Maxygen Inc , Santa Clara CA, described in U S Patent Number 5,837,458, Chang, C -C et al ( 1999) Nat Biotechnol 17 793-797, Chnstians, F C et al (1999) Nat Biotechnol 17 259-264, and Crame ⁇ , A et al ( 1996) Nat.
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner
  • a “vanant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides
  • cDNA sequences derived from human tissues and cell lines were aligned based on nucleotide sequence identity and assembled into "consensus" or "template” sequences which are designated by the template identification numbers (template IDs) in column 2 of Table 1
  • the sequence identification numbers (SEQ ID NO s) conesponding to the template IDs are shown in column 1
  • the template sequences have similanty to GenBank sequences, or "hitsJ as designated by the GI Numbers in column 3
  • the statistical probability of each GenBank hit is indicated by a probability score in column 4, and the functional annotation corresponding to each GenBank hit is listed in column 5
  • the invention incorporates the nucleic acid sequences of these templates as disclosed in the Sequence Listing and the use of these sequences in the diagnosis and treatment of disease states charactenzed by defects in molecules for disease detection and treatment
  • the invention further utilizes these sequences in hybridization and amplification technologies, and in particular, in technologies which assess gene expression patterns conelated with specific cells or tissues and their
  • cDNA was isolated from libraries constructed using RNA derived from normal and diseased human tissues and cell lines
  • the human tissues and cell lines used for cDNA library construction were selected from a broad range of sources to provide a diverse population of cDNAs representative of gene transcnption throughout the human body Descnptions of the human tissues and cell lines used for cDNA library construction are provided in the LIFESEQ database (Incyte Genomics, Inc (Incyte), Palo Alto CA)
  • Human tissues were broadly selected from, for example, cardiovascular, dermatologic, endocrine, gastrointestinal, hematopoietic/immune system, musculoskeletal, neural, reproductive, and urologic sources
  • Cell lines used for cDNA library construction were denved from, for example, leukemic cells, teratocarcinomas, neuroepithehomas, cervical carcinoma, lung fibroblasts, and endothelial cells
  • Such cell lines include, for example, THP-1 , Jurkat, HUVEC, hNT2, WI38, HeLa, and other cell lines commonly used and available from public depositories (American Type Culture Collection, Manassas VA) Pnor to mRNA isolation, cell lines were untreated, treated with a pharmaceutical agent such as 5'-aza-2'-deoxycyt ⁇ d ⁇ ne, treated with an activating agent such as lipopolysacchande in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress
  • Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system (Hamilton Company (Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc. (MJ Research), Watertown MA), and ABI CATALYST 800 thermal cycler (PE Biosystems) Sequencing can be earned out using, for example, the ABI 373 or 377 (PE Biosystems) or MEGABACE 1000 (Molecular Dynamics, Inc (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or other automated and manual sequencing systems well known in the art.
  • nucleotide sequences of the Sequence Listing have been prepared by current, state-of- the-art, automated methods and, as such, may contain occasional sequencing errors or unidentified nucleotides Such unidentified nucleotides are designated by an N These infrequent unidentified bases do not represent a hindrance to practicing the invention for those skilled in the art Several methods employing standard recombinant techniques may be used to correct errors and complete the missing sequence information (See, e g., those described in Ausubel, F M et al. ( 1997) Short
  • Template or “consensus” sequences as follows Sequence chromatograms are processed, ve ⁇ fied, and quality scores are obtained using PHRED Raw sequences are edited using an editing pathway known as Block 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA).
  • BLAST comparisons A series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) are replaced by "n's", or masked, to prevent spurious matches Mitochondnal and nbosomal RNA sequences are also removed
  • RDMS relational database management system
  • assigns edited sequences to existing templates if available
  • additional sequences are added into the RDMS
  • a process is initiated which modifies existing templates or creates new templates from works in progress (i.e., nonfinal assembled sequences) containing queued sequences or the sequences themselves
  • the templates can be merged into bins. If multiple templates exist in one bin, the bin can be split and the templates reannotated.
  • bins are "clone joined" based upon clone information. Clone joining occurs when the 5' sequence of one clone is present in one bin and the 3' sequence from the same clone is present in a different bin, indicating that the two bins should be merged into a single bin. Only bins which share at least two different clones are merged
  • a resultant template sequence may contain either a partial or a full length open reading frame, or all or part of a genetic regulatory element. This vanation is due in part to the fact that the full length cDNAs of many genes are several hundred, and sometimes several thousand, bases in length. With current technology, cDNAs compnsing the coding regions of large genes cannot be cloned because of vector limitations, incomplete reverse transcnption of the mRNA, or incomplete "second strand" synthesis. Template sequences may be extended to include additional contiguous sequences derived from the parent RNA transcnpt using a variety of methods known to those of skill in the art Extension may thus be used to achieve the full length coding sequence of a gene
  • cDNA sequences are analyzed using a variety of programs and algorithms which are well known in the art. (See, e.g , Ausubel, 1997, supra. Chapter 1.1, Meyers, R.A. (Ed ) ( 1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp 856-853, and Table 5 ) These analyses comprise both reading frame determinations, e.g., based on t ⁇ plet codon periodicity for particular organisms (Fickett, J W ( 1982) Nucleic Acids Res. 10:5303-5318), analyses of potential start and stop codons, and homology searches
  • BLAST Altschul, S.F ( 1993) J. Mol. Evol. 36:290-300; Altschul, S F. et al. (1990) J. Mol. Biol. 215:403-410).
  • BLAST is especially useful in determining exact matches and comparing two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user (Karlin, S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:841-845).
  • GenBank e.g., GenBank
  • SwissProt e.g., GenBank
  • BLOCKS e.g., BLOCKS
  • PFAM e.g., PFAM
  • the mddt of the present invention may be used for a variety of diagnostic and therapeutic purposes
  • an mddt may be used to diagnose a particular condition, disease, or disorder associated with disease detection and treatment molecules
  • diseases, and disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinu ⁇ a, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder
  • the expression of mddt may be routinely assessed by hybridization-based methods to determine, for example, the tissue-specificity, disease-specificity, or developmental stage-specificity of mddt expression
  • the level of expression of mddt may be compared among different cell types or tissues, among diseased and normal cell types or tissues, among cell types or tissues at different developmental stages, or among cell types or tissues undergoing various treatments
  • This type of analysis is useful, for example, to assess the relative levels of mddt expression in fully or partiallv differentiated cells or tissues, to determine if changes in mddt expression levels are correlated with the development or progression of specific disease states, and to assess the response of a cell or tissue to a specific therapy, for example, in pharmacological or toxicological studies
  • Methods for the analysis of mddt expression are based on hybridization and amplification technologies and include membrane-based procedures such as northern blot analysis, high-throughput procedures that utilize, for example, microarrays, and PCR-based procedures
  • the mddt, their fragments, or complementary sequences may be used to identify the presence of and/or to determine the degree of similarity between two (or more) nucleic acid sequences
  • the mddt may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropnately selected temperatures and salt concentrations Hybridization with a probe based on the nucleic acid sequence of at least one of the mddt allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or related to the mddt of the Sequence Listing Probes may be selected from non-conserved or unique regions of at least one of the polynucleotides of SEQ ID NO 1 -14 and tested for their ability to identify or amplify the target nucleic acid sequence using standard protocols Polynucleotide sequences that are capable of hybndizing, in particular, to those shown in
  • SEQ ID NO 1-14 and fragments thereof can be identified using vanous conditions of stringency (See, e g , Wahl, G M and S L Berger ( 1987) Methods Enzymol 152 399-407, Kimmel, A R ( 1987) Methods Enzymol 152 507-51 1 ) Hybndization conditions are discussed in “Definitions "
  • a probe for use in Southern or northern hybridization may be denved from a fragment of an mddt sequence, or its complement, that is up to several hundred nucleotides in length and is either single-stranded or double-stranded
  • Such probes may be hybridized in solution to biological materials such as plasmids, bactenal, yeast, or human artificial chromosomes, cleared or sectioned tissues, or to artificial substrates containing mddt
  • Microarrays are particularly suitable for identifying the presence of and detecting the level of expression for multiple genes of interest by examining gene expression correlated with, e g , various stages of development, treatment with a drug or compound, or disease progression
  • An array analogous to a dot or slot blot may be used to arrange and link polynucleotides to the surface of a substrate using one or more of the following mechanical (vacuum), chemical, thermal, or UV bonding procedures
  • Such an array may contain any number of mddt and may be produced by hand
  • Probes may be labeled by either PCR or enzymatic techniques using a vanety of commercially available reporter molecules
  • commercial kits are available for radioactive and chemiluminescent labeling (Amersham Pharmacia Biotech) and for alkaline phosphatase labeling (Life Technologies)
  • mddt may be cloned into commercially available vectors for the production of RNA probes Such probes may be transcribed
  • polynucleotides of SEQ ID NO 1-14 or suitable fragments thereof can be used to isolate full length cDNA sequences utilizing hybridization and/or amplification procedures well known in the art, e g , cDNA library screening, PCR amplification, etc
  • the molecular cloning of such full length cDNA sequences may employ the method of cDNA library screening with probes using the hybridization, stnngency, washing, and probing strategies described above and in Ausubel, supra. Chapters 3, 5, and 6
  • These procedures may also be employed with genomic libraries to isolate genomic sequences of mddt in order to analyze, e g , regulatory elements
  • Genetic Mapping Gene identification and mapping are important in the investigation and treatment of almost all conditions, diseases, and disorders Cancer, cardiovascular disease, Alzheimer's disease, arthntis, diabetes, and mental illnesses are of particular interest Each of these conditions is more complex than the single gene defects of sickle cell anemia or cystic fibrosis, with select groups of genes being predictive of predisposition for a particular condition, disease, or disorder
  • cardiovascular disease may result from malfunctioning receptor molecules that fail to clear cholesterol from the bloodstream
  • diabetes may result when a particular individual's immune system is activated by an infection and attacks the insulin-producing cells of the pancreas.
  • Alzheimer's disease has been linked to a gene on chromosome 21 ; other studies predict a different gene and location. Mapping of disease genes is a complex and reiterative process and generally proceeds from genetic linkage analysis to physical mapping
  • a genetic linkage map traces parts of chromosomes that are inhented in the same pattern as the condition Statistics link the inheritance of particular conditions to particular regions of chromosomes, as defined by RFLP or other markers.
  • RFLP Radio Resource Line
  • mddt sequences may be used to generate hybndization probes useful in chromosomal mapping of naturally occurring genomic sequences.
  • Either coding or noncodmg sequences of mddt may be used, and in some instances, noncoding sequences may be preferable over coding sequences.
  • conservation of an mddt coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries (See, e.g., Harnngton, J.J. et al. ( 1997) Nat. Genet. 15 345-355, Price, C.MJ 1993) Blood Rev 7 127-134; and Trask, B j ( 1991) Trends Genet.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Meyers, supra, pp. 965-968.) Correlation between the location of mddt on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the mddt sequences may also be used to detect polymorphisms that are genetically linked to the inhe ⁇ tance of a particular condition, disease, or disorder.
  • In situ hybndization of chromosomal preparations and genetic mapping techniques may be used for extending existing genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of the corresponding human chromosome is not known.
  • nucleotide sequences of the subject invention may also be used to detect differences in chromosomal architecture due to translocation, inversion, etc , among normal, earner, or affected individuals
  • a disease-associated gene is mapped to a chromosomal region, the gene must be cloned in order to identify mutations or other alterations (e g , translocations or inversions) that may be correlated with disease
  • This process requires a physical map of the chromosomal region containing the disease-gene of interest along with associated markers
  • a physical map is necessary for determining the nucleotide sequence of and order of marker genes on a particular chromosomal region Physical mapping techniques are well known in the art and require the generation of overlapping sets of cloned DNA fragments from a particular organelle, chromosome, or genome These clones are analyzed to reconstruct and catalog their order Once the position of a marker is determined, the DNA from that region is obtained by consulting the catalog and selecting clones from that region The gene of interest is located through positional cloning techniques using hybridization or similar methods
  • the mddt of the present invention may be used to design probes useful in diagnostic assays Such assays, well known to those skilled in the art, may be used to detect or confirm conditions, disorders, or diseases associated with abnormal levels of mddt expression Labeled probes developed from mddt sequences are added to a sample under hybridizing conditions of desired stringency In some instances, mddt, or fragments or oligonucleotides derived from mddt, may be used as primers in amplification steps prior to hybridization The amount of hybridization complex formed is quantified and compared with standards for that cell or tissue If mddt expression vanes significantly from the standard, the assay indicates the presence of the condition, disorder, or disease Qualitative or quantitative diagnostic methods may include northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, enzyme-linked lmmunosorbent assay (ELISA)- ke, pin, or chip-based assays
  • the probes described above may also be used to monitor the progress of conditions, disorders, or diseases associated with abnormal levels of mddt expression, or to evaluate the efficacy of a particular therapeutic treatment
  • the candidate probe may be identified from the mddt that are specific to a given human tissue and have not been observed in GenBank or other genome databases Such a probe may be used in animal studies, prechnical tests, clinical trials, or in monitoring the treatment of an individual patient
  • standard expression is established by methods well known in the art for use as a basis of comparison, samples from patients affected by the disorder or disease are combined with the probe to evaluate any deviation from the standard profile, and a therapeutic agent is administered and effects are monitored to generate a treatment profile Efficacy is evaluated by determining whether the expression progresses toward or returns to the standard normal pattern
  • Treatment profiles may be generated over a penod of several days or several months Statistical methods well known to those skilled in the art may be use to determine the significance of such therapeutic agents
  • the polynucleotides are also useful for identifying individuals from
  • oligonucleotide primers derived from the mddt of the invention may be used to detect single nucleotide polymorphisms (SNPs) SNPs are substitutions, insertions and 0 deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from the polynucleotide sequences encoding MDDT are used to amplify DNA using the polymerase chain reaction (PCR)
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy 5 samples, bodily fluids, and the like SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denatunng gels
  • fSCCP the oligonucleotide primers are fluorescently labeled, which allows detection of the amp mers in high-throughput equipment such as DNA sequencing machines
  • sequence database analysis methods termed in silico SNP o (
  • reagents capable of identifying the source of a particular tissue can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues Panels of such reagents can identify tissue by species and/or by organ type In a similar fashion, these reagents can be used to screen tissue cultures for contamination
  • polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a particular cell type, in the creation of subtracted cDNA libraries which aid in the discovery of novel polynucleotides, in selection and synthesis of oligomers for attachment to an anay or other support, and as an antigen to elicit an immune response
  • mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e g , the neomycin phosphotransferase gene (neo, Capecchi, M R ( 1989) Science 244 1288-1292)
  • the vector integrates into the conesponding region of the host genome by homologous recombination Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in
  • the mddt of the invention can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease
  • knockin technology a region of mddt is injected into animal ES cells, and the injected sequence integrates into the animal cell genome Transformed cells are injected into blastulae, and the blastulae are implanted as descnbed above Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease
  • a mammal inbred to overexpress mddt, resulting, e g , in the secretion of MDDT in its milk may also serve as a convenient source of that protein (Janne, J et al ( 1998) Biotechnol Annu Rev 4 55-74)
  • MDDT encoded by polynucleotides of the present invention may be used to screen for molecules that bind to or are bound by the encoded polypeptides
  • the binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the bound molecule
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e g , receptors), or small molecules
  • the molecule is closely related to the natural ligand of the polypeptide, e g , a ligand or fragment thereof, a natural substrate, or a structural or functional mimetic (See, Cohgan et al , ( 1991 ) Current Protocols in Immunology 1 (2) Chapter 5 )
  • the molecule can be closely related to the natural receptor to which the polypeptide binds, or to at least a fragment of the receptor, e g , the active site
  • the molecule can be rationally designed using known techniques
  • the screening for these molecules involves producing appropnate cells which express the polypeptide, either as a secreted protein or on the cell membrane
  • Preferred cells include cells from mammals, yeast, Drosophila, or E coli Cells expressing the polypeptide or cell membrane fractions which contain the expressed polypeptide are then contacted with a test compound and binding, stimulation, or inhibition of activity of either the polypeptide or the molecule is analyzed
  • An assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label Alternatively, the assay may assess binding in the presence of a labeled competitor
  • the assay can be earned out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical hbranes, or natural product mixtures
  • the assay may also simply compnse the steps of mixing a candidate compound with a solution containing a polypeptide, measunng polypeptide/molecule activity or binding, and companng the polypeptide/molecule activity or binding to a standard
  • an ELISA assay using, e g., a monoclonal or polyclonal antibody, can measure polypeptide level in a sample
  • the antibody can measure polypeptide level by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate
  • the molecules discovered using these assays can be used to treat disease or to b ⁇ ng about a particular result in a patient (e g , blood vessel growth) by activating or inhibiting the polypeptide/molecule Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.
  • a transc ⁇ pt image is the collective pattern of gene expression by a particular tissue or cell type under given conditions and at a given time This pattern of gene expression is defined by the number of expressed genes, their abundance, and their function
  • the mddt of the present invention may be used to develop a transcript image of a tissue or cell type by hybridizing, preferably in a microarray format, the mddt of the present invention to the totality of transcripts or reverse transcripts of a tissue or cell type.
  • the resultant transcript image would provide a profile of gene activity pertaining to disease detection and treatment
  • Transc ⁇ pt images which profile mddt expression may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples.
  • the transcript image may thus reflect mddt expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line
  • Transcript images may be used to profile mddt expression in distinct tissue types This process can be used to determine disease detection and treatment molecule activity in a particular tissue type relative to this activity in a different tissue type
  • Transcript images may be used to generate a profile of mddt expression charactenstic of diseased tissue.
  • Transcript images of tissues before and after treatment may be used for diagnostic purposes, to monitor the progression of disease, and to monitor the efficacy of drug treatments for diseases which affect the activity of disease detection and treatment molecules.
  • Transcript images which profile mddt expression may also be used in conjunction with in vitro model systems and prechnical evaluation of pharmaceuticals.
  • Transcript images of cell lines can be used to assess disease detection and treatment molecule activity and/or to identify cell lines that lack or misregulate this activity. Such cell lines may then be treated with pharmaceutical agents, and a transcript image following treatment may indicate the efficacy of these agents in restoring desired levels of this activity.
  • a similar approach may be used to assess the toxicity of pharmaceutical agents as reflected by undesirable changes in disease detection and treatment molecule activity
  • Candidate pharmaceutical agents may be evaluated by comparing their associated transc ⁇ pt images with those of pharmaceutical agents of known effectiveness
  • Antisense Molecules The polynucleotides of the present invention are useful in antisense technology Antisense technology or therapy relies on the modulation of expression of a target protein through the specific binding of an antisense sequence to a target sequence encoding the target protein or directing its expression (See, e g , Agrawal, S , ed (1996) Antisense Therapeutics, Humana Press Inc , Totawa NJ , Alama, A et al ( 1997) Pharmacol Res 36(3) 171 - 178, Crooke, S T ( 1997) Adv Pharmacol 40 1-49, Sharma, H W and R Narayanan ( 1995) Bioessays 17( 12) 1055-1063, and Lavrosky, Y et al ( 1997) Biochem Mol Med 62( 1 ) 1 1-22 )
  • An antisense sequence is a polynucleotide sequence capable of specifically hybndizing to at least a portion of the target sequence Antisense sequences bind to cellular m
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcnption, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (See, e g , Slater, J E , et al ( 1998) J Allergy Chn Immunol 102(3) 469-475, and Scanlon, K J , et al (1995) 9(13) 1288-1296 )
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (See, e g , Miller, A D ( 1990) Blood 76 271 , Ausubel, F M et al ( 1995) Cunent Protocols in Molecular Biology.
  • the nucleotide sequences encoding MDDT or fragments thereof may be inserted into an appropnate expression vector, i.e., a vector which contains the necessary elements for transc ⁇ ptional and translational control of the inserted coding sequence in a suitable host.
  • an appropnate expression vector i.e., a vector which contains the necessary elements for transc ⁇ ptional and translational control of the inserted coding sequence in a suitable host.
  • Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding MDDT and appropriate transc ⁇ ptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (See, e g , Sambrook, supra. Chapters 4, 8, 16, and 17, and Ausubel, supra. Chapters 9, 10, 13, and 16.)
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding MDDT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosrrud DNA expression vectors, yeast transformed with yeast expression vectors, insect cell systems infected with viral expression vectors (e.g , baculovirus), plant cell systems transformed with viral expression vectors (e.g , cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal (mammalian) cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosrrud DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g , baculovirus)
  • plant cell systems transformed with viral expression vectors e.g , cauliflower
  • Expression vectors derived from retroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di
  • the invention is not limited by the host cell employed. For long term production of recombinant proteins in mammalian systems, stable expression of MDDT in cell lines is preferred.
  • sequences encoding MDDT can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector Any number of selection systems may be used to recover transformed cell lines.
  • expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector Any number of selection systems may be used to recover transformed cell lines.
  • the mddt of the invention may be used for somatic or germline gene therapy Gene therapy may be performed to (I) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-hnked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288.669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deam ase (ADA) deficiency (Blaese, R.M. et al. ( 1995) Science 270 475-480, Bordignon, C. et al ( 1995) Science 270:470-475), cystic fibrosis (Zabner, J.
  • SCID severe combined immunodeficiency
  • ADA adenosine deam ase
  • HBV hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi
  • mddt In the case where a genetic deficiency in mddt expression or regulation causes disease, the expression of mddt from an approp ⁇ ate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
  • diseases or disorders caused by deficiencies in mddt are treated by constructing mammalian expression vectors compnsing mddt and introducing these vectors by mechanical means into mddt-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vitro include (l) direct DNA microinjection into individual cells, (n) ballistic gold particle delivery, (in) liposome-mediated transfection, (IV) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and Anderson, W.F ( 1993) Annu Rev Biochem. 62.191-217; Ivies, Z. ( 1997) Cell 91 :501-510; Boulay, J-L. and Recipon, H. ( 1998) OUT. Opm. Biotechnol 9.445-450).
  • Expression vectors that may be effective for the expression of mddt include, but are not limited to, the PCDNA 3J, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
  • the mddt of the invention may be expressed using (I) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (n) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H. ( 1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551 , Gossen, M.
  • CMV cytomegalovirus
  • RSV40 virus SV40 virus
  • TK thymidine kinase
  • ⁇ -actin genes e.g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H. ( 1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-
  • hposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
  • hposome transformation allows one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and Eb, A.J. ( 1973) Virology 52.456-467), or by electroporation (Neumann, E. et al. ( 1982) EMBO J 1.841-845)
  • the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to mddt expression are treated by constructing a retrovirus vector consisting of (I) the mddt of the invention under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional retrovirus cts-actmg RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commercially available (Stratagene) and are based on published data (Riviere, I. et al. ( 1995) Proc. Natl. Acad. Sci. U.S.A.
  • the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. ( 1987) J. Virol. 61 1647- 1650; Bender, M A. et al. ( 1987) J. Virol 61 J639-1646; Adam, M.A. and Miller, A.D. ( 1988) J Virol. 62.3802-3806; Dull, T et al. ( 1998) J. Virol. 72:8463-8471 , Zufferey, R. et al.
  • VSVg vector producing cell line
  • U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et alJ 1997) J Virol. 71.7020-7029; Bauer, G.
  • an adenovirus-based gene therapy delivery system is used to deliver mddt to cells which have one or more genetic abnormalities with respect to the expression of mddt.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M E.
  • a he ⁇ es-based, gene therapy delivery system is used to deliver mddt to target cells which have one or more genetic abnormalities with respect to the expression of mddt.
  • HSV he ⁇ es simplex virus
  • the use of he ⁇ es simplex virus (HSV)-based vectors may be especially valuable for introducing mddt to cells of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of he ⁇ es-based vectors are well known to those with ordinary skill in the art.
  • a replication-competent he ⁇ es simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of p ⁇ mates (Liu, X. et al ( 1999) Exp.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("He ⁇ es simplex virus strains for gene transfer"), which is hereby inco ⁇ orated by reference.
  • U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transfened to a cell under the control of the approp ⁇ ate promoter for pu ⁇ oses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W F.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deliver mddt to target cells
  • SFV Semliki Forest Virus
  • a subgenomic RNA is generated that normally encodes the viral capsid proteins
  • This subgenorruc RNA replicates to higher levels than the full-length genomic RNA, resulting in the ove ⁇ roduction of capsid proteins relative to the viral proteins with enzymatic activity (e g , protease and polymerase)
  • enzymatic activity e g , protease and polymerase
  • Anti-MDDT antibodies may be used to analyze protein expression levels Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, and Fab fragments
  • polyclonal, monoclonal, chimeric, single chain, and Fab fragments For descriptions of and protocols of antibody technologies, see, e g , Pound J D ( 1998) Immunochemical Protocols.
  • the amino acid sequence encoded by the mddt of the Sequence Listing may be analyzed by approp ⁇ ate software (e g , LASERGENE NAVIGATOR software, DNASTAR) to determine regions of high lmmunogenicity
  • approp ⁇ ate software e g , LASERGENE NAVIGATOR software, DNASTAR
  • the optimal sequences for immunization are selected from the C-terminus, the N-terminus, and those intervening, hydrophihc regions of the polypeptide which are likely to be exposed to the external environment when the polypeptide is in its natural conformation Analysis used to select appropriate epitopes is also described by Ausubel ( 1997, supra.
  • Peptides used for antibody induction do not need to have biological activity, however, they must be antigenic Peptides used to induce specific antibodies may have an amino acid sequence consisting of at five amino acids, preferably at least 10 amino acids, and most preferably 15 amino acids
  • a peptide which mimics an antigenic fragment of the natural polypeptide may be fused with another protein such as keyhole limpet cyanin (KLH, Sigma, St Louis MO) for antibody production
  • KLH keyhole limpet cyanin
  • a peptide encompassing an antigenic region may be expressed from an mddt, synthesized as described above, or purified from human cells
  • mice, goats, and rabbits may be immunized by injection with a peptide
  • va ⁇ ous adjuvants may be used to increase immunological response
  • peptides about 15 residues in length may be synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using fmoc-chemistry and coupled to KLH (Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, 1995, supra) Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1 % bovine serum albumin (BSA), reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti- rabbit IgG Antisera with antipeptide activity are tested for anti-MDDT activity using protocols well known in the art, including ELISA, radioimmunoassay (RIA), and lmmunoblotting
  • isolated and purified peptide may be used to immunize mice (about 100 ⁇ g of peptide) or rabbits (about 1 mg of peptide) Subsequently, the peptide is radioiodinated and used to screen the immunized animals' B-lymphocytes for production of antipeptide antibodies Positive cells are then used to produce hyb ⁇ domas using standard techniques About 20 mg of peptide is sufficient for labeling and screening several thousand clones Hyb ⁇ domas of interest are detected by screening with radioiodinated peptide to identify those fusions producing peptide-specific monoclonal antibody In a typical protocol, wells of a multi-well plate (FAST, Becton-Dickinson,
  • Antibody fragments containing specific binding sites for an epitope may also be generated for example, such fragments include, but are not limited to, the F(ab')2 fragments produced by pepsin digestion of the antibody molecule, and the Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments Alternatively, construction of Fab expression bra ⁇ es in filamentous bacteriophage allows rapid and easy identification of monoclonal fragments with desired specificity (Pound, supra. Chaps 45-47) Antibodies generated against polypeptide encoded by mddt can be used to pu ⁇ fy and characterize full-length MDDT protein and its activity, binding partners, etc
  • Anti-MDDT antibodies may be used in assays to quantify the amount of MDDT found in a particular human cell Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions
  • the peptides and antibodies of the invention may be used with or without modification or labeled by joining them, either covalently or noncovalently, with a reporter molecule
  • Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS) Such immunoassays typically involve the formation of complexes between the MDDT and its specific antibody and the measurement of such complexes These and other assays are described in Pound (supra)
  • RNA was purchased from CLONTECH Laboratories, Inc (Palo Alto CA) or isolated from vanous tissues Some tissues were homogenized and lysed in guanidimum isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as
  • Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis Plasmids were purified using at least one of the following the Magic or WIZARD Minipreps DNA purification system (Promega), the AGTC Mimprep purification kit (Edge BioSystems, Gaithersburg MD), and the QIAWELL 8, QIAWELL 8 Plus, and QIAWELL 8 Ultra plasmid purification systems or the R E A L PREP 96 plasmid purification kit (QIAGEN)
  • Plasmids were resuspended in 0 1 ml of distilled water and stored, with or without lyophihzation, at 4°C
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V B ( 1994) Anal Biochem 216 1-14 ) Host cell lysis and thermal cycling steps were earned out in a single reaction mixture Samples were processed and stored in
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were earned out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (PE Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra. Chapter 1.1) Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII
  • Component sequences from chromatograms were subject to PHRED analysis and assigned a quality score.
  • the sequences having at least a required quality score were subject to vanous preprocessing editing pathways to eliminate, e.g., low quality 3' ends, vector and linker sequences, polyA tails, Alu repeats, mitochondnal and ribosomal sequences, bactenal contamination sequences, and sequences smaller than 50 base pairs.
  • low-information sequences and repetitive elements e.g., dinucleotide repeats, Alu repeats, etc.
  • sequences were then subject to assembly procedures in which the sequences were assigned to gene bins (bins). Each sequence could only belong to one bin. Sequences in each gene bin were assembled to produce consensus sequences (templates). Subsequent new sequences were added to existing bins using BLASTn (v.1 4 WashU) and CROSSMATCH. Candidate pairs were identified as all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using a version of PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation (sense or antisense) of each assembled template was determined based on the number and orientation of its component sequences.
  • Template sequences as disclosed in the sequence listing correspond to sense strand sequences (the "forward” reading frames), to the best determination
  • the complementary (antisense) strands are inherently disclosed herein
  • the component sequences which were used to assemble each template consensus sequence are listed in Table 4, along with their positions along the template nucleotide sequences
  • Bins were compared against each other and those having local similanty of at least 82% were combined and reassembled Reassembled bins having templates of insufficient overlap (less than 95% local identity) were re-split Assembled templates were also subject to analysis by
  • STITCHER/EXON MAPPER algorithms which analyze the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, etc.
  • the template sequences were further analyzed by translating each template in all three forward reading frames and searching each translation against the Pfam database of hidden Markov model-based protein families and domains using the HMMER software package (available to the public from Washington University School of Medicine, St Louis MO) Regions of templates which, when translated, contain similarity to Pfam consensus sequences are reported in Table 2, along with descnptions of Pfam protein domains and families Only those Pfam hits with an E-value of ⁇ 1 x 10 3 are reported (See also World Wide Web site http //pfam wustl edu/ for detailed descriptions of Pfam protein domains and families ) Additionally, the template sequences were translated in all three forward reading frames, and each translation was searched against hidden Markov models for signal peptide and transmembrane domains using the HMMER software package Construction of hidden Markov models and their usage in sequence analysis has been descnbed (See, for example, Eddy, S R ( 1996) Cun Opin Str Biol 6 361-365 ) Regions of templates which
  • HMMER analysis as reported in Tables 2 and 3 may support the results of BLAST analysis as reported in Table 1 or may suggest alternative or additional properties of template-encoded polypeptides not previously uncovered by BLAST or other analyses
  • Template sequences are further analyzed using the bio formatics tools listed in Table 5, or using sequence analysis software known in the art such as MACDNASIS PRO software (Hitachi Software Engineenng, South San Francisco CA) and LASERGENE software (DNASTAR) Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e g , Sambrook, supra, ch 7, Ausubel, 1995, supra, ch 4 and 16 )
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match
  • the product score is a normalized value between 0 and 100, and is calculated as follows the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences)
  • the BLAST score is 5 calculated by assigning a score of +5 for every base that matches in a high-sco ⁇ ng segment pair (HSP), and -4 for every mismatch Two sequences may share more than one HSP (separated by gaps) If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score
  • the product score represents a balance between fractional overlap and quality in a BLAST alignment For example, a product score of 100 is produced only for 100% identity over the 0 entire length of the shorter of the two sequences being compared A product score
  • a tissue distribution profile is determined for each template by compiling the cDNA library tissue classifications of its component cDNA sequences
  • Each component sequence is derived from a cDNA library constructed from a human tissue
  • Each human tissue is classified into one of the following categones cardiovascular system, connective tissue, digestive system, embryonic 0 structures, endocnne system, exocrme glands, genitalia, female, genitalia, male, germ cells, hermc and immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed, or urinary tract
  • Template sequences, component sequences, and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) 5
  • Oligonucleotide pnmers designed using an mddt of the Sequence Listing are used to extend the nucleic acid sequence
  • One pnmer is synthesized to initiate 5' extension of the template, and the other pnmer, to initiate 3' extension of the template
  • the initial primers may be designed using OLIGO 4 06 software (National Biosciences, Inc (National Biosciences), Plymouth MN), or another 5 appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hai ⁇ in structures and p ⁇ mer-pnmer dime ⁇ zations are avoided Selected human cDNA libraries are used to extend the sequence If more than one extension is necessary or desired, additional or nested sets of pnmers are designed High fidelity amplification is obtained by PCR using methods well known in the art PCR is performed in 96
  • Step 3 57°C, 1 min.
  • Step 4 68°C, 2 min, Step 5 Steps 2, 3, and 4 repeated 20 times, Step 6 68 °C, 5 m ⁇ n, Step 7 storage at 4°C
  • the concentration of DNA in each well is determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0 25% (v/v), Molecular Probes) dissolved in IX T ⁇ s-EDTA (TE) and 0 5 ⁇ l of undiluted PCR product into each well of an opaque fluo ⁇ meter plate (Corning Inco ⁇ orated (Corning), Corning NY), allowing the DNA to bind to the reagent
  • the plate is scanned in a FLUOROSKAN II (Labsy stems Oy) to measure the fluorescence of the sample and to quantify the concentration of DNA
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions are successful in
  • the extended nucleotides are desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to re gation into pUC 18 vector (Amersham Pharmacia Biotech)
  • the digested nucleotides are separated on low concentration (0 6 to 0 8%) agarose gels, fragments are excised, and agar digested with AGAR ACE (Promega) Extended clones are rehgated using T4 ligase (New England Biolabs, Inc , Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E coli cells Transformed cells are selected on antibiotic-containing media, individual colonies are picked and cultured overnight at 37 °C in 384- well plates in LB/2x carbenicil
  • the cells are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters Step 1 94°C, 3 mm, Step 2 94°C, 15 sec, Step 3 60°C, 1 min, Step 4 72°C, 2 min, Step 5 steps 2, 3, and 4 repeated 29 times, Step 6 72 °C, 5 min, Step 7 storage at 4°C DNA is quantified by PICOGREEN reagent (Molecular Probes) as described above Samples with low DNA recoveries are reamphfied using the same conditions as descnbed above Samples are diluted with 20% dimethysulfoxide ( 1 2, v/v), and sequenced using DYENAMIC energy transfer sequencing pnmers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems)
  • the mddt is used to obtain regulatory sequences (promoters, introns, and enhancers) using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library
  • Hybridization probes denved from the mddt of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA
  • the labeling of probe nucleotides between 100 and 1000 nucleotides in length is specifically described, but essentially the same procedure may be used with larger cDNA fragments
  • Probe sequences are labeled at room temperature for 30 minutes using a T4 polynucleotide kinase, ⁇ 32 P-ATP, and 0 5X One-Phor-All Plus (Amersham Pharmacia Biotech) buffer and purified using a ProbeQuant G-50 Microcolumn (Amersham Pharmacia Biotech) The probe mixture is diluted to 10 7 dpm/ ⁇ g/ml hybridization buffer and used in a typical membrane-based hybndization analysis
  • the DNA is digested with a restnction endonuclease such as Eco RV and is electrophoresed through a 0 7% agarose gel
  • the DNA fragments are transfened from the agarose to nylon membrane (NYTRAN Plus, Schleicher & Schuell, Inc , Keene NH) using procedures specified by the manufacturer of the membrane
  • Prehyb ⁇ dization is earned out for three or more hours at 68 °C, and hybndization is earned out overnight at 68 °C
  • blots are sequentially washed at room temperature under increasingly stnngent conditions, up to 0 lx saline sodium citrate (SSC) and 0 5% sodium dodecyl sulfate After the blots are placed in a PHOSPHORIMAGER cassette (Molecular Dynamics) or are exposed to autoradiography film, hybridization patterns of standard and experimental lanes are compared Essentially the same procedure is employed when screening RNA
  • the cDNA sequences which were used to assemble SEQ ID NO 1 -14 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algonthm Sequences from these databases that match SEQ ID NO 1 -14 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as PHRAP (Table 5) Radiation hyb ⁇ d and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the clustered sequences have been previously mapped Inclusion of a mapped sequence in a cluster will result in the assignment of all sequences of that cluster, including its particular SEQ ID NO , to that map location
  • the genetic map locations of SEQ ID NO 1-14 are described as ranges, or intervals, of human chromosomes The map position of an interval, in centiMorgans, is measured relative to the terminus of the chro
  • RNA is isolated from tissue samples using the guanidinium thiocyanate method and polyA + RNA is purified using the oligo (dT) cellulose method
  • Each polyA + RNA sample is reverse transcribed using MMLV reverse-transc ⁇ ptase, 0 05 pg/ ⁇ l ohgo-dT primer (21mer), IX first strand buffer, 0 03 u ts/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech)
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng polyA + RNA with GEMBRIGHT kits
  • RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA (W Lei, unpublished) As quantitative controls, the control mRNAs at 0 002 ng,
  • 0 02 ng, 0 2 ng, and 2 ng are diluted into reverse transcnption reaction at ratios of 1 100,000,
  • each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2 5 ml of 0 5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA Probes are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen ( 1 mg ml), 60 ml sodium acetate, and 300 ml of 100% ethanol The probe is then dried to completion using a SpeedVAC (Savant Instruments Inc , Holbrook NY) and resuspended m 14
  • Sequences of the present invention are used to generate anay elements
  • Each anay element is amplified from bacterial cells containing vectors with cloned cDNA inserts
  • PCR amplification uses pnmers complementary to the vector sequences flanking the cDNA insert
  • Anay elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech)
  • Anay elements are applied to the coated glass substrate using a procedure described in US Patent No 5,807,522, inco ⁇ orated herein by reference 1 ⁇ l of the anay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus The apparatus then deposits about 5 nl of anay element sample per slide
  • Microanays are UV-crosslmked using a STRATALTNKER UV-cross nker (Stratagene) Microanays are washed at room temperature once in 0 2% SDS and three times in distilled water Non-specific binding sites are blocked by incubation of microanays in 0 2% casein in phosphate buffered saline (PBS) (Tropix, Inc , Bedford, MA) for 30 minutes at 60° C followed by washes in 0 2% SDS and distilled water as before
  • PBS phosphate buffered saline
  • Hybridization Hybridization reactions contain 9 ⁇ l of probe mixture consisting of 0 2 ⁇ g each of Cy3 and
  • Detection Reporter-labeled hybndization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc , Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5
  • the excitation laser light is focused on the anay using a 20X microscope objective (Nikon, Inc , Melville NY)
  • the slide containing the anay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective
  • the 1 8 cm x 1 8 cm anay used in the present example is scanned with a resolution of 20 micrometers
  • a mixed gas multiline laser excites the two fluorophores sequentially Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, B ⁇ dgewater NJ) conespondmg to the two fluorophores
  • Appropnate filters positioned between the anay and the photomultiplier tubes are used to filter the signals
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5
  • Each anay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously
  • Th sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the probe mix at a known concentration
  • a specific location on the anay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc , Norwood, MA) installed in an IBM-compatible PC computer
  • A/D analog-to-digital
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal)
  • the data is also analyzed quantitatively Where two different fluorophores are excited and measured simultaneously, the data are first conected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore s emission spectrum
  • a g ⁇ d is supenmposed over the fluorescence signal image such that the signal from each spot is centered in each element of the g ⁇ d
  • the fluorescence signal within each element is then integrated to obtain a numerical value conesponding to the average intensity of the signal
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte)
  • oligonucleotides complementary to the mddt are used to detect, decrease, or inhibit expression of the naturally occurnng nucleotide
  • oligonucleotides comprising from about 15 to 30 base pairs is typical in the art However, smaller or larger sequence fragments can also be used
  • Appropnate oligonucleotides are designed from the mddt using OLIGO 4 06 software (National Biosciences) or other appropriate programs and are synthesized using methods standard in the art or ordered from a commercial supplier
  • a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent transcription factor binding to the promoter sequence
  • a complementary oligonucleotide is designed to prevent ribosomal binding and processing of the transcript
  • cDNA is subcloned into an appropnate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element
  • Recombinant vectors are transformed into suitable bacterial hosts, e g , BL21 (DE3)
  • suitable bacterial hosts e g , BL21 (DE3)
  • Antibiotic resistant bacteria express MDDT upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG)
  • IPTG isopropyl beta-D- thiogalactopyranoside
  • Expression of MDDT in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica cahfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculo
  • MDDT is synthesized as a fusion protein with, e g , glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-H ⁇ s, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates GST, a 26- kilodalton enzyme from Schistosoma lapomcum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech) Following purification, the GST moiety can be proteolytically cleaved from MDDT at specifically engineered sites FLAG, an 8-am ⁇ no acid peptide, enables lmmunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak Company, Rochester NY) 6-H ⁇ s, a stretch of six consecutive histidme residues, enables purification on
  • MDDT or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (See, e g , Bolton, A E and W M Hunter ( 1973) Biochem J 133 529-539 )
  • Bolton-Hunter reagent See, e g , Bolton, A E and W M Hunter ( 1973) Biochem J 133 529-539
  • Candidate molecules previously anayed in the wells of a multi-well plate are incubated with the labeled MDDT, washed, and any wells with labeled MDDT complex are assayed
  • Data obtained using different concentrations of MDDT are used to calculate values for the number, affinity, and association of MDDT with the candidate molecules
  • molecules interacting with MDDT are analyzed using the yeast two-hybrid system as described in Fields, S and O Song ( 1989) Nature 340 245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (CLONTECH) MDDT may also be used in the PATHCALLING process (CuraGen Co ⁇ , New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K et al (2000) U S Patent No 6,057, 101)
  • MDDT function is assessed by expressing mddt at physiologically elevated levels in mammalian cell culture systems cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression
  • Vectors of choice include pCMV SPORT (Life Technologies) and pCR3 1 (Invitrogen Co ⁇ oration, Carlsbad CA), both of which contain the cytomegalovirus promoter 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either hposome formulations or electroporation 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected
  • a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector
  • Marker proteins of choice include, e g , Green Fluorescent Protein (GFP, CLONTECH), CD64, or a CD64-GFP fusion protein
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death
  • events include changes in nuclear DNA content as measured by staining of DNA with propidmm iodide, changes in cell size and granulanty as measured by forward light scatter and 90 degree side light scatter, down-regulation of DNA synthesis as measured by decrease in bromodeoxyu ⁇ dine uptake, alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies, and
  • the MDDT amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding peptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are well described in the art (See, e g , Ausubel, 1995, supra. Chapter 1 1 )
  • peptides 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using fmoc-chemistry and coupled to KLH (Sigma) by reaction with N- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (See, e g , Ausubel, supra )
  • Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant
  • Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG
  • Antisera with antipeptide activity are tested for anti-MDDT activity using protocols well known in the art, including ELISA, RIA, and lmmunoblotting XVII. Purification of Naturally Occurring MDDT Using Specific Antibodies
  • Naturally occurnng or recombinant MDDT is substantially purified by lmmunoaffinity chromatography using antibodies specific for MDDT.
  • An lmmunoaffinity column is constructed by covalently coupling anti-MDDT antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) After the coupling, the resin is blocked and washed according to the manufacturer's instructions
  • Media containing MDDT are passed over the lmmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of MDDT (e.g., high ionic strength buffers in the presence of detergent)
  • the column is eluted under conditions that disrupt antibody/MDDT binding (e.g , a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and MDDT is collected

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Abstract

The present invention provides purified disease detection and treatment molecule polynucleotides (mddt). Also encompassed are the polypeptides (MDDT) encoded by mddt. The invention also provides for the use of mddt, or complements, oligonucleotides, or fragments thereof in diagnostic assays. The invention further provides for vectors and host cells containing mddt for the expression of MDDT. The invention additionally provides for the use of isolated and purified MDDT to induce antibodies and to screen libraries of compounds and the use of anti-MDDT antibodies in diagnostic assays. Also provided are microarrays containing mddt and methods of use.

Description

MOLECULES FOR DISEASE DETECTION AND TREATMENT
TECHNICAL FIELD
The present invention relates to molecules for disease detection and treatment and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases associated with disease detection and treatment molecules
BACKGROUND OF THE INVENTION
The human genome is comprised of thousands of genes, many encoding gene products that function in the maintenance and growth of the various cells and tissues in the body Aberrant expression or mutations in these genes and their products is the cause of, or is associated with, a variety of human diseases such as cancer and other cell proliferative disorders The identification of these genes and their products is the basis of an ever-expanding effort to find markers for early detection of diseases, and targets for their prevention and treatment For example, cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body A wide variety of molecules, either aberrantly expressed or mutated, can be the cause of, or involved with, various cancers because tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis Cell proliferation must be regulated to maintain both the number of cells and their spatial organization This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals such as growth factors and other mitogens, and intracellular cues such as DNA damage or nutrient starvation Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors Aberrant expression or mutations in any of these gene products can result in cell proliferative disorders such as cancer Oncogenes are genes generally derived from normal genes that, through abnormal expression or mutation, can effect the transformation of a normal cell to a malignant one (oncogenesis) Oncoproteins, encoded by oncogenes, can affect cell proliferation in a variety of ways and include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins In contrast, tumor-suppressor genes are involved in inhibiting cell proliferation Mutations which cause reduced or loss of function in tumor-suppressor genes result in aberrant cell proliferation and cancer Thus a wide variety of genes and their products have been found that are associated with cell proliferative disorders such as cancer, but many more may exist that are yet to be discovered DNA-based arrays can provide a simple way to explore the expression of a single polymorphic gene or a large number of genes When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene vaπants For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer A cytochrome p450 gene array is useful to determine whether individuals have one of a number of specific mutations that could result in increased drug metabolism, drug resistance or drug toxicity
DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes There is a growing awareness that gene expression is affected in a global fashion A genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes In some cases the interactions may be expected, such as when the genes are part of the same signaling pathway In other cases, such as when the genes participate in separate signaling pathways, the interactions may be totally unexpected Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes
The discovery of new molecules for disease detection and treatment satisfies a need in the art by providing new compositions which are useful in the diagnosis, study, prevention, and treatment of diseases
SUMMARY OF THE INVENTION
The present invention relates to human polynucleotides encoding molecules for disease detection and treatment (mddt) as presented in the Sequence Listing Some of the mddt uniquely identify genes encoding structural, functional, and regulatory molecules for disease detection and treatment
The invention provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) In one alternative, the polynucleotide composes a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14 In another alternative, the polynucleotide comprises at least 60 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 - 14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) The invention further provides a composition for the detection of expression of disease detection and treatment molecule polynucleotides comprising at least one isolated polynucleotide compnsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d), and a detectable label
The invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide compnsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) The method comprises a) hybridizing the sample with a probe compnsing at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof In one alternative, the probe comprises at least 30 contiguous nucleotides In another alternative, the probe compnses at least 60 contiguous nucleotides
The invention further provides a recombinant polynucleotide comprising a promoter sequence operably linked to an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 - 14, b) a naturally occurnng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) In one alternative, the invention provides a cell transformed with the recombinant polynucleotide In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide In a further alternative, the invention provides a method for producing a disease detection and treatment molecule polypeptide, the method comprising a) cultunng a cell under conditions suitable for expression of the disease detection and treatment molecule polypeptide, wherein said cell is transformed with the recombinant polynucleotide, and b) recovering the disease detection and treatment molecule polypeptide so expressed
The invention also provides a purified disease detection and treatment molecule polypeptide (MDDT) encoded by at least one polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14 Additionally, the invention provides an isolated antibody which specifically binds to the disease detection and treatment molecule polypeptide The invention further provides a method of identifying a test compound which specifically binds to the disease detection and treatment molecule polypeptide, the method comprising the steps of a) providing a test compound, b) combining the disease detection and treatment molecule polypeptide with the test compound for a sufficient time and under suitable conditions for binding, and c) detecting binding of the disease detection and treatment molecule polypeptide to the test compound, thereby identifying the test compound which specifically binds the disease detection and treatment molecule polypeptide The invention further provides a microarray wherein at least one element of the microarray is an isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide compnsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1 -14, b) a naturally occurnng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1- 14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) The invention also provides a method of using the microarray for generating a transcnpt image of a sample which contains polynucleotides The method comprises a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybndization complex, and c) quantifying the expression of the polynucleotides in the sample
Additionally, the invention provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide compnses a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) The method compnses a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide The invention further provides a method for detecting a target polynucleotide in a sample for toxicity testing of a compound, said target polynucleotide compnsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, b) a naturally occurnng polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d) The method comprises a) hybridizing the sample with a probe compnsing at least 20 contiguous nucleotides compnsing a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybndization complex is formed between said probe and said target polynucleotide, b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof, and c) companng the presence, absence or amount of said target polynucleotide in a first biological sample and a second biological sample, wherein said first biological sample has been contacted with said compound, and said second sample is a control, whereby a change in presence, absence or amount of said target polynucleotide in said first sample, as compared with said second sample, is indicative of toxic response to said compound
DESCRIPTION OF THE TABLES
Table 1 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with their GenBank hits (GI Numbers), probability scores, and functional annotations corresponding to the GenBank hits
Table 2 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated "start" and "stop" nucleotide positions The reading frames of the polynucleotide segments and the Pfam hits, Pfam descnptions, and E-values corresponding to the polypeptide domains encoded by the polynucleotide segments are indicated
Table 3 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with polynucleotide segments of each template sequence as defined by the indicated "start" and "stop" nucleotide positions The reading frames of the polynucleotide segments are shown, and the polypeptides encoded by the polynucleotide segments constitute either signal peptide (SP) or transmembrane (TM) domains, as indicated
Table 4 shows the sequence identification numbers (SEQ ID NO s) and template identification numbers (template IDs) corresponding to the polynucleotides of the present invention, along with component sequence identification numbers (component IDs) corresponding to each template The component sequences, which were used to assemble the template sequences, are defined by the indicated "start" and "stop" nucleotide positions along each template
Table 5 summarizes the bioinformatics tools which are useful for analysis of the polynucleotides of the present invention The first column of Table 5 lists analytical tools, programs, and algorithms, the second column provides brief descriptions thereof, the third column presents appropπate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences)
DETAILED DESCRIPTION OF THE INVENTION
Before the nucleic acid sequences and methods are presented, it is to be understood that this invention is not limited to the particular machines, methods, and materials descπbed Although particular embodiments are described, machines, methods, and materials similar or equivalent to these embodiments may be used to practice the invention The preferred machines, methods, and matenals set forth are not intended to limit the scope of the invention which is limited only by the appended claims
The singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise All technical and scientific terms have the meanings commonly understood by one of ordinary skill in the art All publications are incorporated by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies which are presented and which might be used in connection with the invention Nothing in the specification is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention
Definitions
As used herein, the lower case "mddt" refers to a nucleic acid sequence, while the upper case "MDDT" refers to an amino acid sequence encoded by mddt A "full-length" mddt refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in human tissue "Adjuvants" are materials such as Freund's adjuvant, mineral gels (aluminum hydroxide), and surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol) which may be administered to increase a host's immunological response
"Allele" refers to an alternative form of a nucleic acid sequence Alleles result from a "mutation," a change or an alternative reading of the genetic code Any given gene may have none, one, or many allelic forms Mutations which give rise to alleles include deletions, additions, or substitutions of nucleotides Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence The present invention encompasses allelic mddt "Amino acid sequence" refers to a peptide, a polypeptide, or a protein of either natural or synthetic origin The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a fragment, epitope, vaπant, or derivative of a protein expressed by a nucleic acid sequence.
"Amplification" refers to the production of additional copies of a sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art
"Antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic determinant Antibodies that bind MDDT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen The polypeptide or peptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired Commonly used earners that are chemically coupled to peptides include bovine serum albumin, thyroglobulm, and keyhole limpet hemocyanin (KLH) The coupled peptide is then used to immunize the animal
"Antisense sequence" refers to a sequence capable of specifically hybπdizing to a target sequence The antisense sequence may include DNA, RNA, or any nucleic acid mimic or analog such as peptide nucleic acid (PNA), oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracιl, or 7-deaza-2'-deoxyguanosme. "Antisense sequence" refers to a sequence capable of specifically hybridizing to a target sequence The antisense sequence can be DNA, RNA, or any nucleic acid mimic or analog
"Antisense technology" refers to any technology which relies on the specific hybridization of an antisense sequence to a target sequence
A "bin" is a portion of computer memory space used by a computer program for storage of data, and bounded in such a manner that data stored in a bin may be retrieved by the program
"Biologically active" refers to an amino acid sequence having a structural, regulatory, or biochemical function of a naturally occurnng amino acid sequence
"Clone joining" is a process for combining gene bins based upon the bins' containing sequence information from the same clone. The sequences may assemble into a primary gene transcript as well as one or more splice variants
"Complementary" descnbes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing (5'-A-G-T-3T pairs with its complement 3'-T-C-A-5')
A "component sequence" is a nucleic acid sequence selected by a computer program such as PHRED and used to assemble a consensus or template sequence from one or more component sequences A "consensus sequence" or "template sequence" is a nucleic acid sequence which has been assembled from overlapping sequences, using a computer program for fragment assembly such as the GELVIEW fragment assembly system (Genetics Computer Group (GCG), Madison WI) or using a relational database management system (RDMS)
"Conservative amino acid substitutions" are those substitutions that, when made, least interfere with the properties of the original protein, 1 e , the structure and especially the function of the protein is conserved and not significantly changed by such substitutions The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions
Oπεinal Residue Conservative Substitution
Ala Gly, Ser
Gin Asn, Glu, His
He Leu, Val
Leu He, Val
Lys Arg, Gin, Glu
Met Leu, He
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain
"Deletion" refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or am o acid residue, respectively, is absent
"Derivative" refers to the chemical modification of a nucleic acid sequence, such as by replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray "E-value" refers to the statistical probability that a match between two sequences occurred by chance
A "fragment" is a unique portion of mddt or MDDT which is identical in sequence to but shorter in length than the parent sequence A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/ amino acid residue For example, a fragment may comprise from 10 to 1000 contiguous amino acid residues or nucleotides A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues or nucleotides in length Fragments may be preferentially selected from certain regions of a molecule For example, a polypeptide fragment may compnse a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing and the figures, may be encompassed by the present embodiments A fragment of mddt compnses a region of unique polynucleotide sequence that specifically identifies mddt, for example, as distinct from any other sequence in the same genome A fragment of mddt is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish mddt from related polynucleotide sequences The precise length of a fragment of mddt and the region of mddt to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment A fragment of MDDT is encoded by a fragment of mddt A fragment of MDDT comprises a region of unique amino acid sequence that specifically identifies MDDT For example, a fragment of MDDT is useful as an lmmunogenic peptide for the development of antibodies that specifically recognize MDDT The precise length of a fragment of MDDT and the region of MDDT to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment
A "full length" nucleotide sequence is one containing at least a start site for translation to a protein sequence, followed by an open reading frame and a stop site, and encoding a "full length" polypeptide
"Hit" refers to a sequence whose annotation will be used to descnbe a given template Criteria for selecting the top hit are as follows if the template has one or more exact nucleic acid matches, the top hit is the exact match with highest percent identity If the template has no exact matches but has significant protein hits, the top hit is the protein hit with the lowest E-value If the template has no significant protein hits, but does have significant non-exact nucleotide hits, the top hit is the nucleotide hit with the lowest E-value "Homology" refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of an mddt or between a reference amino acid sequence and a fragment of an MDDT
"Hybndization" refers to the process by which a strand of nucleotides anneals with a complementary strand through base pairing Specific hybndization is an indication that two nucleic acid sequences share a high degree of identity Specific hybndization complexes form under defined annealing conditions, and remain hybndized after the "washing" step The defined hybridization conditions include the annealing conditions and the washing step(s), the latter of which is particularly important in determining the stnngency of the hybndization process, with more stnngent conditions allowing less non-specific binding, I e , binding between pairs of nucleic acid probes that are not perfectly matched Permissive conditions for annealing of nucleic acid sequences are routinely determinable and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency
Generally, stnngency of hybridization is expressed with reference to the temperature under which the wash step is earned out Generally, such wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe An equation for calculating Tm and conditions for nucleic acid hybridization is well known and can be found in Sambrook et al , 1989, Molecular Cloning A Laboratory Manual, 2nd ed , vol 1 -3, Cold Spring Harbor Press, Plainview NY, specifically see volume 2, chapter 9
High stnngency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0 2 x SSC and about 0 1 % SDS, for 1 hour Alternatively, temperatures of about 65°C, 60°C, or 55°C may be used SSC concentration may be varied from about 0 2 to 2 x SSC, with SDS being present at about 0 1 %
Typically, blocking reagents are used to block non-specific hybndization Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200 μg/ml Useful variations on these conditions will be readily apparent to those skilled in the art Hybndization, particularly under high stringency conditions, may be suggestive of evolutionary similanty between the nucleotides Such similarity is strongly indicative of a similar role for the nucleotides and their resultant proteins Other parameters, such as temperature, salt concentration, and detergent concentration may be varied to achieve the desired stnngency Denaturants, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as RNA DNA hybridizations Appropnate hybndization conditions are routinely determinable by one of ordinary skill in the art "lmmunogenic" descnbes the potential for a natural, recombinant, or synthetic peptide, epitope, polypeptide, or protein to induce antibody production appropnate animals, cells, or cell lines
"Insertion" or "addition" refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or residue, respectively, is added to the sequence
"Labeling" refers to the covalent or noncovalent joining of a polynucleotide, polypeptide, or antibody with a reporter molecule capable of producing a detectable or measurable signal
"Microarray" is any aπangement of nucleic acids, amino acids, antibodies, etc , on a substrate The substrate may be a solid support such as beads, glass, paper, nitrocellulose, nylon, or an appropriate membrane
"Linkers" are short stretches of nucleotide sequence which may be added to a vector or an mddt to create restnction endonuclease sites to facilitate cloning "Polylinkers" are engineered to incorporate multiple restriction enzyme sites and to provide for the use of enzymes which leave 5' or 3' overhangs (e g , BamHI, EcoRI, and Hindlll) and those which provide blunt ends (e g , EcoRV, SnaBI, and Stul)
"Naturally occurring" refers to an endogenous polynucleotide or polypeptide that may be isolated from viruses or prokaryotic or eukaryotic cells
"Nucleic acid sequence" refers to the specific order of nucleotides joined by phosphodiester bonds in a linear, polymenc arrangement Depending on the number of nucleotides, the nucleic acid sequence can be considered an oligomer, oligonucleotide, or polynucleotide The nucleic acid can be DNA, RNA, or any nucleic acid analog, such as PNA, may be of genomic or synthetic origin, may be either double-stranded or single-stranded, and can represent either the sense or antisense (complementary) strand
"Oligomer" refers to a nucleic acid sequence of at least about 6 nucleotides and as many as about 60 nucleotides, preferably about 15 to 40 nucleotides, and most preferably between about 20 and 30 nucleotides, that may be used in hybridization or amplification technologies Oligomers may be used as, e g , pnmers for PCR, and are usually chemically synthesized
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence Generally, operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame
"Peptide nucleic acid" (PNA) refers to a DNA mimic in which nucleotide bases are attached to a pseudopeptide backbone to increase stability PNAs, also designated antigene agents, can prevent gene expression by targeting complementary messenger RNA The phrases "percent identity" and "% identity", as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algonthm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI) CLUSTAL V is described in Higgins, D.G. and Sharp, P.M ( 1989) CABIOS 5: 151-153 and in Higgins, D.G. et al. ( 1992)
CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows. Ktuple=2, gap penalty=5, wιndow=4, and "diagonals saved"=4 The "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequence pairs Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S F. et al. ( 1990) J Mol. Biol. 215 403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http7/www. ncbi.nlm.nih.gov/BLAST/ The BLAST software suite includes various sequence analysis programs including "blastn," that is used to determine alignment between a known polynucleotide sequence and other sequences on a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www ncbi.nlm nih.gov/gorf/bl2/. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below) BLAST programs are commonly used with gap and other parameters set to default settings For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1
Penalty for mismatch: -2 Open Gap: 5 and Extension Gap. 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter on
Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein
The phrases "percent identity" and "% identity", as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm Methods of polypeptide sequence alignment are well-known Some alignment methods take into account conservative amino acid substitutions Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity of the substituted residue, thus preserving the structure (and therefore function) of the folded polypeptide
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3 12e sequence alignment program (described and referenced above) For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows Ktuple= 1 , gap penalty=3, wιndow=5, and "diagonals saved"=5 The PAM250 matrix is selected as the default residue weight table As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polypeptide sequence pairs
Alternatively the NCBI BLAST software suite may be used For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2 0 9 (May-07-1999) with blastp set at default parameters Such default parameters may be, for example
Matrix BLOSUM62 Open Gap 11 and Extension Gap 1 penalty
Gap x drop-off 50
Expect 10
Filter on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured
"Post-translational modification" of an MDDT may involve hpidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art These processes may occur synthetically or biochemically Biochemical modifications will vary by cell type depending on the enzymatic milieu and the MDDT
"Probe" refers to mddt or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes "Pnmers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing The primer may then be extended along the target DNA strand by a DNA polymerase enzyme Pnmer pairs can be used for amplification (and identification) of a nucleic acid sequence, e g , by the polymerase chain reaction (PCR) Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the figures and Sequence Listing, may be used
Methods for preparing and using probes and pnmers are descnbed in the references, for example Sambrook et al , 1989, Molecular Cloning A Laboratory Manual, 2nd ed , vol 1-3, Cold Spnng Harbor Press, Plainview NY, Ausubel et al J987, Current Protocols in Molecular Biology. Greene Publ Assoc & Wiley-Intersciences, New York NY, Innis et al , 1990, PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA PCR pnmer pairs can be denved from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0 5, 1991 , Whitehead Institute for Biomedical Research, Cambndge MA) Oligonucleotides for use as pnmers are selected using software known in the art for such purpose For example, OLIGO 4 06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases Similar primer selection programs have incorporated additional features for expanded capabilities For example, the PπmOU pnmer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope The Pπmer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispnming library," in which sequences to avoid as primer binding sites are user-specified Pπmer3 is useful, in particular, for the selection of oligonucleotides for microarrays (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs ) The PnmeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs pnmers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybndization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids Methods of oligonucleotide selection are not limited to those described above "Purified" refers to molecules, either polynucleotides or polypeptides that are isolated or separated from their natural environment and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other compounds with which they are naturally associated
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e g , by genetic engineering techniques such as those described in Sambrook, supra The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell
Alternatively, such recombinant nucleic acids may be part of a viral vector, e g , based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal "Regulatory element" refers to a nucleic acid sequence from nontranslated regions of a gene, and includes enhancers, promoters, introns, and 3' untranslated regions, which interact with host proteins to carry out or regulate transcription or translation
"Reporter" molecules are chemical or biochemical moieties used for labeling a nucleic acid, an amino acid, or an antibody They include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cof actors, inhibitors, magnetic particles, and other moieties known in the art
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymme are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyπbose
"Sample" is used in its broadest sense Samples may contain nucleic or amino acids, antibodies, or other materials, and may be derived from any source (e g , bodily fluids including, but not limited to, saliva, blood, and urine, chromosome(s), organelles, or membranes isolated from a cell, genomic DNA, RNA, or cDNA in solution or bound to a substrate, and cleared cells or tissues or blots or imprints from such cells or tissues)
"Specific binding" or "specifically binding" refers to the interaction between a protein or peptide and its agonist, antibody, antagonist, or other binding partner The interaction is dependent upon the presence of a particular structure of the protein, e g , the antigenic determinant or epitope, recognized by the binding molecule For example, if an antibody is specific for epitope "A," the presence of a polypeptide containing epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody
"Substitution" refers to the replacement of at least one nucleotide or ammo acid by a different nucleotide or amino acid
"Substrate" refers to any suitable rigid or semi-rigid support including, e g , membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles or capillaries The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound A "transcript image" refers to the collective pattern of gene expression by a particular tissue or cell type under given conditions at a given time
"Transformation" refers to a process by which exogenous DNA enters a recipient cell Transformation may occur under natural or artificial conditions using vanous methods well known in the art Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell The method is selected based on the host cell being transformed
"Transformants" include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as cells which transiently express inserted DNA or RNA
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al ( 1989), supra
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 25% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters Such a pair of nucleic acids may show, for example, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or even at least 98% or greater sequence identity over a certain defined length The variant may result in "conservative" amino acid changes which do not affect structural and/or chemical properties A variant may be described as, for example, an "allelic" (as defined above), "splice," "species," or "polymorphic" variant A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons dunng mRNA processing The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule Species variants are polynucleotide sequences that vary from one species to another The resulting polypeptides generally will have significant amino acid identity relative to each other A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state
In an alternative, variants of the polynucleotides of the present invention may be generated through recombinant methods One possible method is a DNA shuffling technique such as MOLECULARBREEDING (Maxygen Inc , Santa Clara CA, described in U S Patent Number 5,837,458, Chang, C -C et al ( 1999) Nat Biotechnol 17 793-797, Chnstians, F C et al (1999) Nat Biotechnol 17 259-264, and Crameπ, A et al ( 1996) Nat. Biotechnol 14 315-319) to alter or improve the biological properties of MDDT, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner
A "vanant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2 0 9 (May-07- 1999) set at default parameters Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides
THE INVENTION
In a particular embodiment, cDNA sequences derived from human tissues and cell lines were aligned based on nucleotide sequence identity and assembled into "consensus" or "template" sequences which are designated by the template identification numbers (template IDs) in column 2 of Table 1 The sequence identification numbers (SEQ ID NO s) conesponding to the template IDs are shown in column 1 The template sequences have similanty to GenBank sequences, or "hitsJ as designated by the GI Numbers in column 3 The statistical probability of each GenBank hit is indicated by a probability score in column 4, and the functional annotation corresponding to each GenBank hit is listed in column 5 The invention incorporates the nucleic acid sequences of these templates as disclosed in the Sequence Listing and the use of these sequences in the diagnosis and treatment of disease states charactenzed by defects in molecules for disease detection and treatment The invention further utilizes these sequences in hybridization and amplification technologies, and in particular, in technologies which assess gene expression patterns conelated with specific cells or tissues and their responses in vivo or in vitro to pharmaceutical agents, toxins, and other treatments In this manner, the sequences of the present invention are used to develop a transcnpt image for a particular cell or tissue
Deπvation of Nucleic Acid Sequences cDNA was isolated from libraries constructed using RNA derived from normal and diseased human tissues and cell lines The human tissues and cell lines used for cDNA library construction were selected from a broad range of sources to provide a diverse population of cDNAs representative of gene transcnption throughout the human body Descnptions of the human tissues and cell lines used for cDNA library construction are provided in the LIFESEQ database (Incyte Genomics, Inc (Incyte), Palo Alto CA) Human tissues were broadly selected from, for example, cardiovascular, dermatologic, endocrine, gastrointestinal, hematopoietic/immune system, musculoskeletal, neural, reproductive, and urologic sources
Cell lines used for cDNA library construction were denved from, for example, leukemic cells, teratocarcinomas, neuroepithehomas, cervical carcinoma, lung fibroblasts, and endothelial cells Such cell lines include, for example, THP-1 , Jurkat, HUVEC, hNT2, WI38, HeLa, and other cell lines commonly used and available from public depositories (American Type Culture Collection, Manassas VA) Pnor to mRNA isolation, cell lines were untreated, treated with a pharmaceutical agent such as 5'-aza-2'-deoxycytιdιne, treated with an activating agent such as lipopolysacchande in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress
Sequencing of the cDNAs
Methods for DNA sequencing are well known in the art Conventional enzymatic methods employ the Klenow fragment of DNA polymerase I, SEQUENASE DNA polymerase (U S Biochemical Coφoration, Cleveland OH), Taq polymerase (PE Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Inc (Amersham Pharmacia Biotech), Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies Inc (Life Technologies), Gaithersburg MD), to extend the nucleic acid sequence from an oligonucleotide pnmer annealed to the DNA template of interest Methods have been developed for the use of both single-stranded and double- stranded templates Chain termination reaction products may be electrophoresed on urea- polyacrylanude gels and detected either by autoradiography (for radioisotope-labeled nucleotides) or by fluorescence (for fluorophore-labeled nucleotides) Automated methods for mechanized reaction preparation, sequencing, and analysis using fluorescence detection methods have been developed. Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system (Hamilton Company (Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc. (MJ Research), Watertown MA), and ABI CATALYST 800 thermal cycler (PE Biosystems) Sequencing can be earned out using, for example, the ABI 373 or 377 (PE Biosystems) or MEGABACE 1000 (Molecular Dynamics, Inc (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or other automated and manual sequencing systems well known in the art. The nucleotide sequences of the Sequence Listing have been prepared by current, state-of- the-art, automated methods and, as such, may contain occasional sequencing errors or unidentified nucleotides Such unidentified nucleotides are designated by an N These infrequent unidentified bases do not represent a hindrance to practicing the invention for those skilled in the art Several methods employing standard recombinant techniques may be used to correct errors and complete the missing sequence information (See, e g., those described in Ausubel, F M et al. ( 1997) Short
Protocols in Molecular Biology, John Wiley & Sons, New York NY, and Sambrook, J. et al. ( 1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY )
Assembly of cDNA Sequences Human polynucleotide sequences may be assembled using programs or algorithms well known in the art Sequences to be assembled are related, wholly or in part, and may be derived from a single or many different transcπpts Assembly of the sequences can be performed using such programs as PHRAP (Phils Revised Assembly Program) and the GELVIEW fragment assembly system (GCG), or other methods known in the art Alternatively, cDNA sequences are used as "component" sequences that are assembled into
"template" or "consensus" sequences as follows Sequence chromatograms are processed, veπfied, and quality scores are obtained using PHRED Raw sequences are edited using an editing pathway known as Block 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte Genomics, Palo Alto, CA). A series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) are replaced by "n's", or masked, to prevent spurious matches Mitochondnal and nbosomal RNA sequences are also removed The processed sequences are then loaded into a relational database management system (RDMS) which assigns edited sequences to existing templates, if available When additional sequences are added into the RDMS, a process is initiated which modifies existing templates or creates new templates from works in progress (i.e., nonfinal assembled sequences) containing queued sequences or the sequences themselves After the new sequences have been assigned to templates, the templates can be merged into bins. If multiple templates exist in one bin, the bin can be split and the templates reannotated.
Once gene bins have been generated based upon sequence alignments, bins are "clone joined" based upon clone information. Clone joining occurs when the 5' sequence of one clone is present in one bin and the 3' sequence from the same clone is present in a different bin, indicating that the two bins should be merged into a single bin. Only bins which share at least two different clones are merged
A resultant template sequence may contain either a partial or a full length open reading frame, or all or part of a genetic regulatory element. This vanation is due in part to the fact that the full length cDNAs of many genes are several hundred, and sometimes several thousand, bases in length. With current technology, cDNAs compnsing the coding regions of large genes cannot be cloned because of vector limitations, incomplete reverse transcnption of the mRNA, or incomplete "second strand" synthesis. Template sequences may be extended to include additional contiguous sequences derived from the parent RNA transcnpt using a variety of methods known to those of skill in the art Extension may thus be used to achieve the full length coding sequence of a gene
Analysis of the cDNA Sequences
The cDNA sequences are analyzed using a variety of programs and algorithms which are well known in the art. (See, e.g , Ausubel, 1997, supra. Chapter 1.1, Meyers, R.A. (Ed ) ( 1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp 856-853, and Table 5 ) These analyses comprise both reading frame determinations, e.g., based on tπplet codon periodicity for particular organisms (Fickett, J W ( 1982) Nucleic Acids Res. 10:5303-5318), analyses of potential start and stop codons, and homology searches
Computer programs known to those of skill in the art for performing computer-assisted searches for amino acid and nucleic acid sequence similarity, include, for example, Basic Local
Alignment Search Tool (BLAST; Altschul, S.F ( 1993) J. Mol. Evol. 36:290-300; Altschul, S F. et al. (1990) J. Mol. Biol. 215:403-410). BLAST is especially useful in determining exact matches and comparing two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user (Karlin, S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:841-845). Using an appropnate search tool (e.g., BLAST or HMM), GenBank, SwissProt, BLOCKS, PFAM and other databases may be searched for sequences containing regions of homology to a query mddt or MDDT of the present invention.
Other approaches to the identification, assembly, storage, and display of nucleotide and polypeptide sequences are provided in "Relational Database for Stonng Biomolecule Information/' U S S N 08/947,845, filed October 9, 1997, "Project-Based Full-Length Biomolecular Sequence Database," U S S N 08/81 1 ,758, filed March 6, 1997, and "Relational Database and System for Storing Information Relating to Biomolecular Sequences," U S S N 09/034,807, filed March 4, 1998, all of which are incorporated by reference herein in their entirety Protein hierarchies can be assigned to the putative encoded polypeptide based on, e g , motif,
BLAST, or biological analysis Methods for assigning these hierarchies are descnbed, for example, in "Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Data," U S S N 08/812,290, filed March 6, 1997, incorporated herein by reference
Human Disease Detection and Treatment Molecule Sequences
The mddt of the present invention may be used for a variety of diagnostic and therapeutic purposes For example, an mddt may be used to diagnose a particular condition, disease, or disorder associated with disease detection and treatment molecules Such conditions, diseases, and disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuπa, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus and an autoimmune/inflammatory disorder, such as actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondyhtis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mel tus, emphysema, erythroblastosis feta s, erythema nodosum, atrophic gastntis, glomerulonephπtis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuπa, hepatitis, hypereosinophiha, lrntable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthema gravis, myocardial or pencardial inflammation, myelofibrosis, osteoarthntis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthntis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, pnmary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma The mddt can be used to detect the presence of, or to quantify the amount of, an mddt-related polynucleotide in a sample This information is then compared to information obtained from appropnate reference samples, and a diagnosis is established Alternatively, a polynucleotide complementary to a given mddt can inhibit or inactivate a therapeutically relevant gene related to the mddt
Analysis of mddt Expression Patterns
The expression of mddt may be routinely assessed by hybridization-based methods to determine, for example, the tissue-specificity, disease-specificity, or developmental stage-specificity of mddt expression For example, the level of expression of mddt may be compared among different cell types or tissues, among diseased and normal cell types or tissues, among cell types or tissues at different developmental stages, or among cell types or tissues undergoing various treatments This type of analysis is useful, for example, to assess the relative levels of mddt expression in fully or partiallv differentiated cells or tissues, to determine if changes in mddt expression levels are correlated with the development or progression of specific disease states, and to assess the response of a cell or tissue to a specific therapy, for example, in pharmacological or toxicological studies Methods for the analysis of mddt expression are based on hybridization and amplification technologies and include membrane-based procedures such as northern blot analysis, high-throughput procedures that utilize, for example, microarrays, and PCR-based procedures
Hybridization and Genetic Analysis
The mddt, their fragments, or complementary sequences, may be used to identify the presence of and/or to determine the degree of similarity between two (or more) nucleic acid sequences The mddt may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropnately selected temperatures and salt concentrations Hybridization with a probe based on the nucleic acid sequence of at least one of the mddt allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or related to the mddt of the Sequence Listing Probes may be selected from non-conserved or unique regions of at least one of the polynucleotides of SEQ ID NO 1 -14 and tested for their ability to identify or amplify the target nucleic acid sequence using standard protocols Polynucleotide sequences that are capable of hybndizing, in particular, to those shown in
SEQ ID NO 1-14 and fragments thereof, can be identified using vanous conditions of stringency (See, e g , Wahl, G M and S L Berger ( 1987) Methods Enzymol 152 399-407, Kimmel, A R ( 1987) Methods Enzymol 152 507-51 1 ) Hybndization conditions are discussed in "Definitions "
A probe for use in Southern or northern hybridization may be denved from a fragment of an mddt sequence, or its complement, that is up to several hundred nucleotides in length and is either single-stranded or double-stranded Such probes may be hybridized in solution to biological materials such as plasmids, bactenal, yeast, or human artificial chromosomes, cleared or sectioned tissues, or to artificial substrates containing mddt Microarrays are particularly suitable for identifying the presence of and detecting the level of expression for multiple genes of interest by examining gene expression correlated with, e g , various stages of development, treatment with a drug or compound, or disease progression An array analogous to a dot or slot blot may be used to arrange and link polynucleotides to the surface of a substrate using one or more of the following mechanical (vacuum), chemical, thermal, or UV bonding procedures Such an array may contain any number of mddt and may be produced by hand or by using available devices, materials, and machines Microarrays may be prepared, used, and analyzed using methods known in the art (See, e g ,
Brennan, T M et al (1995) U S Patent No 5,474,796, Schena, M et al (1996) Proc Natl Acad Sci USA 93 10614-10619, Baldeschweιler et al (1995) PCT application W095/251 1 16, Shalon, D et al (1995) PCT application WO95/35505, Heller, R A et al (1997) Proc Natl Acad Sci USA 94 2150- 2155, and Heller, M J et al (1997) U S Patent No 5,605,662 ) Probes may be labeled by either PCR or enzymatic techniques using a vanety of commercially available reporter molecules For example, commercial kits are available for radioactive and chemiluminescent labeling (Amersham Pharmacia Biotech) and for alkaline phosphatase labeling (Life Technologies) Alternatively, mddt may be cloned into commercially available vectors for the production of RNA probes Such probes may be transcribed in the presence of at least one labeled nucleotide (e g , 32P-ATP, Amersham Pharmacia Biotech)
Additionally the polynucleotides of SEQ ID NO 1-14 or suitable fragments thereof can be used to isolate full length cDNA sequences utilizing hybridization and/or amplification procedures well known in the art, e g , cDNA library screening, PCR amplification, etc The molecular cloning of such full length cDNA sequences may employ the method of cDNA library screening with probes using the hybridization, stnngency, washing, and probing strategies described above and in Ausubel, supra. Chapters 3, 5, and 6 These procedures may also be employed with genomic libraries to isolate genomic sequences of mddt in order to analyze, e g , regulatory elements
Genetic Mapping Gene identification and mapping are important in the investigation and treatment of almost all conditions, diseases, and disorders Cancer, cardiovascular disease, Alzheimer's disease, arthntis, diabetes, and mental illnesses are of particular interest Each of these conditions is more complex than the single gene defects of sickle cell anemia or cystic fibrosis, with select groups of genes being predictive of predisposition for a particular condition, disease, or disorder For example, cardiovascular disease may result from malfunctioning receptor molecules that fail to clear cholesterol from the bloodstream, and diabetes may result when a particular individual's immune system is activated by an infection and attacks the insulin-producing cells of the pancreas. In some studies, Alzheimer's disease has been linked to a gene on chromosome 21 ; other studies predict a different gene and location. Mapping of disease genes is a complex and reiterative process and generally proceeds from genetic linkage analysis to physical mapping
As a condition is noted among members of a family, a genetic linkage map traces parts of chromosomes that are inhented in the same pattern as the condition Statistics link the inheritance of particular conditions to particular regions of chromosomes, as defined by RFLP or other markers. (See, for example, Lander, E. S. and Botstein, D. (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.) Occasionally, genetic markers and their locations are known from previous studies More often, however, the markers are simply stretches of DNA that differ among individuals. Examples of genetic linkage maps can be found in various scientific journals or at the Online Mendehan Inheritance in Man (OMIM) World Wide Web site.
In another embodiment of the invention, mddt sequences may be used to generate hybndization probes useful in chromosomal mapping of naturally occurring genomic sequences. Either coding or noncodmg sequences of mddt may be used, and in some instances, noncoding sequences may be preferable over coding sequences For example, conservation of an mddt coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries (See, e.g., Harnngton, J.J. et al. ( 1997) Nat. Genet. 15 345-355, Price, C.MJ 1993) Blood Rev 7 127-134; and Trask, B j ( 1991) Trends Genet. 7J49-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Meyers, supra, pp. 965-968.) Correlation between the location of mddt on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. The mddt sequences may also be used to detect polymorphisms that are genetically linked to the inheπtance of a particular condition, disease, or disorder.
In situ hybndization of chromosomal preparations and genetic mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending existing genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of the corresponding human chromosome is not known. These new marker sequences can be mapped to human chromosomes and may provide valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques Once a disease or syndrome has been crudely correlated by genetic linkage with a particular genomic region, e g , ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (See, e g , Gatti, R A et al ( 1988) Nature 336 577-580 ) The nucleotide sequences of the subject invention may also be used to detect differences in chromosomal architecture due to translocation, inversion, etc , among normal, earner, or affected individuals
Once a disease-associated gene is mapped to a chromosomal region, the gene must be cloned in order to identify mutations or other alterations (e g , translocations or inversions) that may be correlated with disease This process requires a physical map of the chromosomal region containing the disease-gene of interest along with associated markers A physical map is necessary for determining the nucleotide sequence of and order of marker genes on a particular chromosomal region Physical mapping techniques are well known in the art and require the generation of overlapping sets of cloned DNA fragments from a particular organelle, chromosome, or genome These clones are analyzed to reconstruct and catalog their order Once the position of a marker is determined, the DNA from that region is obtained by consulting the catalog and selecting clones from that region The gene of interest is located through positional cloning techniques using hybridization or similar methods
Diagnostic Uses
The mddt of the present invention may be used to design probes useful in diagnostic assays Such assays, well known to those skilled in the art, may be used to detect or confirm conditions, disorders, or diseases associated with abnormal levels of mddt expression Labeled probes developed from mddt sequences are added to a sample under hybridizing conditions of desired stringency In some instances, mddt, or fragments or oligonucleotides derived from mddt, may be used as primers in amplification steps prior to hybridization The amount of hybridization complex formed is quantified and compared with standards for that cell or tissue If mddt expression vanes significantly from the standard, the assay indicates the presence of the condition, disorder, or disease Qualitative or quantitative diagnostic methods may include northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, enzyme-linked lmmunosorbent assay (ELISA)- ke, pin, or chip-based assays
The probes described above may also be used to monitor the progress of conditions, disorders, or diseases associated with abnormal levels of mddt expression, or to evaluate the efficacy of a particular therapeutic treatment The candidate probe may be identified from the mddt that are specific to a given human tissue and have not been observed in GenBank or other genome databases Such a probe may be used in animal studies, prechnical tests, clinical trials, or in monitoring the treatment of an individual patient In a typical process, standard expression is established by methods well known in the art for use as a basis of comparison, samples from patients affected by the disorder or disease are combined with the probe to evaluate any deviation from the standard profile, and a therapeutic agent is administered and effects are monitored to generate a treatment profile Efficacy is evaluated by determining whether the expression progresses toward or returns to the standard normal pattern Treatment profiles may be generated over a penod of several days or several months Statistical methods well known to those skilled in the art may be use to determine the significance of such therapeutic agents The polynucleotides are also useful for identifying individuals from minute biological samples, for example, by matching the RFLP pattern of a sample's DNA to that of an individual's DNA The polynucleotides of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome These sequences can be used to prepare PCR pnmers for amplifying and isolating such selected DNA, which can then be sequenced Using this technique, an individual can be identified through a unique set of DNA sequences Once a unique ID database is established for an individual, positive identification of that individual can be made from extremely small tissue samples
In a particular aspect, oligonucleotide primers derived from the mddt of the invention may be used to detect single nucleotide polymorphisms (SNPs) SNPs are substitutions, insertions and 0 deletions that are a frequent cause of inherited or acquired genetic disease in humans Methods of
SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding MDDT are used to amplify DNA using the polymerase chain reaction (PCR) The DNA may be derived, for example, from diseased or normal tissue, biopsy 5 samples, bodily fluids, and the like SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denatunng gels In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amp mers in high-throughput equipment such as DNA sequencing machines Additionally, sequence database analysis methods, termed in silico SNP o (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into common consensus sequences These computer- based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the 5 high throughput MASSARRAY system (Sequenom, Inc , San Diego CA) DNA-based identification techniques are critical in forensic technology DNA sequences taken from very small biological samples such as tissues, e g , hair or skin, or body fluids, e g , blood, saliva, semen, etc , can be amplified using, e g , PCR, to identify individuals (See, e g , Erhch, H (1992) PCR Technology, Freeman and Co , New York, NY) Similarly, polynucleotides of the present invention can be used as polymorphic markers
There is also a need for reagents capable of identifying the source of a particular tissue Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues Panels of such reagents can identify tissue by species and/or by organ type In a similar fashion, these reagents can be used to screen tissue cultures for contamination
The polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a particular cell type, in the creation of subtracted cDNA libraries which aid in the discovery of novel polynucleotides, in selection and synthesis of oligomers for attachment to an anay or other support, and as an antigen to elicit an immune response
Disease Model Systems Using mddt
The mddt of the invention or their mammalian ho ologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells Such techniques are well known in the art and are useful for the generation of animal models of human disease (See, e g , U S Patent Number 5, 175,383 and U S Patent Number 5,767,337 ) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e g , the neomycin phosphotransferase gene (neo, Capecchi, M R ( 1989) Science 244 1288-1292) The vector integrates into the conesponding region of the host genome by homologous recombination Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J D ( 1996) Chn Invest 97 1999-2002, Wagner, K U et al ( 1997) Nucleic Acids Res 25 4323-4330) Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains Transgenic animals thus generated may be tested with potential therapeutic or toxic agents The mddt of the invention may also be manipulated in vitro in ES cells denved from human blastocysts Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J A et al ( 1998) Science 282 1 145-1 147)
The mddt of the invention can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease With knockin technology, a region of mddt is injected into animal ES cells, and the injected sequence integrates into the animal cell genome Transformed cells are injected into blastulae, and the blastulae are implanted as descnbed above Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease Alternatively, a mammal inbred to overexpress mddt, resulting, e g , in the secretion of MDDT in its milk, may also serve as a convenient source of that protein (Janne, J et al ( 1998) Biotechnol Annu Rev 4 55-74)
Screening Assays
MDDT encoded by polynucleotides of the present invention may be used to screen for molecules that bind to or are bound by the encoded polypeptides The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the bound molecule Examples of such molecules include antibodies, oligonucleotides, proteins (e g , receptors), or small molecules
Preferably, the molecule is closely related to the natural ligand of the polypeptide, e g , a ligand or fragment thereof, a natural substrate, or a structural or functional mimetic (See, Cohgan et al , ( 1991 ) Current Protocols in Immunology 1 (2) Chapter 5 ) Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or to at least a fragment of the receptor, e g , the active site In either case, the molecule can be rationally designed using known techniques Preferably, the screening for these molecules involves producing appropnate cells which express the polypeptide, either as a secreted protein or on the cell membrane Preferred cells include cells from mammals, yeast, Drosophila, or E coli Cells expressing the polypeptide or cell membrane fractions which contain the expressed polypeptide are then contacted with a test compound and binding, stimulation, or inhibition of activity of either the polypeptide or the molecule is analyzed
An assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label Alternatively, the assay may assess binding in the presence of a labeled competitor
Additionally, the assay can be earned out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical hbranes, or natural product mixtures The assay may also simply compnse the steps of mixing a candidate compound with a solution containing a polypeptide, measunng polypeptide/molecule activity or binding, and companng the polypeptide/molecule activity or binding to a standard Preferably, an ELISA assay using, e g., a monoclonal or polyclonal antibody, can measure polypeptide level in a sample The antibody can measure polypeptide level by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate
All of the above assays can be used in a diagnostic or prognostic context. The molecules discovered using these assays can be used to treat disease or to bπng about a particular result in a patient (e g , blood vessel growth) by activating or inhibiting the polypeptide/molecule Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.
Transcript Imaging
Another embodiment relates to the use of mddt to develop a transcript image of a tissue or cell type A transcπpt image is the collective pattern of gene expression by a particular tissue or cell type under given conditions and at a given time This pattern of gene expression is defined by the number of expressed genes, their abundance, and their function Thus the mddt of the present invention may be used to develop a transcript image of a tissue or cell type by hybridizing, preferably in a microarray format, the mddt of the present invention to the totality of transcripts or reverse transcripts of a tissue or cell type. The resultant transcript image would provide a profile of gene activity pertaining to disease detection and treatment
Transcπpt images which profile mddt expression may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect mddt expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line Transcript images may be used to profile mddt expression in distinct tissue types This process can be used to determine disease detection and treatment molecule activity in a particular tissue type relative to this activity in a different tissue type Transcript images may be used to generate a profile of mddt expression charactenstic of diseased tissue. Transcript images of tissues before and after treatment may be used for diagnostic purposes, to monitor the progression of disease, and to monitor the efficacy of drug treatments for diseases which affect the activity of disease detection and treatment molecules.
Transcript images which profile mddt expression may also be used in conjunction with in vitro model systems and prechnical evaluation of pharmaceuticals. Transcript images of cell lines can be used to assess disease detection and treatment molecule activity and/or to identify cell lines that lack or misregulate this activity. Such cell lines may then be treated with pharmaceutical agents, and a transcript image following treatment may indicate the efficacy of these agents in restoring desired levels of this activity. A similar approach may be used to assess the toxicity of pharmaceutical agents as reflected by undesirable changes in disease detection and treatment molecule activity Candidate pharmaceutical agents may be evaluated by comparing their associated transcπpt images with those of pharmaceutical agents of known effectiveness
Antisense Molecules The polynucleotides of the present invention are useful in antisense technology Antisense technology or therapy relies on the modulation of expression of a target protein through the specific binding of an antisense sequence to a target sequence encoding the target protein or directing its expression (See, e g , Agrawal, S , ed (1996) Antisense Therapeutics, Humana Press Inc , Totawa NJ , Alama, A et al ( 1997) Pharmacol Res 36(3) 171 - 178, Crooke, S T ( 1997) Adv Pharmacol 40 1-49, Sharma, H W and R Narayanan ( 1995) Bioessays 17( 12) 1055-1063, and Lavrosky, Y et al ( 1997) Biochem Mol Med 62( 1 ) 1 1-22 ) An antisense sequence is a polynucleotide sequence capable of specifically hybndizing to at least a portion of the target sequence Antisense sequences bind to cellular mRNA and/or genomic DNA, affecting translation and/or transcription Antisense sequences can be DNA, RNA, or nucleic acid mimics and analogs (See, e g , Rossi, J J et al ( 1991 ) Antisense Res Dev 1 (3) 285-288, Lee, R et al ( 1998) Biochemistry 37(3) 900- 1010, Pardπdge, W M et al ( 1995) Proc Natl Acad Sci USA 92( 12) 5592-5596, and Nielsen, P E and Haaima, G ( 1997) Chem Soc Rev 96 73-78 ) Typically, the binding which results in modulation of expression occurs through hybndization or binding of complementary base pairs Antisense sequences can also bind to DNA duplexes through specific interactions in the major groove of the double helix The polynucleotides of the present invention and fragments thereof can be used as antisense sequences to modify the expression of the polypeptide encoded by mddt The antisense sequences can be produced ex vivo, such as by using any of the ABI nucleic acid synthesizer senes (PE Biosystems) or other automated systems known in the art Antisense sequences can also be produced biologically, such as by transforming an appropriate host cell with an expression vector containing the sequence of interest (See, e g , Agrawal, supra )
In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcnption, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (See, e g , Slater, J E , et al ( 1998) J Allergy Chn Immunol 102(3) 469-475, and Scanlon, K J , et al (1995) 9(13) 1288-1296 ) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (See, e g , Miller, A D ( 1990) Blood 76 271 , Ausubel, F M et al ( 1995) Cunent Protocols in Molecular Biology. John Wiley & Sons. New York NY, Uckert, W and W Walther ( 1994) Pharmacol Ther 63(3) 323-347 ) Other gene delivery mechanisms include posome-deπved systems, artificial viral envelopes, and other systems known in the art (See, e g , Rossi, J J. ( 1995) Br Med. Bull 51 ( l ).217-225, Boado, R.J. et al. ( 1998) J. Pharm. Sci. 87( 1 1) 1308-1315; and Morns, M.C. et al. ( 1997) Nucleic Acids Res. 25(14):2730- 2736.)
Expression
In order to express a biologically active MDDT, the nucleotide sequences encoding MDDT or fragments thereof may be inserted into an appropnate expression vector, i.e., a vector which contains the necessary elements for transcπptional and translational control of the inserted coding sequence in a suitable host. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding MDDT and appropriate transcπptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (See, e g , Sambrook, supra. Chapters 4, 8, 16, and 17, and Ausubel, supra. Chapters 9, 10, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and express sequences encoding MDDT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosrrud DNA expression vectors, yeast transformed with yeast expression vectors, insect cell systems infected with viral expression vectors (e.g , baculovirus), plant cell systems transformed with viral expression vectors (e.g , cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal (mammalian) cell systems. (See, e.g., Sambrook, supra; Ausubel, 1995, supra. Van Heeke, G. and S.M. Schuster ( 1989) J. Biol Chem. 264:5503-5509; Bitter, G.A et al. ( 1987) Methods Enzymol. 153:516-544, Scorer, CA. et al. ( 1994) Bio/Technology 12.181-184, Engelhard, E.K. et al. ( 1994) Proc Natl. Acad Sci. USA 91 J224-3227; Sandig, V. et al. ( 1996) Hum. Gene Ther. 7 1937-1945; Takamatsu, N. ( 1987) EMBO J 6.307-31 1 , Coruzzi, G. et al. ( 1984) EMBO J 3: 1671 -1680; Brog e, R. et al. ( 1984) Science 224:838-843, Winter, J. et al. ( 1991 ) Results Probl. Cell Differ 17:85-105; The McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York NY, pp. 191-196, Logan, J. and T. Shenk ( 1984) Proc. Natl. Acad. Sci. USA 81 :3655-3659; and Harπngton, J J. et al. (1997) Nat. Genet. 15J45-355 ) Expression vectors derived from retroviruses, adenoviruses, or heφes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di
Nicola, M. et al. ( 1998) Cancer Gen. Ther. 5(6) 350-356; Yu, M. et al., ( 1993) Proc. Natl. Acad. Sci USA 90(13):6340-6344; Buller, R.M. et al. ( 1985) Nature 317(6040) 813-815; McGregor, D.P et al (1994) Mol. Immunol. 31(3).219-226; and Verma, I.M. and N. Somia ( 1997) Nature 389.239-242 ) The invention is not limited by the host cell employed. For long term production of recombinant proteins in mammalian systems, stable expression of MDDT in cell lines is preferred. For example, sequences encoding MDDT can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector Any number of selection systems may be used to recover transformed cell lines. (See, e.g., Wigler, M. et al (1977) Cell 1 1.223-232; Lowy, I. et al. ( 1980) Cell 22:817-823., Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77J567-3570; Colbere-Garapin, F. et al. (1981 ) J. Mol Biol 150 1-14, Hartman, S.C and R.C.Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051 , Rhodes, CA. ( 1995) Methods Mol Biol. 55: 121-131.)
Therapeutic Uses of mddt
The mddt of the invention may be used for somatic or germline gene therapy Gene therapy may be performed to (I) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-hnked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288.669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deam ase (ADA) deficiency (Blaese, R.M. et al. ( 1995) Science 270 475-480, Bordignon, C. et al ( 1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. ( 1993) Cell 75:207- 216; Crystal, R G. et al. ( 1995) Hum. Gene Therapy 6.643-666; Crystal, R.G. et al ( 1995) Hum. Gene Therapy 6.667-703), thalassemias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R.G. ( 1995) Science 270-404-410, Verma, I M and Somia, N ( 1997) Nature 389.239-242)), (n) express a conditionally lethal gene product (e g , in the case of cancers which result from unregulated cell proliferation), or (in) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. ( 1988) Nature 335:395-396, Poeschla, E. et al ( 1996) Proc Natl. Acad. Sci. USA 93J 1395-1 1399), hepatitis B or C virus (HBV, HCV), fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in mddt expression or regulation causes disease, the expression of mddt from an appropπate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency In a further embodiment of the invention, diseases or disorders caused by deficiencies in mddt are treated by constructing mammalian expression vectors compnsing mddt and introducing these vectors by mechanical means into mddt-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (l) direct DNA microinjection into individual cells, (n) ballistic gold particle delivery, (in) liposome-mediated transfection, (IV) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and Anderson, W.F ( 1993) Annu Rev Biochem. 62.191-217; Ivies, Z. ( 1997) Cell 91 :501-510; Boulay, J-L. and Recipon, H. ( 1998) OUT. Opm. Biotechnol 9.445-450).
Expression vectors that may be effective for the expression of mddt include, but are not limited to, the PCDNA 3J, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). The mddt of the invention may be expressed using (I) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (n) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and Bujard, H. ( 1992) Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551 , Gossen, M. et al., ( 1995) Science 268 1766-1769; Rossi, F.M V. and Blau, H.M. ( 1998) Curr Opin. Biotechnol 9 451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND, Invitrogen), the FK506/rapamycιn inducible promoter; or the RU486/mιfepπstone inducible promoter (Rossi, F M.V. and Blau, H.M supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding MDDT from a normal individual.
Commercially available hposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and Eb, A.J. ( 1973) Virology 52.456-467), or by electroporation (Neumann, E. et al. ( 1982) EMBO J 1.841-845) The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to mddt expression are treated by constructing a retrovirus vector consisting of (I) the mddt of the invention under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional retrovirus cts-actmg RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. ( 1995) Proc. Natl. Acad. Sci. U.S.A. 92:6733-6737), incoφorated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. ( 1987) J. Virol. 61 1647- 1650; Bender, M A. et al. ( 1987) J. Virol 61 J639-1646; Adam, M.A. and Miller, A.D. ( 1988) J Virol. 62.3802-3806; Dull, T et al. ( 1998) J. Virol. 72:8463-8471 , Zufferey, R. et al. ( 1998) J Virol 72:9873-9880) U.S. Patent Number 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incoφorated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et alJ 1997) J Virol. 71.7020-7029; Bauer, G. et al (1997) Blood 89:2259-2267, Bonyhadi, M.L. ( 1997) j. Virol. 71 :4707-4716; Ranga, U. et al. ( 1998) Proc. Natl. Acad. Sci. U.S.A. 95: 1201-1206; Su, L. ( 1997) Blood 89:2283-2290)
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver mddt to cells which have one or more genetic abnormalities with respect to the expression of mddt. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M E. et al ( 1995) Transplantation 27.263-268) Potentially useful adenoviral vectors are descnbed in U S Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incoφorated by reference For adenoviral vectors, see also Antinozzi, P.A. et al. ( 1999) Annu. Rev. Nutr. 19J 1 1-544 and Verma, I.M and Somia, N. (1997) Nature 18:389:239-242, both incoφorated by reference herein. In another alternative, a heφes-based, gene therapy delivery system is used to deliver mddt to target cells which have one or more genetic abnormalities with respect to the expression of mddt. The use of heφes simplex virus (HSV)-based vectors may be especially valuable for introducing mddt to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of heφes-based vectors are well known to those with ordinary skill in the art. A replication-competent heφes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of pπmates (Liu, X. et al ( 1999) Exp. Eye Res.169 385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("Heφes simplex virus strains for gene transfer"), which is hereby incoφorated by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transfened to a cell under the control of the appropπate promoter for puφoses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W F. et al. 1999 J. Virol. 73:519-532 and Xu, H. et al., ( 1994) Dev. Biol 163: 152-161 , hereby incoφorated by reference. The manipulation of cloned heφesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large heφesvirus genomes, the growth and propagation of heφesvirus, and the infection of cells with heφesvirus are techniques well known to those of ordinary skill in the art. In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver mddt to target cells The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H and Li, K-J (1998) OUT Opin Biotech 9 464-469) During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins This subgenorruc RNA replicates to higher levels than the full-length genomic RNA, resulting in the oveφroduction of capsid proteins relative to the viral proteins with enzymatic activity (e g , protease and polymerase) Similarly, inserting mddt into the alphavirus genome in place of the capsid-coding region results in the production of a large number of mddt RNAs and the synthesis of high levels of MDDT in vector transduced cells While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21 ) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S A et al ( 1997) Virology 228 74-83) The wide host range of alphaviruses will allow the introduction of MDDT into a variety of cell types The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art
Antibodies
Anti-MDDT antibodies may be used to analyze protein expression levels Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, and Fab fragments For descriptions of and protocols of antibody technologies, see, e g , Pound J D ( 1998) Immunochemical Protocols. Humana Press, Totowa, NJ The amino acid sequence encoded by the mddt of the Sequence Listing may be analyzed by appropπate software (e g , LASERGENE NAVIGATOR software, DNASTAR) to determine regions of high lmmunogenicity The optimal sequences for immunization are selected from the C-terminus, the N-terminus, and those intervening, hydrophihc regions of the polypeptide which are likely to be exposed to the external environment when the polypeptide is in its natural conformation Analysis used to select appropriate epitopes is also described by Ausubel ( 1997, supra. Chapter 1 1 7) Peptides used for antibody induction do not need to have biological activity, however, they must be antigenic Peptides used to induce specific antibodies may have an amino acid sequence consisting of at five amino acids, preferably at least 10 amino acids, and most preferably 15 amino acids A peptide which mimics an antigenic fragment of the natural polypeptide may be fused with another protein such as keyhole limpet cyanin (KLH, Sigma, St Louis MO) for antibody production A peptide encompassing an antigenic region may be expressed from an mddt, synthesized as described above, or purified from human cells
Procedures well known in the art may be used for the production of antibodies Various hosts including mice, goats, and rabbits, may be immunized by injection with a peptide Depending on the host species, vaπous adjuvants may be used to increase immunological response
In one procedure, peptides about 15 residues in length may be synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using fmoc-chemistry and coupled to KLH (Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (Ausubel, 1995, supra) Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1 % bovine serum albumin (BSA), reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti- rabbit IgG Antisera with antipeptide activity are tested for anti-MDDT activity using protocols well known in the art, including ELISA, radioimmunoassay (RIA), and lmmunoblotting
In another procedure, isolated and purified peptide may be used to immunize mice (about 100 μg of peptide) or rabbits (about 1 mg of peptide) Subsequently, the peptide is radioiodinated and used to screen the immunized animals' B-lymphocytes for production of antipeptide antibodies Positive cells are then used to produce hybπdomas using standard techniques About 20 mg of peptide is sufficient for labeling and screening several thousand clones Hybπdomas of interest are detected by screening with radioiodinated peptide to identify those fusions producing peptide-specific monoclonal antibody In a typical protocol, wells of a multi-well plate (FAST, Becton-Dickinson,
Palo Alto, CA) are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species IgG) antibodies at 10 mg/ml The coated wells are blocked with 1 % BSA and washed and exposed to supernatants from hybπdomas After incubation, the wells are exposed to radiolabeled peptide at 1 mg/ml Clones producing antibodies bind a quantity of labeled peptide that is detectable above background Such clones are expanded and subjected to 2 cycles of cloning Cloned hybπdomas are injected into pπstane-treated mice to produce ascites, and monoclonal antibody is punfied from the ascitic fluid by affinity chromatography on protein A (Amersham Pharmacia Biotech) Several procedures for the production of monoclonal antibodies, including in vitro production, are descnbed in Pound (supra) Monoclonal antibodies with antipeptide activity are tested for anti-MDDT activity using protocols well known in the art, including ELISA, RIA, and lmmunoblotting
Antibody fragments containing specific binding sites for an epitope may also be generated For example, such fragments include, but are not limited to, the F(ab')2 fragments produced by pepsin digestion of the antibody molecule, and the Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments Alternatively, construction of Fab expression braπes in filamentous bacteriophage allows rapid and easy identification of monoclonal fragments with desired specificity (Pound, supra. Chaps 45-47) Antibodies generated against polypeptide encoded by mddt can be used to puπfy and characterize full-length MDDT protein and its activity, binding partners, etc
Assays Using Antibodies
Anti-MDDT antibodies may be used in assays to quantify the amount of MDDT found in a particular human cell Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions The peptides and antibodies of the invention may be used with or without modification or labeled by joining them, either covalently or noncovalently, with a reporter molecule
Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art Examples include ELISA, RIA, and fluorescent activated cell sorting (FACS) Such immunoassays typically involve the formation of complexes between the MDDT and its specific antibody and the measurement of such complexes These and other assays are described in Pound (supra)
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever The disclosures of all patents, applications, and publications mentioned above and below, in particular U S Provisional Application No 60/137,412, filed June 3, 1999, U S Provisional Application No 60/147,542, filed August 5, 1999, U S Provisional Application No 60/147,501 , filed August 5, 1999, U S Provisional Application No 60/147,500, filed August 5, 1999 are hereby expressly incoφorated by reference
EXAMPLES
I. Construction of cDNA Libraries
RNA was purchased from CLONTECH Laboratories, Inc (Palo Alto CA) or isolated from vanous tissues Some tissues were homogenized and lysed in guanidimum isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as
TRIZOL (Life Technologies), a monophasic solution of phenol and guamdine isothiocyanate The resulting lysates were centπfuged over CsCl cushions or extracted with chloroform RNA was precipitated with either isopropanol or sodium acetate and ethanol, or by other routine methods Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA punty In most cases, RNA was treated with DNase For most libranes, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega Coφoration (Promega), Madison WI), OLIGOTEX latex particles (QIAGEN, Inc (QIAGEN), Valencia CA), or an OLIGOTEX mRNA punfication kit (QIAGEN) Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e g , the POLY(A)PURE mRNA purification kit (Ambion, Inc , Austin TX) In some cases, Stratagene was provided with RNA and constructed the coπesponding cDNA libranes Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene Cloning Systems, Inc (Stratagene), La Jolla CA) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art (See, e g , Ausubel, 1997, supra. Chapters 5 1 through 6 6 ) Reverse transcπption was initiated using oligo d(T) or random pnmers Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes For most libraries, the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis cDNAs were ligated into compatible restriction enzyme sites of the polyhnker of a suitable plasmid, e g , PBLUESCRIPT plasmid (Stratagene), pSPORTl plasmid (Life Technologies), or pINCY (Incyte) Recombinant plasmids were transformed into competent E coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies
II. Isolation of cDN A Clones
Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis Plasmids were purified using at least one of the following the Magic or WIZARD Minipreps DNA purification system (Promega), the AGTC Mimprep purification kit (Edge BioSystems, Gaithersburg MD), and the QIAWELL 8, QIAWELL 8 Plus, and QIAWELL 8 Ultra plasmid purification systems or the R E A L PREP 96 plasmid purification kit (QIAGEN)
Following precipitation, plasmids were resuspended in 0 1 ml of distilled water and stored, with or without lyophihzation, at 4°C
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V B ( 1994) Anal Biochem 216 1-14 ) Host cell lysis and thermal cycling steps were earned out in a single reaction mixture Samples were processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometπcally using PICOGREEN dye (Molecular Probes, Inc (Molecular Probes), Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland) III. Sequencing and Analysis cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 thermal cycler (PE Biosystems) or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific Coφ., Sunnyvale CA) or the MICROLAB 2200 liquid transfer system (Hamilton). cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were earned out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (PE Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra. Chapter 1.1) Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII
IV. Assembly and Analysis of Sequences
Component sequences from chromatograms were subject to PHRED analysis and assigned a quality score. The sequences having at least a required quality score were subject to vanous preprocessing editing pathways to eliminate, e.g., low quality 3' ends, vector and linker sequences, polyA tails, Alu repeats, mitochondnal and ribosomal sequences, bactenal contamination sequences, and sequences smaller than 50 base pairs. In particular, low-information sequences and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) were replaced by "n's", or masked, to prevent spurious matches.
Processed sequences were then subject to assembly procedures in which the sequences were assigned to gene bins (bins). Each sequence could only belong to one bin. Sequences in each gene bin were assembled to produce consensus sequences (templates). Subsequent new sequences were added to existing bins using BLASTn (v.1 4 WashU) and CROSSMATCH. Candidate pairs were identified as all BLAST hits having a quality score greater than or equal to 150. Alignments of at least 82% local identity were accepted into the bin. The component sequences from each bin were assembled using a version of PHRAP. Bins with several overlapping component sequences were assembled using DEEP PHRAP. The orientation (sense or antisense) of each assembled template was determined based on the number and orientation of its component sequences. Template sequences as disclosed in the sequence listing correspond to sense strand sequences (the "forward" reading frames), to the best determination The complementary (antisense) strands are inherently disclosed herein The component sequences which were used to assemble each template consensus sequence are listed in Table 4, along with their positions along the template nucleotide sequences
Bins were compared against each other and those having local similanty of at least 82% were combined and reassembled Reassembled bins having templates of insufficient overlap (less than 95% local identity) were re-split Assembled templates were also subject to analysis by
STITCHER/EXON MAPPER algorithms which analyze the probabilities of the presence of splice variants, alternatively spliced exons, splice junctions, differential expression of alternative spliced genes across tissue types or disease states, etc These resulting bins were subject to several rounds of the above assembly procedures Once gene bins were generated based upon sequence alignments, bins were clone joined based upon clone information If the 5' sequence of one clone was present one bin and the 3' sequence from the same clone was present in a different bin, it was likely that the two bins actually belonged together in a single bin The resulting combined bins underwent assembly procedures to regenerate the consensus sequences The final assembled templates were subsequently annotated using the following procedure
Template sequences were analyzed using BLASTn (v2 0, NCBI) versus gbpπ (GenBank version 1 16) "Hits" were defined as an exact match having from 95% local identity over 200 base pairs through 100% local identity over 100 base pairs, or a homolog match having an E-value, i e a probability score, of < 1 x 108 The hits were subject to frameshift FASTx versus GENPEPT (GenBank version 1 16) (See Table 5) In this analysis, a homolog match was defined as having an E-value of < 1 x 10 8 The assembly method used above was described in "System and Methods for Analyzing Biomolecular Sequences," U S S N 09/276,534, filed March 25, 1999, and the LIFESEQ Gold user manual (Incyte) both incoφorated by reference herein
Following assembly, template sequences were subjected to motif, BLAST, and functional analyses, and categorized in protein hierarchies using methods descnbed in, e g , "Database System Employing Protein Function Hierarchies for Viewing Biomolecular Sequence Data," U S S N 08/812,290, filed March 6, 1997, "Relational Database for Storing Biomolecule Information;' U S S N 08/947,845, filed October 9, 1997, "Project-Based Full-Length Biomolecular Sequence Database," U S S N 08/81 1 ,758, filed March 6, 1997, and "Relational Database and System for Storing Information Relating to Biomolecular Sequences," U S S N 09/034,807, filed March 4, 1998, all of which are incoφorated by reference herein
The template sequences were further analyzed by translating each template in all three forward reading frames and searching each translation against the Pfam database of hidden Markov model-based protein families and domains using the HMMER software package (available to the public from Washington University School of Medicine, St Louis MO) Regions of templates which, when translated, contain similarity to Pfam consensus sequences are reported in Table 2, along with descnptions of Pfam protein domains and families Only those Pfam hits with an E-value of < 1 x 10 3 are reported (See also World Wide Web site http //pfam wustl edu/ for detailed descriptions of Pfam protein domains and families ) Additionally, the template sequences were translated in all three forward reading frames, and each translation was searched against hidden Markov models for signal peptide and transmembrane domains using the HMMER software package Construction of hidden Markov models and their usage in sequence analysis has been descnbed (See, for example, Eddy, S R ( 1996) Cun Opin Str Biol 6 361-365 ) Regions of templates which, when translated, contain similanty to signal peptide or transmembrane domain consensus sequences are reported in Table 3 Only those signal peptide or transmembrane hits with a cutoff score of 1 1 bits or greater are reported A cutoff score of 1 1 bits or greater corresponds to at least about 91 -94% true-positives in signal peptide prediction, and at least about 75% true-positives in transmembrane domain prediction
The results of HMMER analysis as reported in Tables 2 and 3 may support the results of BLAST analysis as reported in Table 1 or may suggest alternative or additional properties of template-encoded polypeptides not previously uncovered by BLAST or other analyses
Template sequences are further analyzed using the bio formatics tools listed in Table 5, or using sequence analysis software known in the art such as MACDNASIS PRO software (Hitachi Software Engineenng, South San Francisco CA) and LASERGENE software (DNASTAR) Template sequences may be further queried against public databases such as the GenBank rodent, mammalian, vertebrate, prokaryote, and eukaryote databases
V. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e g , Sambrook, supra, ch 7, Ausubel, 1995, supra, ch 4 and 16 )
Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LEFESEQ (Incyte Pharmaceuticals) This analysis is much faster than multiple membrane-based hybridizations In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar The basis of the search is the product score, which is defined as
BLAST Score x Percent Identity 5 x minimum { length(Seq l)Jength(Seq 2) ] The product score takes into account both the degree of similarity between two sequences and the length of the sequence match The product score is a normalized value between 0 and 100, and is calculated as follows the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences) The BLAST score is 5 calculated by assigning a score of +5 for every base that matches in a high-scoπng segment pair (HSP), and -4 for every mismatch Two sequences may share more than one HSP (separated by gaps) If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score The product score represents a balance between fractional overlap and quality in a BLAST alignment For example, a product score of 100 is produced only for 100% identity over the 0 entire length of the shorter of the two sequences being compared A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap
5 VI. Tissue Distribution Profiling
A tissue distribution profile is determined for each template by compiling the cDNA library tissue classifications of its component cDNA sequences Each component sequence, is derived from a cDNA library constructed from a human tissue Each human tissue is classified into one of the following categones cardiovascular system, connective tissue, digestive system, embryonic 0 structures, endocnne system, exocrme glands, genitalia, female, genitalia, male, germ cells, hermc and immune system, liver, musculoskeletal system, nervous system, pancreas, respiratory system, sense organs, skin, stomatognathic system, unclassified/mixed, or urinary tract Template sequences, component sequences, and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) 5
VII. Transcript Image Analysis
Transcript images are generated as descnbed in Seilhamer et al , "Comparative Gene Transcnpt Analysis," U S Patent Number 5,840,484, incoφorated herein by reference
o VIII. Extension of Polynucleotide Sequences and Isolation of a Full-length cDNA
Oligonucleotide pnmers designed using an mddt of the Sequence Listing are used to extend the nucleic acid sequence One pnmer is synthesized to initiate 5' extension of the template, and the other pnmer, to initiate 3' extension of the template The initial primers may be designed using OLIGO 4 06 software (National Biosciences, Inc (National Biosciences), Plymouth MN), or another 5 appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in haiφin structures and pπmer-pnmer dimeπzations are avoided Selected human cDNA libraries are used to extend the sequence If more than one extension is necessary or desired, additional or nested sets of pnmers are designed High fidelity amplification is obtained by PCR using methods well known in the art PCR is performed in 96-well plates using the PTC-200 thermal cycler (MJ Research) The reaction mix contains DNA template, 200 nmol of each pnmer, reaction buffer containing Mg,+, (NH4)2S04, and β- mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for pnmer pair PCI A and PCI B Step 1 94°C, 3 min, Step 2 94°C, 15 sec, Step 3 60°C, 1 min, Step 4 68°C, 2 nun, Step 5 Steps 2, 3, and 4 repeated 20 times, Step 6 68 °C, 5 nun, Step 7 storage at 4°C In the alternative, the parameters for pnmer pair T7 and SK+ are as follows Step 1 94CC, 3 min, Step 2 94°C, 15 sec. Step 3 57°C, 1 min. Step 4 68°C, 2 min, Step 5 Steps 2, 3, and 4 repeated 20 times, Step 6 68 °C, 5 mιn, Step 7 storage at 4°C The concentration of DNA in each well is determined by dispensing 100 μl PICOGREEN quantitation reagent (0 25% (v/v), Molecular Probes) dissolved in IX Tπs-EDTA (TE) and 0 5 μl of undiluted PCR product into each well of an opaque fluoπmeter plate (Corning Incoφorated (Corning), Corning NY), allowing the DNA to bind to the reagent The plate is scanned in a FLUOROSKAN II (Labsy stems Oy) to measure the fluorescence of the sample and to quantify the concentration of DNA A 5 μl to 10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions are successful in extending the sequence
The extended nucleotides are desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to re gation into pUC 18 vector (Amersham Pharmacia Biotech) For shotgun sequencing, the digested nucleotides are separated on low concentration (0 6 to 0 8%) agarose gels, fragments are excised, and agar digested with AGAR ACE (Promega) Extended clones are rehgated using T4 ligase (New England Biolabs, Inc , Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E coli cells Transformed cells are selected on antibiotic-containing media, individual colonies are picked and cultured overnight at 37 °C in 384- well plates in LB/2x carbenicilhn liquid media
The cells are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters Step 1 94°C, 3 mm, Step 2 94°C, 15 sec, Step 3 60°C, 1 min, Step 4 72°C, 2 min, Step 5 steps 2, 3, and 4 repeated 29 times, Step 6 72 °C, 5 min, Step 7 storage at 4°C DNA is quantified by PICOGREEN reagent (Molecular Probes) as described above Samples with low DNA recoveries are reamphfied using the same conditions as descnbed above Samples are diluted with 20% dimethysulfoxide ( 1 2, v/v), and sequenced using DYENAMIC energy transfer sequencing pnmers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems)
In like manner, the mddt is used to obtain regulatory sequences (promoters, introns, and enhancers) using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library
IX. Labeling of Probes and Southern Hybridization Analyses
Hybridization probes denved from the mddt of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA The labeling of probe nucleotides between 100 and 1000 nucleotides in length is specifically described, but essentially the same procedure may be used with larger cDNA fragments Probe sequences are labeled at room temperature for 30 minutes using a T4 polynucleotide kinase, γ32P-ATP, and 0 5X One-Phor-All Plus (Amersham Pharmacia Biotech) buffer and purified using a ProbeQuant G-50 Microcolumn (Amersham Pharmacia Biotech) The probe mixture is diluted to 107 dpm/μg/ml hybridization buffer and used in a typical membrane-based hybndization analysis
The DNA is digested with a restnction endonuclease such as Eco RV and is electrophoresed through a 0 7% agarose gel The DNA fragments are transfened from the agarose to nylon membrane (NYTRAN Plus, Schleicher & Schuell, Inc , Keene NH) using procedures specified by the manufacturer of the membrane Prehybπdization is earned out for three or more hours at 68 °C, and hybndization is earned out overnight at 68 °C To remove non-specific signals, blots are sequentially washed at room temperature under increasingly stnngent conditions, up to 0 lx saline sodium citrate (SSC) and 0 5% sodium dodecyl sulfate After the blots are placed in a PHOSPHORIMAGER cassette (Molecular Dynamics) or are exposed to autoradiography film, hybridization patterns of standard and experimental lanes are compared Essentially the same procedure is employed when screening RNA
X. Chromosome Mapping of mddt
The cDNA sequences which were used to assemble SEQ ID NO 1 -14 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algonthm Sequences from these databases that match SEQ ID NO 1 -14 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as PHRAP (Table 5) Radiation hybπd and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the clustered sequences have been previously mapped Inclusion of a mapped sequence in a cluster will result in the assignment of all sequences of that cluster, including its particular SEQ ID NO , to that map location The genetic map locations of SEQ ID NO 1-14 are described as ranges, or intervals, of human chromosomes The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination ) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters
XI. Microarray Analysis
Probe Preparation from Tissue or Cell Samples Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and polyA+ RNA is purified using the oligo (dT) cellulose method Each polyA+ RNA sample is reverse transcribed using MMLV reverse-transcπptase, 0 05 pg/μl ohgo-dT primer (21mer), IX first strand buffer, 0 03 u ts/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech) The reverse transcription reaction is performed in a 25 ml volume containing 200 ng polyA+ RNA with GEMBRIGHT kits
(Incyte) Specific control polyA+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA (W Lei, unpublished) As quantitative controls, the control mRNAs at 0 002 ng,
0 02 ng, 0 2 ng, and 2 ng are diluted into reverse transcnption reaction at ratios of 1 100,000,
1 10,000, 1 1000, 1 100 (w/w) to sample mRNA respectively The control mRNAs are diluted into reverse transcription reaction at ratios of 1 3, 3 1 , 1 10, 10 1 , 1 25, 25 1 (w/w) to sample mRNA differential expression patterns After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2 5 ml of 0 5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA Probes are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen ( 1 mg ml), 60 ml sodium acetate, and 300 ml of 100% ethanol The probe is then dried to completion using a SpeedVAC (Savant Instruments Inc , Holbrook NY) and resuspended m 14 μl 5X SSC/0 2% SDS Microarray Preparation
Sequences of the present invention are used to generate anay elements Each anay element is amplified from bacterial cells containing vectors with cloned cDNA inserts PCR amplification uses pnmers complementary to the vector sequences flanking the cDNA insert Anay elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech)
Purified anay elements are immobilized on polymer-coated glass slides Glass microscope slides (Corning) are cleaned by ultrasound in 0 1% SDS and acetone, with extensive distilled water washes between and after treatments Glass slides are etched in 4% hydrofluoric acid (VWR
Scientific Products Coφoration (VWR), West Chester, PA), washed extensively in distilled water, and coated with 0 05% aminopropyl silane (Sigma) in 95% ethanol Coated slides are cured in a 1 10°C oven
Anay elements are applied to the coated glass substrate using a procedure described in US Patent No 5,807,522, incoφorated herein by reference 1 μl of the anay element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus The apparatus then deposits about 5 nl of anay element sample per slide
Microanays are UV-crosslmked using a STRATALTNKER UV-cross nker (Stratagene) Microanays are washed at room temperature once in 0 2% SDS and three times in distilled water Non-specific binding sites are blocked by incubation of microanays in 0 2% casein in phosphate buffered saline (PBS) (Tropix, Inc , Bedford, MA) for 30 minutes at 60° C followed by washes in 0 2% SDS and distilled water as before
Hybridization Hybridization reactions contain 9 μl of probe mixture consisting of 0 2 μg each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0 2% SDS hybridization buffer The probe mixture is heated to 65° C for 5 minutes and is ahquoted onto the microanay surface and covered with an 1 8 cm2 covershp The anays are transfened to a wateφroof chamber having a cavity just slightly larger than a microscope slide The chamber is kept at 100% humidity internally by the addition of 140 μl of 5x SSC in a corner of the chamber The chamber containing the anays is incubated for about 6 5 hours at 60° C The anays are washed for 10 mm at 45° C in a first wash buffer ( 1 X SSC, 0 1 % SDS), three times for 10 minutes each at 45° C in a second wash buffer (0 IX SSC), and dried
Detection Reporter-labeled hybndization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc , Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5 The excitation laser light is focused on the anay using a 20X microscope objective (Nikon, Inc , Melville NY) The slide containing the anay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective The 1 8 cm x 1 8 cm anay used in the present example is scanned with a resolution of 20 micrometers
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bπdgewater NJ) conespondmg to the two fluorophores Appropnate filters positioned between the anay and the photomultiplier tubes are used to filter the signals The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5 Each anay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously Th sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the probe mix at a known concentration A specific location on the anay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of 1 100,000 When two probes from different sources (e g , representing test and control cells), each labeled with a different fluorophore, are hybridized to a single anay for the puφose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc , Norwood, MA) installed in an IBM-compatible PC computer The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal) The data is also analyzed quantitatively Where two different fluorophores are excited and measured simultaneously, the data are first conected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore s emission spectrum
A gπd is supenmposed over the fluorescence signal image such that the signal from each spot is centered in each element of the gπd The fluorescence signal within each element is then integrated to obtain a numerical value conesponding to the average intensity of the signal The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte)
XII. Complementary Nucleic Acids Sequences complementary to the mddt are used to detect, decrease, or inhibit expression of the naturally occurnng nucleotide The use of oligonucleotides comprising from about 15 to 30 base pairs is typical in the art However, smaller or larger sequence fragments can also be used Appropnate oligonucleotides are designed from the mddt using OLIGO 4 06 software (National Biosciences) or other appropriate programs and are synthesized using methods standard in the art or ordered from a commercial supplier To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent transcription factor binding to the promoter sequence To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding and processing of the transcript
XIII. Expression of MDDT
Expression and punfication of MDDT is accomplished using bacterial or virus-based expression systems For expression of MDDT in bacteria, cDNA is subcloned into an appropnate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element Recombinant vectors are transformed into suitable bacterial hosts, e g , BL21 (DE3) Antibiotic resistant bacteria express MDDT upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG) Expression of MDDT in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica cahfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding MDDT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcπption Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases Infection of the latter requires additional genetic modifications to baculovirus (See e g , Engelhard, supra, and Sandig, supra )
In most expression systems, MDDT is synthesized as a fusion protein with, e g , glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-Hιs, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates GST, a 26- kilodalton enzyme from Schistosoma lapomcum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech) Following purification, the GST moiety can be proteolytically cleaved from MDDT at specifically engineered sites FLAG, an 8-amιno acid peptide, enables lmmunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak Company, Rochester NY) 6-Hιs, a stretch of six consecutive histidme residues, enables purification on metal-chelate resins (QIAGEN) Methods for protein expression and purification are discussed in Ausubel ( 1995, supra, Chapters 10 and 16) Puπfied MDDT obtained by these methods can be used directly in the following activity assay
XIV. Demonstration of MDDT Activity
MDDT, or biologically active fragments thereof, are labeled with 125I Bolton-Hunter reagent (See, e g , Bolton, A E and W M Hunter ( 1973) Biochem J 133 529-539 ) Candidate molecules previously anayed in the wells of a multi-well plate are incubated with the labeled MDDT, washed, and any wells with labeled MDDT complex are assayed Data obtained using different concentrations of MDDT are used to calculate values for the number, affinity, and association of MDDT with the candidate molecules
Alternatively, molecules interacting with MDDT are analyzed using the yeast two-hybrid system as described in Fields, S and O Song ( 1989) Nature 340 245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (CLONTECH) MDDT may also be used in the PATHCALLING process (CuraGen Coφ , New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K et al (2000) U S Patent No 6,057, 101)
XV. Functional Assays
MDDT function is assessed by expressing mddt at physiologically elevated levels in mammalian cell culture systems cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression Vectors of choice include pCMV SPORT (Life Technologies) and pCR3 1 (Invitrogen Coφoration, Carlsbad CA), both of which contain the cytomegalovirus promoter 5-10 μg of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either hposome formulations or electroporation 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected
Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector Marker proteins of choice include, e g , Green Fluorescent Protein (GFP, CLONTECH), CD64, or a CD64-GFP fusion protein Flow cytometry (FCM), an automated laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death These events include changes in nuclear DNA content as measured by staining of DNA with propidmm iodide, changes in cell size and granulanty as measured by forward light scatter and 90 degree side light scatter, down-regulation of DNA synthesis as measured by decrease in bromodeoxyuπdine uptake, alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies, and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface Methods in flow cytometry are discussed in Ormerod, M G ( 1994) Flow Cytometry, Oxford, New York NY The influence of MDDT on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding MDDT and either CD64 or CD64-GFP CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG) Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Inc , Lake Success NY) mRNA can be purified from the cells using methods well known by those of skill in the art Expression of mRNA encoding MDDT and other genes of interest can be analyzed by northern analysis or microanay techniques
XVI. Production of Antibodies MDDT substantially purified using polyacrylamide gel electrophoresis (PAGE, see, e g ,
Hanmgton, M G ( 1990) Methods Enzymol 182 488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols
Alternatively, the MDDT amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding peptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are well described in the art (See, e g , Ausubel, 1995, supra. Chapter 1 1 )
Typically, peptides 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using fmoc-chemistry and coupled to KLH (Sigma) by reaction with N- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (See, e g , Ausubel, supra ) Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG Antisera with antipeptide activity are tested for anti-MDDT activity using protocols well known in the art, including ELISA, RIA, and lmmunoblotting XVII. Purification of Naturally Occurring MDDT Using Specific Antibodies
Naturally occurnng or recombinant MDDT is substantially purified by lmmunoaffinity chromatography using antibodies specific for MDDT. An lmmunoaffinity column is constructed by covalently coupling anti-MDDT antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) After the coupling, the resin is blocked and washed according to the manufacturer's instructions
Media containing MDDT are passed over the lmmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of MDDT (e.g., high ionic strength buffers in the presence of detergent) The column is eluted under conditions that disrupt antibody/MDDT binding (e.g , a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and MDDT is collected
All publications and patents mentioned in the above specification are herein incoφorated by reference Vanous modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention Although the invention has been described in connection with specific prefened embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims
TABLE
SEQ ID NO Template ID Gl Number Probability Score Annotation
2 2277093 g6468308 0 Novel human gene mapping to chomosome 1
3 2377032 g4103857 300E 73 fetal globm inducing factor (Mus musculus)
4 240091 1 g4038076 100E-141 Impact (Mus musculus)
5 2430966 g2181494 100E 22 H sapiens telomeπc DNA sequence clone 18QTEL072 read 18QTELOO072 seq
7 4053134 g6807590 800E 27 Novel human gene mapping to chomosome 1
13 3316421 j g3879938 100E 28 Similarity to Human ADP/ATP carrier protein (SW ADT .JHUMAN) cDNA EST
EMBL D71893 comes from this gene cDNA EST EMBL D74630 comes from this gene cDNA EST yk394fl 3 comes from this gene cDNA EST yk394f 1 5 comes from
K x
TABLE 2
SEQ ID NO Template ID Start Stop Frame Pfam Hit Pfam Description E-value
1 222197 6 607 801 forward 1 zf-DHHC DHHC zinc finger domain 2 40E-34
2 227709 3 76 432 forward 1 ArfGap Putative GTP-ase activating protein for Arf 1 40E-52
3 237703 2 890 988 forward 2 ank Ank repeat 8 80E-10
4 240091 1 366 905 forward 3 UPF0029 Uncharacteπzed protein family UPF0029 3 30E-12
5 243096 ό 208 1056 forward 1 MMR.HSRl GTPase of unknown function 6 40E-41
6 244366 6 61 1 805 forward 2 zf-DHHC DHHC zinc finger domain 1 60E-32
7 405313 4 156 509 forward 3 ArfGap Putative GTP-ase activating protein for Arf 1 30E-52
8 436857 2 400 1593 forward 1 Peptιdase_M20 Peptidase family M20/M25/M40 8 90E-40
9 247285 1 j 372 461 forward 3 WW WW domain 3 80E-04
10 254510 1 j 123 314 forward 3 KRAB KRAB box 3 90E-24
1 1 284125 2 j 1069 1 185 forward 1 WD40 WD domain G-beta repeat 8 lOE-10
12 331554 4 j 697 789 forward 1 PH PH domain 4 80E-04
13 331642 1 j 446 757 forward 2 mιto_carr Mitochondnal carrier proteins 1 40E-05
13 331642 1 j 831 929 forward 3 mιto_carr Mitochondnal carrier proteins 4 20E-04
14 445594 2 j 155 283 forward 2 KRAB KRAB box 4 20E-19
TABLE 3
SEQ ID NO: Template ID Start Stop Frame Domain Type
1 222197.6 317 406 forward 2 SP
1 222197.6 901 984 forward 1 TM
2 227709.3 563 649 forward 2 SP
5 243096.6 3096 3182 forward 3 SP
6 244366.6 2801 2878 forward 2 TM
7 405313.4 2256 2333 forward 3 TM
7 405313.4 1503 1589 forward 3 TM
TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
1 222197.6 3989355H1 1 122
1 222197.6 3989355R6 1 462
1 222197.6 g 1189739 58 533
1 222197.6 g 1123521 56 494
1 222197.6 3417884H2 105 341
1 222197.6 3398916H1 111 329
1 222197.6 696738H1 228 480
1 222197.6 3387328H1 248 542
1 222197.6 3387328F6 248 705
1 222197.6 640954H1 499 771
1 222197.6 640954R1 499 841
1 222197.6 2674395H1 544 647
1 222197.6 4871937H1 609 809
1 222197.6 6014949H1 680 954
1 222197.6 1310167H1 729 952
1 222197.6 1310167F6 729 1153
1 222197.6 3422058H1 733 986
1 222197.6 1429773H1 748 1016
1 222197.6 1429773F6 748 1211
1 222197.6 4725459H1 770 889
1 222197.6 2692245H1 774 1025
1 222197.6 2692245F6 774 1300
1 222197.6 2658283H1 818 1051
1 222197.6 4402233H1 847 1083
1 222197.6 673783H1 847 1089
1 222197.6 487422H1 871 1123
1 222197.6 3928678H1 898 1175
1 222197.6 2641613F6 1019 1494
1 222197.6 2641613H1 1019 1259
1 222197.6 2770396H1 1027 1273
1 222197.6 2599469H1 1040 1311
1 222197.6 486115H1 1181 1456
1 222197.6 1626615H1 1247 1456
1 222197.6 1626615F6 1247 1728
1 222197.6 383522H1 1260 1526
1 222197.6 3355867H1 1261 1532
1 222197.6 3617236H1 1286 1573
1 222197.6 3510978H1 1300 1567
1 222197.6 1568105H1 1325 1446
1 222197.6 1571377H1 1325 1550
1 222197.6 3806389H1 1368 1628
1 222197.6 g774888 1369 1729
1 222197.6 2995341 HI 1382 1634
1 222197.6 5547807H1 1412 1611
1 222197.6 619375H1 1501 1738
1 222197.6 g1962367 1501 1997
1 222197.6 2695323H1 1517 1790
1 222197.6 3142880H1 1518 1792
1 222197.6 3805357H1 1518 1820
1 222197.6 1962884H1 1538 1808 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
222197.6 1807088F6 1555 2037
222197.6 1400677H1 1628 1894
222197.6 811652H1 1650 1954
222197.6 304811OH1 1665 1918
222197.6 3048110F6 1665 1992
222197.6 3048102H1 1665 1965
222197.6 5059358H1 1668 1965
222197.6 2150138H1 1671 1925
222197.6 039720H1 1711 1970
222197.6 2434795H1 3030 3132
222197.6 2130701H1 3039 3136
222197.6 2647674H1 1740 1841
222197.6 3448915H1 1810 2065
222197.6 4377215H1 1841 2043
222197.6 4317587H1 1841 1916
222197.6 1851036H1 1841 2033
222197.6 5108761H1 1861 2107
222197.6 2893265H1 1873 2139
222197.6 1568060H1 1878 2083
222197.6 1568004H1 1878 2097
222197.6 3531152H1 1889 2207
222197.6 2851378H1 1922 2260
222197.6 1931202H1 1922 2196
222197.6 2132091 R6 1936 2208
222197.6 2132091 HI 1936 2096
222197.6 5288578H1 1941 2067
222197.6 g2110737 1944 2226
222197.6 2207507H1 1996 2250
222197.6 2345452H1 1996 2256
222197.6 3414767H1 2025 2265
222197.6 1306171 F6 2042 2378
222197.6 1306171H1 2042 2284
222197.6 4588038H1 2066 2349
222197.6 4587760H1 2066 2221
222197.6 1389765H1 2108 2367
222197.6 gl 137612 2112 2425
222197.6 2663717H1 2146 2388
222197.6 3321090H1 2152 2435
222197.6 3840347H1 2151 2339
222197.6 2415559F6 2175 2618
222197.6 2415559H1 2175 2420
222197.6 3146224H1 2178 2430
222197.6 4201740H1 2178 2451
222197.6 3713261H1 2194 2446
222197.6 5900620H1 2195 2484
222197.6 1239238H1 2205 2358
222197.6 1965353R6 2235 2691
222197.6 1965353H1 2235 2500
222197.6 1471606H1 2273 2483
222197.6 3929839H1 2278 2578 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
222197.6 1521966H1 2278 2474
222197.6 1449385H1 2291 2533
222197.6 2381896H1 2307 2560
222197.6 2381895H1 2307 2559
222197.6 4643037H1 2326 2554
222197.6 3703243H1 2351 2650
222197.6 4295130H1 2350 2618
222197.6 4296184H1 2350 2589
222197.6 5841522H2 2396 2675
222197.6 3790395F6 2413 2974
222197.6 3811615H1 2413 2745
222197.6 2353349H1 2414 2513
222197.6 569817H1 2413 2660
222197.6 1621792H1 2413 2625
222197.6 520835H1 2417 2637
222197.6 g2161759 2433 2797
222197.6 1463438H1 2433 2622
222197.6 1621792T6 2437 3096
222197.6 3475011 HI 2442 2682
222197.6 1969764H1 2447 2686
222197.6 4188958H1 2451 2774
222197.6 2134836H1 2458 2578
222197.6 4054390H1 2467 2749
222197.6 5185060H1 2468 2694
222197.6 4058390H1 2468 2580
222197.6 4024007H1 2469 2784
222197.6 5597388H1 2493 2771
222197.6 3934968H1 2512 2787
222197.6 2770396T6 2518 3095
222197.6 993964H1 2526 2698
222197.6 1807088T6 2531 3099
222197.6 2132091T6 2530 3101
222197.6 1965395T6 2532 3098
222197.6 1805709H1 2532 2781
222197.6 4466288H1 2537 2803
222197.6 3020435H1 2537 2821
222197.6 g2355832 2538 3035
222197.6 1672661 HI 2554 2667
222197.6 1881147H1 2554 2807
222197.6 5098316H1 2573 2856
222197.6 1429773T6 2573 3089
222197.6 1626615T6 2584 3091
222197.6 1479854T6 2587 3117
222197.6 3935053H1 2598 2897
222197.6 3930918H1 2598 2915
222197.6 1654064H1 2608 2850
222197.6 2951301 HI 2619 2908
222197.6 g4223642 2627 3028
222197.6 2752320H1 2628 2928
222197.6 g2161260 2634 3031 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
1 222197.6 1306171T6 2636 3099
1 222197.6 3387328T6 2640 3092
1 222197.6 81 1652T6 2639 3097
1 222197.6 1959841 HI 2652 2915
1 222197.6 1959841T6 2652 3094
1 222197.6 1959841R6 2652 31 10
1 222197.6 g4265714 2655 3143
1 222197.6 g4186863 2662 3139
1 222197.6 g3249761 2663 3146
1 222197.6 g41 14970 2663 3136
1 222197.6 705080H1 2666 2908
1 222197.6 2045743H1 2673 2971
1 222197.6 2415559T6 2675 3097
1 222197.6 g4394362 2678 3067
1 222197.6 g2617967 2680 3140
1 222197.6 g 1548565 2685 3028
1 222197.6 3790395T6 2684 3120
1 222197.6 g4393109 2684 3136
1 222197.6 1915761 H1 2689 2948
1 222197.6 g2153774 2702 3136
1 222197.6 996350H1 2729 2980
1 222197.6 996350R1 2729 3028
1 222197.6 996350T1 2729 2992
1 222197.6 997484H1 2731 3034
1 222197.6 480176T6 2736 2990
1 222197.6 480176R6 2736 3028
1 222197.6 912187H1 2744 3042
1 222197.6 1313558H1 2744 3006
1 222197.6 g6561 8 2763 3141
1 222197.6 505701 1 HI 2784 3089
1 222197.6 2260766H1 2792 3062
1 222197.6 g3737413 2803 3139
1 222197.6 1686080H1 2810 3041
1 222197.6 g821623 2838 3148
1 222197.6 2641613T6 2833 3094
1 222197.6 2328044H1 2845 3113
1 222197.6 g4371777 2859 3141
1 222197.6 g 1516072 2860 3144
1 222197.6 g2433045 2865 3091
1 222197.6 2648474H1 2866 3123
1 222197.6 2434653H1 2871 3069
1 222197.6 1878079H1 2881 3147
1 222197.6 g4109641 2896 3139
1 222197.6 2428514H1 2909 3098
1 222197.6 4146636H1 2909 3172
1 222197.6 4703838H1 2929 3139
1 222197.6 3125420H1 2934 3139
1 222197.6 5942277H1 2971 3137
2 227709.3 783646H1 1577 1867
2 227709.3 231421 1 H1 1585 1834 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
2 227709.3 342368H1 1595 1832
2 227709.3 1833241 R6 1595 2004
2 227709.3 •1833241 HI 1595 1853
2 227709.3 154117H1 1609 1752
2 227709.3 4938186H1 1617 1905
2 227709.3 4912871H1 1628 1918
2 227709.3 2243937H1 1653 1871
2 227709.3 876350H1 1530 1682
2 227709.3 4959782H1 1532 1785
2 227709.3 1839309H1 1663 1974
2 227709.3 1378304H1 1695 1940
2 227709.3 1660925H1 1696 1936
2 227709.3 1894917H1 1696 1912
2 227709.3 2394914H1 1716 1815
2 227709.3 3035674H1 1714 2034
2 227709.3 808578H1 1714 2019
2 227709.3 3035136H1 1714 2035
2 227709.3 808578R1 1714 2351
2 227709.3 3852902H1 1716 1984
2 227709.3 3122052H1 1729 2075
2 227709.3 6107590H1 1729 2046
2 227709.3 5925422H1 1735 2039
2 227709.3 4378762H1 1746 2059
2 227709.3 3781168H1 1763 1958
2 227709.3 2469542H1 1763 2006
2 227709.3 4585384H1 1765 2071
2 227709.3 3772159H1 1772 2087
2 227709.3 1436901F6 1787 2216
2 227709.3 1436902H1 1787 2081
2 227709.3 1436902F1 1787 2417
2 227709.3 732765H1 1796 2043
2 227709.3 531886H1 1796 2064
2 227709.3 732765R1 1796 2359
2 227709.3 323492H1 1796 2072
2 227709.3 1755142H1 1811 2072
2 227709.3 2088594H1 1817 2083
2 227709.3 1531927H1 1820 2036
2 227709.3 1281210H1 1828 1963
2 227709.3 618185H1 1834 2133
2 227709.3 072066H1 1833 2072
2 227709.3 920524H1 1837 2173
2 227709.3 g2030053 1840 2258
2 227709.3 g681548 1845 2248
2 227709.3 gl 190789 1847 2173
2 227709.3 3052574H1 1853 2158
2 227709.3 4546684H1 1856 1963
2 227709.3 g1846206 1855 2184
2 227709.3 1231442H1 1856 2170
2 227709.3 4546692H1 1856 1961
2 227709.3 1231220H1 1856 2108 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
2 227709.3 5693680H1 1861 2153
2 227709.3 2040451 HI 1861 2194
2 227709.3 5781387H1 1861 2129
2 227709 J 3506145H1 1861 2181
2 227709.3 3479633H1 1859 2214
2 227709.3 2196589H1 1861 2123
2 227709.3 3872090H1 1867 2086
2 227709.3 1210943R1 1867 2217
2 227709.3 072676H1 1867 2104
2 227709.3 3120279H1 1867 2163
2 227709 J 1210943H1 1867 2127
2 227709 J 5877032H1 1869 2133
2 227709.3 5469466H1 1875 2177
2 227709.3 030331 HI 1877 2151
2 227709.3 g 1970048 1878 2203
2 227709.3 2257012H1 1883 2139
2 227709.3 705952H1 1891 2209
2 227709 J 2564249H1 1891 2202
2 227709.3 5712256H1 1902 2222
2 227709.3 3798289H1 1903 2214
2 227709.3 456952H1 1903 2171
2 227709.3 693406H1 1910 2219
2 227709.3 2403540H1 1915 2217
2 227709.3 6095157H1 1918 2217
2 227709.3 4257073H1 1928 2230
2 227709.3 074494H1 1936 2178
2 227709 J 073044H1 1936 2246
2 227709.3 073608H1 1936 2217
2 227709.3 5882532H1 1937 2217
2 227709.3 073991 HI 1936 2227
2 227709.3 073890H1 1936 2119
2 227709.3 073335H1 1936 2162
2 227709.3 5882935H1 1938 2217
2 227709.3 5883716H1 1938 2217
2 227709.3 5881208H1 1939 2217
2 227709.3 5888519H1 1939 2212
2 227709.3 5890218H1 1939 2212
2 227709.3 4783188H1 1938 2225
2 227709.3 2317709H1 1941 2222
2 227709.3 1876721H1 1952 2217
2 227709.3 2469335H1 1954 2223
2 227709.3 734056H1 1953 2076
2 227709.3 2938267H1 1957 2217
2 227709.3 3166569H1 1957 2217
2 227709.3 4591368H1 1966 2227
2 227709.3 2397855H1 1966 2240
2 227709.3 6105204H1 1965 2217
2 227709.3 874849H1 1968 2217
2 227709.3 4458852H1 1967 2217
2 227709.3 874849R1 1968 2621 TABLE 4
ID NO: Template ID Component ID Start Stop
2 227709.3 1896188H1 1989 2217
2 227709.3 2470872T6 1988 2644
2 227709.3 1831904H1 1994 2217
2 227709.3 2433378H1 2003 2173
2 227709.3 4893472H1 2003 2337
2 227709.3 2395264H1 2016 2217
2 227709.3 4203434H1 2018 2350
2 227709.3 4886606H1 2025 2329
2 227709.3 4886606F6 2025 2094
2 227709.3 504991 HI 2036 2217
2 227709.3 3123928H1 2035 2317
2 227709.3 372978R6 2539 2691
2 227709.3 2356168H1 2576 2698
2 227709.3 213804H1 2632 2687
2 227709.3 3347292H1 461 695
2 227709.3 5013953H1 518 793
2 227709.3 4327103H1 527 781
2 227709.3 3295045H1 569 811
2 227709.3 4012389H1 621 883
2 227709.3 1377181F1 637 1043
2 227709.3 1377181H1 637 880
2 227709.3 5883912H1 663 856
2 227709.3 5886832H1 663 927
2 227709.3 5881250H1 664 943
2 227709.3 377379H1 687 946
2 227709.3 2098327H1 697 942
2 227709.3 3511206H1 728 873
2 227709.3 4031927H1 736 993
2 227709.3 2448606H1 738 977
2 227709.3 2473593T6 743 1332
2 227709.3 2444327H1 744 975
2 227709.3 388684H1 774 1038
2 227709.3 924593R1 778 1154
2 227709.3 924593H1 778 1044
2 227709.3 2707537T6 791 1332
2 227709.3 1842323T6 803 1331
2 227709.3 1386726H1 813 1109
2 227709.3 1436357H1 816 1076
2 227709.3 1436357F1 816 1379
2 227709.3 1842323H1 818 1009
2 227709.3 1842323R6 818 1347
2 227709.3 2757452H1 862 1137
2 227709.3 338917H1 874 1099
2 227709.3 g1382744 934 1319
2 227709.3 g2880866 952 1325
2 227709.3 5289932H1 954 1212
2 227709.3 736987R6 967 1219
2 227709.3 g2955000 966 1369
2 227709.3 736987H1 967 1187
2 227709.3 4792654H1 971 1250 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
2 227709.3 270359H1 989 1337
2 227709.3 2532882H1 985 1263
2 227709.3 3246655H1 990 1254
2 227709.3 g2106694 1001 1373
2 227709.3 6210707H1 1069 1397
2 227709.3 340933H1 1086 1262
2 227709.3 4638914H1 1094 1344
2 227709.3 6211794H1 1106 1397
2 227709.3 1220278H1 1148 1408
2 227709.3 4762916H1 1193 1486
2 227709.3 6208765H1 1202 1504
2 227709.3 3167466H1 1208 1497
2 227709.3 4541633H1 1218 1466
2 227709.3 1389793H1 1 242
2 227709.3 1221361H1 146 324
2 227709.3 6210207H1 1217 1524
2 227709.3 6208587H1 1217 1448
2 227709.3 432063H1 149 454
2 227709.3 2197686H1 1241 1389
2 227709.3 3871923H1 142 426
2 227709.3 2473593H1 205 438
2 227709.3 014086H1 1244 1539
2 227709.3 g1809628 1244 1573
2 227709.3 g570725 1274 1510
2 227709.3 013903H1 1285 1535
2 227709.3 3159468H1 1288 1590
2 227709.3 5219130H1 1303 1575
2 227709.3 2473593F6 205 346
2 227709.3 862771 HI 1304 1579
2 227709.3 336851OH1 1323 1609
2 227709.3 2473008H1 1340 1591
2 227709.3 3633133H1 1353 1666
2 227709.3 2470872F6 267 453
2 227709.3 2472485H1 1367 1615
2 227709.3 3940725H1 1371 1561
2 227709.3 1839612H1 1376 1668
2 227709.3 1839635H1 1376 1702
2 227709.3 2440859H1 1388 1640
2 227709.3 2560406H1 1389 1677
2 227709.3 g776447 1411 1595
2 227709.3 g892952 1432 1807
2 227709.3 4753851 HI 1460 1734
2 227709.3 855862R1 1460 2074
2 227709.3 855862H1 1460 1682
2 227709.3 5436709H1 1470 1708
2 227709.3 3872292H1 1474 1684
2 227709.3 2470872H1 267 518
2 227709.3 4738304H2 363 616
2 227709.3 634613H1 376 613
2 227709.3 489031OH1 1474 1746 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
2 227709.3 964305H1 396 658
2 227709.3 gl 303518 1483 2011
2 227709.3 4081986H1 1497 1695
2 227709.3 2731973H1 1501 1745
2 227709.3 964305R1 396 999
2 227709.3 4984432H1 398 674
2 227709.3 4323306H1 407 691
2 227709.3 656683H1 431 693
2 227709.3 656689H1 431 723
2 227709.3 g774837 1512 1815
2 227709.3 g573087 1512 1840
2 227709.3 3038419H1 1514 1800
2 227709.3 2830914H1 1515 1788
2 227709.3 5206976H2 1530 1806
2 227709.3 073335T6 2039 2650
2 227709.3 g3173537 2054 2217
2 227709.3 862087T1 2058 2649
2 227709.3 862087H1 2058 2344
2 227709.3 g1955564 2058 2421
2 227709.3 2469827T6 2058 2648
2 227709.3 2395978H1 2063 2318
2 227709.3 1833241T6 2074 2648
2 227709.3 1226609H1 2075 2353
2 227709.3 1436901T6 2075 2641
2 227709.3 2827153H1 2083 2449
2 227709.3 1878980H1 2083 2374
2 227709.3 2473240T6 2083 2643
2 227709.3 2431389H1 2087 2329
2 227709.3 2400824H1 2087 2358
2 227709.3 1879688F6 2095 2530
2 227709.3 1879688H1 2095 2389
2 227709.3 1879688T6 2096 2653
2 227709.3 2195425H1 2098 2400
2 227709.3 g1962618 2102 2694
2 227709.3 2325847H1 2112 2374
2 227709.3 4468886H1 2112 2414
2 227709.3 3940725T6 2118 2654
2 227709.3 2917539H1 2119 2418
2 227709.3 1347063H1 2130 2371
2 227709.3 554568H1 2137 2387
2 227709.3 323431 HI 2148 2442
2 227709.3 450205H1 2150 2378
2 227709.3 1448863H1 2150 2423
2 227709.3 2472138T6 2186 2646
2 227709.3 736987T6 2206 2648
2 227709.3 g3932020 2237 2687
2 227709.3 1676401 HI 2247 2475
2 227709.3 406441 HI 2259 2522
2 227709.3 334657H1 2259 2512
2 227709.3 5888253H1 2261 2496 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
2 227709.3 3102430H1 2261 2530
2 227709.3 6106355H1 2261 2524
2 227709.3 g3182016 2261 2693
2 227709 J 604792H1 2261 2486
2 227709.3 2428425H1 2261 2433
2 227709.3 5883372H1 2261 2444
2 227709.3 g4264131 2262 2702
2 227709.3 633186H1 2262 2544
2 227709 J 3993266H1 2261 2540
2 227709.3 2017401 HI 2263 2427
2 227709.3 2424057H1 2268 2536
2 227709.3 404680H1 2273 2496
2 227709.3 3153874H1 2288 2593
2 227709 J g4291477 2288 2694
2 227709.3 4061504H1 2288 2546
2 227709.3 g2968506 2289 2694
2 227709.3 3123730H1 2289 2606
2 227709.3 3123955H1 2289 2589
2 227709.3 g4115080 2293 2694
2 227709.3 308633H1 2295 2534
2 227709.3 2397634H1 2297 2495
2 227709.3 g4332177 2302 2694
2 227709.3 g4267824 2303 2694
2 227709.3 308633F1 2303 2687
2 227709.3 308633R1 2303 2687
2 227709.3 2371681H1 2309 2551
2 227709.3 2421774H1 2309 2547
2 227709.3 g1810042 2311 2687
2 227709.3 g3958397 2313 2677
2 227709.3 4695290H1 2316 2582
2 227709.3 2756565H1 2320 2630
2 227709.3 go12404 2323 2687
2 227709.3 2445854H1 2327 2587
2 227709.3 449703H1 2332 2458
2 227709.3 2877181H1 2334 2622
2 227709.3 1211271R1 2334 2687
2 227709.3 1211271T1 2334 2649
2 227709.3 1211271H1 2334 2608
2 227709.3 g616409 2341 2660
2 227709.3 2017458H1 2340 2619
2 227709.3 1534375H1 2342 2572
2 227709.3 3145020H1 2344 2683
2 227709.3 1539734H1 2358 2600
2 227709.3 3786174H1 2363 2661
2 227709.3 1454741 FI 2367 2687
2 227709.3 1454741 HI 2367 2632
2 227709.3 2717951 HI 2372 2548
2 227709.3 1359816H1 2374 2618
2 227709.3 1359816F1 2374 2694
2 227709.3 g564645 2385 2694 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
2 227709.3 gll54316 2388 2698
2 227709.3 g891561 2393 2707
2 227709.3 2473312H1 2393 2648
2 227709.3 g2992779 2394 2643
2 227709.3 1218580T6 2395 2649
2 227709.3 1218580T1 2395 2649
2 227709.3 g824343 2396 2719
2 227709.3 1218580R6 2395 2691
2 227709.3 1218573H1 2395 2641
2 227709.3 286961 HI 2403 2690
2 227709.3 4367190H1 2408 2684
2 227709.3 2399122H1 2420 2672
2 227709.3 5882896H1 2423 2687
2 227709.3 g967282 2422 2700
2 227709.3 5883904H1 2423 2687
2 227709.3 5883908H1 2423 2687
2 227709.3 5882375H1 2424 2567
2 227709.3 gll91305 2429 2710
2 227709.3 794290H1 2437 2664
2 227709.3 520742H1 2442 2678
2 227709.3 g646174 2443 2687
2 227709.3 1538631 HI 2443 2651
2 227709.3 1722285H1 2444 2677
2 227709.3 g1202716 2469 2701
2 227709.3 862903T1 2482 2648
2 227709.3 095638H1 2484 2694
2 227709.3 862903R1 2484 2694
2 227709.3 3124846H1 2490 2692
2 227709 J 5906470H1 2497 2691
2 227709.3 2535162H1 2525 2651
2 227709.3 4460277H1 2533 2694
2 227709.3 372978T6 2539 2649
2 227709.3 372978H1 2539 2686
3 237703.2 g1963754 1 374
3 237703.2 gl 137733 95 407
3 237703.2 g843567 124 414
3 237703.2 3070350H1 189 477
3 237703.2 3070350F6 189 709
3 237703.2 1439542H1 413 686
3 237703.2 3203352H1 437 711
3 237703.2 g2013304 598 954
3 237703.2 3799002H1 701 1010
3 237703.2 044160T6 958 1453
3 237703.2 824258H1 1020 1254
3 237703.2 g1894392 1031 1472
3 237703.2 3491432H1 1052 1315
3 237703.2 2601554F6 1059 1602
3 237703.2 2601554H1 1060 1336
3 237703.2 4617816H1 1073 1337
3 237703.2 4058186H1 1111 1197 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
3 237703.2 5554248H1 1145 1355
3 237703.2 5554148H1 1145 1386
3 237703.2 g3594366 1199 1611
3 237703.2 5122547HI 1278 1516
3 237703.2 2278690H1 1379 1654
3 237703.2 2278690R6 1379 1879
3 237703.2 5564683H1 1402 1658
3 237703.2 950519H1 1436 1678
3 237703.2 950519R6 1436 1723
3 237703.2 gl 123529 1437 1805
3 237703.2 5260937H1 1514 1744
3 237703.2 3386649H1 1527 1744
3 237703.2 5272970H1 1532 1780
3 237703.2 3808872H1 1533 1838
3 237703.2 950519T6 1554 2019
3 237703.2 319252H1 1592 1986
3 237703.2 4411457H1 1669 1947
3 237703.2 g1933240 1669 2147
3 237703.2 2601554T6 1698 2313
3 237703.2 824257T6 1699 2313
3 237703.2 2859138T6 1776 2312
3 237703.2 1872409F6 1788 2150
3 237703.2 1872409H1 1788 2062
3 237703.2 1572418H1 1798 1997
3 237703.2 1872409T6 1805 2312
3 237703.2 530381 HI 1810 1963
3 237703.2 5583255H1 1811 2075
3 237703.2 126942H1 1816 2025
3 237703.2 2278690T6 1829 2311
3 237703.2 1211984H1 1832 2066
3 237703.2 2703527H1 1832 2106
3 237703.2 3253906H1 1885 2159
3 237703.2 g3934221 1896 2349
3 237703.2 1620273H1 1897 2117
3 237703.2 g2819399 1908 2351
3 237703.2 g3895924 1936 2349
3 237703.2 g2881190 1962 2270
3 237703.2 040587H1 1971 2158
3 237703.2 g3147053 1973 2349
3 237703.2 g843522 2009 2349
3 237703.2 g1844904 2023 2349
3 237703.2 g2881790 2024 2349
3 237703.2 g2820075 2030 2349
3 237703.2 g2237723 2051 2350
3 237703.2 238512H1 2085 2313
3 237703.2 292954H1 2182 2320
3 237703.2 g2013921 2238 2522
3 237703.2 g1980268 2386 2742
4 240091.1 2898155H1 1 289
4 240091.1 2434264H1 3 215 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
4 240091.1 5075278H1 3 127
4 240091.1 2647594H1 1720 1770
4 240091.1 4785026H1 1803 2073
4 240091.1 4785001 HI 1803 2069
4 240091.1 3600323H1 1258 1557
4 240091.1 2448218F6 1267 1485
4 240091.1 2448218H1 1267 1508
4 240091.1 1391961T6 1267 1730
4 240091.1 083349H1 1309 1464
4 240091.1 070643H1 1309 1543
4 240091.1 3712971T6 1317 1744
4 240091.1 3399693H1 1384 1606
4 240091.1 g3000643 1400 1562
4 240091.1 g3659260 1446 1772
4 240091.1 3491102H1 1447 1559
4 240091.1 2286846H1 1496 1696
4 240091.1 4575130H1 1513 1756
4 240091.1 2584803H1 1539 1770
4 240091.1 2584803F6 1539 1770
4 240091.1 4399541 HI 1590 1833
4 240091.1 489490H1 1599 1844
4 240091.1 5541073H1 1605 1804
4 240091.1 797486H1 1609 1772
4 240091.1 2435453H1 3 231
4 240091.1 2434264R6 3 491
4 240091.1 527914H1 4 275
4 240091.1 4382936H1 10 241
4 240091.1 4210908H1 20 292
4 240091.1 3615238H1 47 340
4 240091.1 3615238F6 47 528
4 240091.1 2733107H1 54 275
4 240091.1 494380H1 62 307
4 240091.1 1391961 F6 66 475
4 240091.1 1391961H1 66 318
4 240091.1 580112H1 359 558
4 240091.1 1232706F6 389 842
4 240091.1 1232706H1 389 629
4 240091.1 3487133H1 432 698
4 240091.1 g4244249 511 981
4 240091.1 447619H1 580 799
4 240091.1 5782214H1 670 964
4 240091.1 4913546F6 830 1249
4 240091.1 4913546H1 830 1108
4 240091.1 3892111 HI 834 1130
4 240091.1 4742244H1 836 1102
4 240091.1 g1484624 867 1316
4 240091.1 2376485F6 901 1205
4 240091.1 2376485H1 901 1124
4 240091.1 2376485T6 902 1167
4 240091.1 1849607H1 907 1198 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
4 240091.1 4030992H1 952 1198
4 240091.1 2764886H1 975 1201
4 240091.1 4665206H1 1138 1401
4 240091.1 1232706T6 1176 1729
4 240091.1 2434264T6 1210 1746
4 240091.1 4424318H1 1223 1459
5 243096.6 g3228919 1006 1482
5 243096.6 g3597815 1016 1479
5 243096.6 g3933703 1018 1479
5 243096.6 g4372582 1022 1487
5 243096.6 4103071H1 1030 1104
5 243096.6 3478526H1 1032 1183
5 243096.6 g4457447 1045 1481
5 243096.6 g4222324 1075 1480
5 243096.6 631209R6 1076 1478
5 243096.6 g3118595 1075 1478
5 243096.6 g2577165 1076 1480
5 243096.6 g2185952 1078 1493
5 243096.6 g2063697 1079 1482
5 243096.6 222052H1 1079 1215
5 243096.6 222052F1 1078 1479
5 243096.6 222052R1 1078 1479
5 243096.6 g3737532 1083 1509
5 243096.6 1849724T6 1095 1440
5 243096.6 g4110131 1115 1481
5 243096.6 631209T6 1116 1438
5 243096.6 2446727T6 1119 1438
5 243096.6 g3155321 1129 1475
5 243096.6 g3178176 1148 1494
5 243096.6 948177H1 1149 1428
5 243096.6 948177R1 1149 1488
5 243096.6 1942176H1 1163 1441
5 243096.6 1942176R6 1163 1418
5 243096.6 1942168H1 1163 1440
5 243096.6 5884138H1 1164 1433
5 243096.6 1418514T6 1209 1430
5 243096.6 1418514H1 1216 1431
5 243096.6 1418364H1 1216 1468
5 243096.6 1418514F6 1216 1479
5 243096.6 632343H1 1218 1458
5 243096.6 g4107711 1220 1526
5 243096.6 g4457962 1227 1483
5 243096.6 g2837785 1228 1479
5 243096.6 g819991 1238 1496
5 243096.6 g564440 1237 1488
5 243096.6 g816379 1251 1540
5 243096.6 g885380 1252 1488
5 243096.6 g768804 1261 1481
5 243096.6 6093263H1 1263 1492
5 243096.6 g645318 1286 1488 TABLE 4
ID NO: Template ID Component ID Start Stop
5 243096.6 g566867 1292 1526
5 243096.6 g816311 1302 1679
5 243096.6 g671079 1296 1488
5 243096.6 g2219072 1297 1472
5 243096.6 g2539665 1300 1479
5 243096.6 g670466 1300 1526
5 243096.6 2474482H1 1313 1542
5 243096.6 g4328047 1314 1487
5 243096.6 g2205935 1335 1454
5 243096.6 g832021 1367 1678
5 243096.6 g2205936 1375 1472
5 243096.6 g2789365 1393 1479
5 243096.6 g873007 1418 1540
5 243096.6 g900098 1419 1488
5 243096.6 g567639 1424 1667
5 243096.6 1918362H1 1473 1731
5 243096.6 4727611 HI 1576 1854
5 243096.6 g822245 1648 1976
5 243096.6 g812869 1651 2012
5 243096.6 g830918 1654 2012
5 243096.6 1414612H1 1662 1890
5 243096.6 4761250H1 1662 1939
5 243096.6 g678372 1665 1972
5 243096.6 g561207 1665 1955
5 243096.6 g2002379 1665 2002
5 243096.6 g709471 1665 1866
5 243096.6 4761242H1 1664 1939
5 243096.6 g518391 1678 1933
5 243096.6 4595686H1 1707 1981
5 243096.6 1511493H1 1792 1996
5 243096.6 1511493F6 1792 2244
5 243096.6 1512376H1 1792 2009
5 243096.6 g2003356 1922 2087
5 243096.6 4697285H1 2203 2446
5 243096.6 4941432H1 2381 2673
5 243096.6 1230891 HI 2413 2508
5 243096.6 1522037H1 2421 2625
5 243096.6 3749969H1 2502 2799
5 243096.6 2125142H1 2527 2795
5 243096.6 2125142F6 2527 2841
5 243096.6 121562H1 2620 2806
5 243096.6 856668H1 2745 2933
5 243096.6 5882521 HI 2758 3030
5 243096.6 5888582H1 2759 2976
5 243096.6 5882569H1 2760 3030
5 243096.6 g775350 2793 3137
5 243096.6 g705857 2790 3138
5 243096.6 g2002380 2803 3138
5 243096.6 5927949H1 2845 3140
5 243096.6 1511493T6 2883 3500 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
5 243096.6 1335311 HI 2951 3205
5 243096.6 1613745H1 2965 3179
5 243096.6 3472823H1 3005 3245
5 243096.6 g570224 3048 3318
5 243096.6 g4095588 3134 3545
5 243096.6 g831152 3143 3366
5 243096.6 g4286632 3205 3476
5 243096.6 5907720H1 3208 3501
5 243096.6 g4187457 3286 3557
5 243096.6 g3842315 3295 3465
5 243096.6 g4005713 3303 3465
5 243096.6 g4006389 3305 3465
5 243096.6 g4006377 3305 3559
5 243096.6 g4006150 3305 3537
5 243096.6 g4006070 3315 3542
5 243096.6 g4187003 3315 3554
5 243096.6 g4188554 3315 3537
5 243096.6 g4006771 3315 3537
5 243096.6 g4072007 3316 3542
5 243096.6 g4017934 3316 3537
5 243096.6 g4150328 3316 3465
5 243096.6 g4005644 3316 3537
5 243096.6 5840086H1 3345 3553
5 243096.6 5289394H1 3472 3737
5 243096.6 g710217 3508 3789
5 243096.6 g694295 3619 3781
5 243096.6 g2206232 3623 3794
5 243096.6 g2206104 3656 3795
5 243096.6 2897215H1 1 249
5 243096.6 3541808H1 181 397
5 243096.6 2352032H1 32 249
5 243096.6 2446727F6 44 104
5 243096.6 3123367H1 44 356
5 243096.6 4385825H1 181 379
5 243096.6 2446727H1 44 308
5 243096.6 2905666H1 45 326
5 243096.6 2767616H1 46 308
5 243096.6 4521271H1 214 473
5 243096.6 1725750H1 47 209
5 243096.6 g1965606 235 621
5 243096.6 3117919H1 47 328
5 243096.6 2762827H1 49 309
5 243096.6 5395762H1 245 510
5 243096.6 5585677H1 251 484
5 243096.6 3416289H1 253 507
5 243096.6 5407275H1 257 511
5 243096.6 5407149H1 257 520
5 243096.6 3452689H1 49 240
5 243096.6 4819033H1 292 515
5 243096.6 483458H1 50 302 TABLE 4
ID NO: Template ID Component ID Start Stop
5 243096.6 1941753H1 291 537
5 243096.6 485741 HI 50 296
5 243096.6 g766717 307 480
5 243096.6 2215901HI 51 147
5 243096.6 2990593H1 73 383
5 243096.6 4017755H1 76 378
5 243096.6 2558416H1 81 363
5 243096.6 870507R1 82 682
5 243096.6 870507H1 82 339
5 243096.6 3692401 HI 82 285
5 243096.6 2387055H1 83 336
5 243096.6 1641267H1 329 555
5 243096.6 2483682H1 84 331
5 243096.6 4977750H1 89 382
5 243096.6 g4244257 343 817
5 243096.6 3165660H1 89 373
5 243096.6 4043243H1 89 406
5 243096.6 3500940H1 347 625
5 243096.6 3580985H1 91 415
5 243096.6 1518953F6 93 405
5 243096.6 g672832 92 414
5 243096.6 g574622 92 418
5 243096.6 2206923H1 355 624
5 243096.6 2681788H1 92 287
5 243096.6 g672843 92 444
5 243096.6 790680R1 365 938
5 243096.6 790680H1 365 584
5 243096.6 3500449H1 386 701
5 243096.6 1518953H1 92 280
5 243096.6 3510335H1 92 396
5 243096.6 4401867H1 393 653
5 243096.6 1624276H1 395 583
5 243096.6 3099469H1 92 415
5 243096.6 g873107 93 484
5 243096.6 g874944 93 492
5 243096.6 2201243H1 411 667
5 243096.6 4907323H2 97 377
5 243096.6 2215590H1 421 665
5 243096.6 1647105H1 102 323
5 243096.6 3337242H1 105 332
5 243096.6 3328567H1 421 709
5 243096.6 5165830H1 113 391
5 243096.6 1919378R6 432 865
5 243096.6 2078775H1 114 391
5 243096.6 1919378H1 432 700
5 243096.6 1798353H1 115 371
5 243096.6 5109893H1 447 675
5 243096.6 3581083H1 116 378
5 243096.6 2202470H1 456 711
5 243096.6 164221OH1 461 676 TABLE 4
ID NO: Template ID Component ID Start Stop
5 243096.6 2828817H1 122 399
5 243096.6 1642206H1 461 676
5 243096.6 gό69309 136 449
5 243096.6 4932616H1 463 618
5 243096.6 g571269 136 492
5 243096.6 1753890H1 464 690
5 243096.6 756217H1 464 706
5 243096.6 3030389H1 464 764
5 243096.6 g677690 136 462
5 243096.6 1754027H1 464 704
5 243096.6 3893562H1 139 449
5 243096.6 g885379 139 482
5 243096.6 026083H1 509 692
5 243096.6 2758492H1 143 413
5 243096.6 836506R1 513 1092
5 243096.6 5294950H1 147 395
5 243096.6 836506H1 513 759
5 243096.6 1520256H1 517 685
5 243096.6 173031 HI 157 390
5 243096.6 5197820H2 161 419
5 243096.6 g2240993 522 940
5 243096.6 g766746 161 395
5 243096.6 3728525H1 572 854
5 243096.6 gό77072 160 502
5 243096.6 2828115T6 574 950
5 243096.6 g2816446 612 874
5 243096.6 4536339H1 624 877
5 243096.6 2670757H1 629 868
5 243096.6 4994228H1 697 1004
5 243096.6 793596H1 697 946
5 243096.6 g2058963 697 941
5 243096.6 1560602H1 719 948
5 243096.6 1535660H1 719 898
5 243096.6 g2058866 730 936
5 243096.6 2316449H1 764 1045
5 243096.6 686656H1 771 1028
5 243096.6 3728525T1 783 1432
5 243096.6 3659224H2 789 1073
5 243096.6 639411 HI 798 1050
5 243096.6 5332257H1 820 1063
5 243096.6 2084254H1 837 1136
5 243096.6 2652025T6 859 1424
5 243096.6 961879T6 859 1437
5 243096.6 5088178T6 858 1465
5 243096.6 1849724F6 860 1441
5 243096.6 1849724H1 860 1135
5 243096.6 5395762T1 884 1440
5 243096.6 1919378T6 880 1452
5 243096.6 2663785H1 891 1144
5 243096.6 3625012H1 904 1051 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
5 243096.6 2683022H1 931 1212
5 243096.6 3499439H1 933 1219
5 243096.6 4005888H1 935 1211
5 243096.6 1682469T7 939 1485
5 243096.6 2351530H1 941 1130
5 243096.6 g2063947 951 1207
5 243096.6 1668866H1 960 1201
5 243096.6 1667633H1 960 1220
5 243096.6 g2358923 963 1060
5 243096.6 3085780H1 971 1082
5 243096.6 g814507 161 443
5 243096.6 g830479 161 470
5 243096.6 g816410 162 519
6 244366.6 1889554H1 493 750
6 244366.6 1889554F6 493 939
6 244366.6 4298324H1 1 255
6 244366.6 853003H1 12 225
6 244366.6 g2178494 517 912
6 244366.6 853003R6 26 483
6 244366.6 3327565H1 555 787
6 244366.6 2263295H1 278 532
6 244366.6 5401026H1 604 816
6 244366.6 1285225H1 606 862
6 244366.6 2674162H1 661 904
6 244366.6 3101288H1 295 585
6 244366.6 3295139H1 815 1056
6 244366.6 6002940H1 886 1170
6 244366.6 3101288F6 295 694
6 244366.6 6002740H1 904 1170
6 244366.6 3246058F6 941 1282
6 244366.6 3246058H1 941 1192
6 244366.6 3887233H1 959 1240
6 244366.6 2431320H1 972 1192
6 244366.6 1513444H1 978 1189
6 244366.6 2813740H1 1071 1363
6 244366.6 2815664H1 1071 1274
6 244366.6 2813707H1 1071 1359
6 244366.6 3492628H1 1138 1414
6 244366.6 2183893H1 1190 1450
6 244366.6 5641164H1 1238 1485
6 244366.6 580082H1 1239 1487
6 244366.6 3155135H1 1254 1487
6 244366.6 3075416H1 1282 1565
6 244366.6 3559024H1 1351 1639
6 244366.6 3451987H1 1525 1785
6 244366.6 4378692H1 1597 1817
6 244366.6 g2162961 1742 2237
6 244366.6 3890528H1 1764 1919
6 244366.6 5017346H1 3006 3272
6 244366.6 1690531 HI 2938 3105 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
6 244366.6 5890083H1 3007 3133
6 244366.6 4614333H1 3009 3145
6 244366.6 2783117H1 2979 3239
6 244366.6 3037603H1 3012 3292
6 244366.6 032789H1 3013 3234
6 244366.6 5262767H2 3029 3291
6 244366.6 2626903H1 3031 3283
6 244366.6 1403785H1 3034 3328
6 244366.6 3321234H1 2980 3254
6 244366.6 003727H1 3043 3400
6 244366.6 003176H1 3043 3543
6 244366.6 003684H1 3043 3424
6 244366.6 003185H1 3043 3550
6 244366.6 003182H1 3043 3493
6 244366.6 003701 HI 3043 3471
6 244366.6 003615H1 3043 3429
6 244366.6 003188H1 3043 3483
6 244366.6 003127H1 3043 3453
6 244366.6 003465H1 3043 3433
6 244366.6 003422H1 3043 3380
6 244366.6 5290617H1 3002 3300
6 244366.6 003521 HI 3043 3549
6 244366.6 003294H1 3043 3411
6 244366.6 003642H1 3043 3400
6 244366.6 003646H1 3043 3405
6 244366.6 003660H1 3043 3392
6 244366.6 5138512H1 3079 3394
6 244366.6 094605H1 3081 3258
6 244366.6 1726602H1 3114 3335
6 244366.6 1623474T6 3120 3724
6 244366.6 2381350H1 3122 3380
6 244366.6 768275H1 3130 3388
6 244366.6 3495251 HI 3143 3350
6 244366.6 3705742H1 3161 3533
6 244366.6 4744563H1 3175 3471
6 244366.6 3101288T6 3197 3715
6 244366.6 5561382H1 3201 3503
6 244366.6 074734H1 3202 3442
6 244366.6 073329H1 3202 3403
6 244366.6 1975441T6 3222 3706
6 244366.6 3477744H1 3244 3416
6 244366.6 2112529T6 3250 3723
6 244366.6 g3933445 3274 3756
6 244366.6 4861989H1 3274 3567
6 244366.6 1737024F6 3292 3734
6 244366.6 g2584374 3285 3757
6 244366.6 1735490H1 3292 3561
6 244366.6 1737024H1 3292 3554
6 244366.6 393035H1 3303 3590
6 244366.6 2158031F6 3307 3758 TABLE 4
ID NO: Template ID Component ID Start Stop
6 244366.6 g4438605 3308 3758
6 244366.6 2554055H1 3325 3624
6 244366.6 g2934389 3326 3756
6 244366.6 g4391414 3341 3756
6 244366.6 4460645H1 3349 3601
6 244366.6 g2208607 3359 3756
6 244366.6 g2318342 3366 3756
6 244366.6 g1062585 3370 3743
6 244366.6 g4081771 3372 3761
6 244366.6 g1925212 3381 3757
6 244366.6 4726758H1 3390 3662
6 244366.6 g2410378 3392 3763
6 244366.6 867419H1 3398 3679
6 244366.6 go16070 3402 3764
6 244366.6 g2163418 3405 3756
6 244366.6 g2555602 3413 3760
6 244366.6 g561365 3428 3756
6 244366.6 1889554T6 3433 3725
6 244366.6 g2336915 3444 3756
6 244366.6 gόlόl 15 3445 3756
6 244366.6 g4435130 3469 3756
6 244366.6 g4525507 3502 3757
6 244366.6 2158031H1 3523 3756
6 244366.6 g2401624 3528 3765
6 244366.6 g4268526 3559 3756
6 244366.6 2009370H1 3666 3756
6 244366.6 218073H1 3682 3756
6 244366.6 2350763H1 3699 3760
6 244366.6 1647483H1 2520 2769
6 244366.6 2432662H1 2526 2757
6 244366.6 901941R1 2538 3096
6 244366.6 901941 HI 2538 2895
6 244366.6 901981H1 2538 2858
6 244366.6 2052263H1 2545 2857
6 244366.6 3483573H1 2553 2851
6 244366.6 3565807H1 2558 2822
6 244366.6 g2278841 2560 2917
6 244366.6 g2178439 2563 2917
6 244366.6 g2153824 2565 2917
6 244366.6 g1329145 2568 2874
6 244366.6 2198515H1 2647 2908
6 244366.6 2200581 HI 2647 2725
6 244366.6 g1548506 2680 3207
6 244366.6 2321185H1 2694 2917
6 244366.6 3244071T6 2568 2798
6 244366.6 2936492H1 2700 2917
6 244366.6 600642H1 2586 2891
6 244366.6 g1124072 2711 2850
6 244366.6 g1833465 2712 2856
6 244366.6 g4327019 2736 2851 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
6 244366.6 3165778H1 2588 2925
6 244366.6 g2139164 2594 2835
6 244366.6 633565H1 2753 2917
6 244366.6 1520269F6 2766 3147
6 244366.6 1520026H1 2766 2917
6 244366.6 1520269H1 2766 2917
6 244366.6 g4095144 2611 2917
6 244366.6 1237708H1 2767 3016
6 244366.6 2768411 HI 2770 3013
6 244366.6 gl321416 2615 2858
6 244366.6 2416809H1 2800 2917
6 244366.6 1570846H1 2811 3018
6 244366.6 3898114H1 2621 2859
6 244366.6 874422H1 2824 3131
6 244366.6 g1349372 2825 2952
6 244366.6 2815235H1 2842 3116
6 244366.6 4897079H1 2925 3201
6 244366.6 1231478H1 2632 2861
6 244366.6 2152887H1 2927 3042
6 244366.6 3510024H1 2635 2917
6 244366.6 1975441F6 2938 3306
6 244366.6 2189605H1 2938 3203
6 244366.6 1975441 HI 2938 3088
6 244366.6 3470739H1 2938 3153
6 244366.6 g1844965 2643 2917
6 244366.6 1623474H1 2155 2382
6 244366.6 1338343H1 1799 2055
6 244366.6 2022520H1 2157 2423
6 244366.6 805131H1 2200 2397
6 244366.6 795024H1 2202 2393
6 244366.6 1338343F6 1799 2241
6 244366.6 1297158H1 1810 2050
6 244366.6 3354386H1 2223 2491
6 244366.6 2540345H1 2224 2461
6 244366.6 2313137H1 1887 2152
6 244366.6 2805024H1 2233 2536
6 244366.6 2454581T6 2281 2827
6 244366.6 g570404 1961 2245
6 244366.6 2773281 HI 2282 2528
6 244366.6 3246058T6 2284 2807
6 244366.6 3332425T6 2286 2817
6 244366.6 3321733H1 1988 2109
6 244366.6 g2153937 2324 2754
6 244366.6 1464642H1 1995 2224
6 244366.6 g1319564 2331 2938
6 244366.6 3555988H1 2005 2304
6 244366.6 3384030H1 2043 2317
6 244366.6 g1898453 2332 2760
6 244366.6 g1062443 2337 2746
6 244366.6 3188982H1 2348 2686 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
6 244366.6 2255759H1 2045 2318
6 244366.6 2112529H1 2355 2621
6 244366.6 3693731 HI 2364 2662
6 244366.6 1864803H1 2069 2351
6 244366.6 853003T6 2385 2815
6 244366.6 g1925211 2137 2615
6 244366.6 2641982H1 2385 2598
6 244366.6 2516658H1 2397 2535
6 244366.6 1864803T6 2414 2898
6 244366.6 1338343T6 2421 2814
6 244366.6 1398258H1 2440 2681
6 244366.6 1829874H1 2471 2738
6 244366.6 589137H1 2478 2685
6 244366.6 4855638H1 2147 2411
6 244366.6 1698592H1 2500 2726
6 244366.6 3524562H1 2154 2348
6 244366.6 gl319504 2523 2952
6 244366.6 1623474F6 2155 2682
7 405313.4 4640462H1 573 837
7 405313.4 g1774849 595 979
7 405313.4 4721077H1 54 194
7 405313.4 5944975H1 61 370
7 405313.4 1948647H1 596 828
7 405313.4 1592016H1 86 282
7 405313.4 1948647R6 596 1136
7 405313.4 g4070751 686 1137
7 405313.4 2384959H1 86 263
7 405313.4 4571373H1 715 978
7 405313.4 g954058 893 1203
7 405313.4 1559555H1 903 1120
7 405313.4 1559555F6 903 1363
7 405313.4 go17633 914 1316
7 405313.4 1302977H1 86 257
7 405313.4 4215272H1 919 1195
7 405313.4 g1492868 88 230
7 405313.4 4643815H1 962 1212
7 405313.4 965308H1 968 1255
7 405313.4 965308R1 968 1622
7 405313.4 1321926T6 132 483
7 405313.4 5136028H1 993 1266
7 405313.4 g4264253 155 609
7 405313.4 g3739298 156 610
7 405313.4 4306178H1 1008 1207
7 405313.4 g1237752 1009 1175
7 405313.4 4551446H1 1047 1310
7 405313.4 g4522654 210 522
7 405313.4 1628853H1 1049 1219
7 405313.4 1627193H1 1049 1261
7 405313.4 1316291H1 349 522
7 405313.4 1628853F6 1049 1649 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
7 405313.4 1283759H1 1053 1330
7 405313.4 4312209H1 1067 1367
7 405313.4 4103086H1 1115 1239
7 405313.4 g1984348 1147 1472
7 405313.4 g2540589 1168 1616
7 405313.4 g1492809 369 527
7 405313.4 3584223H1 1177 1354
7 405313.4 102569H1 458 607
7 405313.4 4829437H1 540 737
7 405313.4 3674811 HI 1186 1495
7 405313.4 1733325H1 1192 1412
7 405313.4 g1984560 561 804
7 405313.4 g2010449 2002 2335
7 405313.4 2682711 HI 2014 2309
7 405313.4 g4535531 2016 2337
7 405313.4 g1773873 2022 2340
7 405313.4 g4137010 2025 2346
7 405313.4 4111488H1 2026 2288
7 405313.4 2153069H1 2034 2315
7 405313.4 870657R1 2041 2613
7 405313.4 870657H1 2041 2250
7 405313.4 5433876H1 2063 2317
7 405313.4 g3162264 2073 2338
7 405313.4 g3057393 2080 2337
7 405313.4 g3872586 2081 2335
7 405313.4 2081907T6 2084 2288
7 405313.4 659926H1 2097 2337
7 405313.4 056422H1 2097 2317
7 405313.4 3486469H1 2105 2337
7 405313.4 817086R1 2127 2337
7 405313.4 817086H1 2127 2388
7 405313.4 81708611 2127 2280
7 405313.4 g3597649 2142 2335
7 405313.4 3988965H1 2205 2499
7 405313.4 2664989H1 1633 1850
7 405313.4 3962192H1 1645 1776
7 405313.4 2290557H1 1645 1921
7 405313.4 4115475H1 1647 1861
7 405313.4 g670108 1649 1960
7 405313.4 g570685 1650 1964
7 405313.4 2213032H1 1653 1919
7 405313.4 1667188H1 1653 1775
7 405313.4 2285586H1 1653 1841
7 405313.4 990792H1 1669 1968
7 405313.4 1283707T6 1682 2306
7 405313.4 3781966H1 1686 2020
7 405313.4 2402093H1 1700 1946
7 405313.4 3666248H1 1707 1866
7 405313.4 1948647T6 1711 2289
7 405313.4 5681549H1 1746 2012 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
7 405313.4 2885455T6 1752 2292
7 405313.4 1301039H1 1790 2095
7 405313.4 1669180T6 1799 2303
7 405313.4 1669180H1 1808 2038
7 405313.4 3436329H1 1815 1956
7 405313.4 g1624740 1828 2218
7 405313.4 1559555T6 1831 2302
7 405313.4 3106278H1 1846 2121
7 405313.4 2323323T6 1848 2308
7 405313.4 1914641 HI 1848 2062
7 405313.4 1628853T6 1854 2300
7 405313.4 g2946487 1855 2335
7 405313.4 2081907F6 1854 2313
7 405313.4 2081917H1 1854 2015
7 405313.4 g672957 1859 2199
7 405313.4 2916179H1 1878 2177
7 405313.4 3556793T6 1881 2306
7 405313.4 g2783325 1885 2345
7 405313.4 1268187F1 1888 2344
7 405313.4 1268187H1 1888 2152
7 405313.4 1268187F6 1888 2203
7 405313.4 1268187T6 1890 2317
7 405313.4 g616527 1892 2244
7 405313.4 g3593850 1896 2335
7 405313.4 g573001 1897 2264
7 405313.4 g815353 1898 2253
7 405313.4 g4083770 1902 2335
7 405313.4 g4281927 1912 2337
7 405313.4 g2753877 1930 2345
7 405313.4 2199211 HI 1930 2188
7 405313.4 2375908H1 1934 2181
7 405313.4 g3797979 1954 2337
7 405313.4 2653312H1 1963 2221
7 405313.4 g4265408 1967 2342
7 405313.4 g3919084 1969 2335
7 405313.4 g4085496 1970 2334
7 405313.4 g668546 1976 2156
7 405313.4 2149388H1 1985 2274
7 405313.4 2601556H1 1995 2280
7 405313.4 g2789326 2898 3179
7 405313.4 g646138 2913 3179
7 405313.4 g888680 2916 3206
7 405313.4 g646137 2924 3179
7 405313.4 g645108 2927 3179
7 405313.4 g917579 2933 3178
7 405313.4 g3051580 2943 3179
7 405313.4 g3764150 2943 3179
7 405313.4 g2903435 2947 3179
7 405313.4 g1225232 2947 3179
7 405313.4 g1087854 2947 3111
79 bi s TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
7 405313.4 gl647814 2998 3213
7 405313.4 g917686 3058 3210
7 405313.4 1226453T1 3087 3168
7 405313.4 1226453H1 3087 3167
7 405313.4 g3174481 3089 3178
7 405313.4 g646728 2878 3179
7 405313.4 g884856 2884 3211
7 405313.4 gδ15354 2895 3216
7 405313.4 2714769H1 2231 2345
7 405313.4 1440914H1 2252 2421
7 405313.4 1440914F6 2252 2675
7 405313.4 4311940H1 2263 2546
7 405313.4 6073069H1 2266 2498
7 405313.4 549605H1 2285 2554
7 405313.4 2157285H1 2287 2523
7 405313.4 2323179H1 2324 2460
7 405313.4 2323108H1 2324 2581
7 405313.4 g2197270 2337 2696
7 405313.4 2294826H1 2444 2517
7 405313.4 g1043992 2444 2683
7 405313.4 386011 OH1 2444 2682
7 405313.4 3246952H1 2472 2725
7 405313.4 1956178H1 2480 2773
7 405313.4 4312886H1 2495 2794
7 405313.4 g770052 2623 2930
7 405313.4 1415784H1 2660 2922
7 405313.4 g884855 2666 3025
7 405313.4 2014171H1 2668 2943
7 405313.4 g888679 2667 3021
7 405313.4 2224791 HI 2686 2955
7 405313.4 4106915H1 2695 2996
7 405313.4 g2217789 2772 3181
7 405313.4 g2874275 2772 3179
7 405313.4 1440914R1 2773 3179
7 405313.4 g4075424 2775 3179
7 405313.4 g4328896 2776 3179
7 405313.4 287748H1 2778 3143
7 405313.4 3496950H1 2802 3087
7 405313.4 2750652H1 2806 3105
7 405313.4 g765774 2820 3182
7 405313.4 g3895056 2831 3179
7 405313.4 g4450984 2833 3179
7 405313.4 g2099917 2853 3348
7 405313.4 g2018248 2860 2990
7 405313.4 g564562 2869 3179
7 405313.4 g2459191 2869 3206
7 405313.4 g1099005 2521 2798
7 405313.4 2731146H1 2559 2794
7 405313.4 gl 198836 2578 2840
7 405313.4 2229784H1 2603 2852
80 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
7 405313.4 2229548H1 2603 2858
7 405313.4 1321926H1 1 235
7 405313.4 1321926F6 1 388
7 405313.4 1337104H1 8 265
7 405313.4 2682113H1 8 281
7 405313.4 g2754255 1191 1654
7 405313.4 4541436H1 1206 1470
7 405313.4 g2558152 1209 1673
7 405313.4 g2540638 1218 1616
7 405313.4 1282095H1 1225 1484
7 405313.4 1283707H1 1225 1511
7 405313.4 1282048H1 1225 1506
7 405313.4 1283707F6 1226 1659
7 405313.4 3280293H1 1234 1511
7 405313.4 5398765H1 1241 1391
7 405313.4 5072115H1 1293 1556
7 405313.4 5219645H1 1319 1443
7 405313.4 1893001 HI 1317 1616
7 405313.4 3696826H1 1319 1614
7 405313.4 5920192H1 1319 1647
7 405313.4 g3770003 1322 1609
7 405313.4 5665259H1 1327 1555
7 405313.4 3151495H1 1326 1625
7 405313.4 3357256H2 1345 1500
7 405313.4 692886H1 1353 1604
7 405313.4 5400503H1 1388 1623
7 405313.4 2323323H1 1395 1657
7 405313.4 2323323R6 1395 1895
7 405313.4 4622312H1 1404 1718
7 405313.4 5373496H1 1438 1700
7 405313.4 624120H1 1456 1728
7 405313.4 2535460H1 1469 1750
7 405313.4 2115405H1 1505 1801
7 405313.4 g954059 1516 1748
7 405313.4 2292561 HI 1538 1790
7 405313.4 2556287H1 1580 1842
7 405313.4 g866975 1610 1953
7 405313.4 861911R1 1619 2200
7 405313.4 861911H1 1619 1876
7 405313.4 g873285 1626 2006
8 436857.2 232864F1 1467 1959
8 436857.2 2704880T6 1506 1925
8 436857.2 g4373224 1522 1968
8 436857.2 g4112872 1522 1954
8 436857.2 g4390509 1556 1962
8 436857.2 5858881 HI 1624 1888
8 436857.2 5267222H1 1653 1883
8 436857.2 1477850T6 1680 2225
8 436857.2 4617960T6 1761 2215
8 436857.2 g917596 1901 2223 TABLE 4
SEQ ID NO: Template ID Component ID Start Stop
8 436857.2 3270104H1 1906 2161
8 436857.2 5782044H1 2003 2209
8 436857.2 g4268407 2010 2240
8 436857.2 g3051680 2063 2241
8 436857.2 481468H1 2063 2250
8 436857.2 5683178H1 1 212
8 436857.2 1477850H1 56 278
8 436857.2 1477850F6 56 488
8 436857.2 4619212H1 138 290
8 436857.2 g 1978924 207 556
8 436857.2 3758509H1 206 498
8 436857.2 4617960H1 323 550
8 436857.2 4718961 HI 323 555
8 436857.2 4617960F6 323 761
8 436857.2 1992924H1 366 651
8 436857.2 g4269060 528 627
8 436857.2 4255690H1 599 830
8 436857.2 4761770H1 727 1004
8 436857.2 4613106H1 795 1034
8 436857.2 g2000739 795 1025
8 436857.2 2704880H1 883 1176
8 436857.2 2704880F6 883 1313
8 436857.2 2707669H1 961 1264
8 436857.2 805609H1 1054 1283
8 436857.2 805609R1 1054 1630
8 436857.2 4135963H1 1113 1415
8 436857.2 4294249H1 1152 1398
8 436857.2 5450335H1 1156 1421
8 436857.2 5373082H1 1203 1419
8 436857.2 4190554H1 1247 1412
82

Claims

CLAIMS What is claimed is:
1. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of: a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-14, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-14, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a) through d).
2. An isolated polynucleotide of claim 1, comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-14.
3. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 1.
4. A composition for the detection of expression of disease detection and treatment molecule polynucleotides comprising at least one of the polynucleotides of claim 1 and a detectable label.
5. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 1 , the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
6. A method for detecting a target polynucleotide in a sample, said target polynucleotide comprising a sequence of a polynucleotide of claim 1, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide, and
83 b) detecting the presence or absence of said hybndization complex, and, optionally, if present, the amount thereof
7 A method of claim 5, wherein the probe comprises at least 30 contiguous nucleotides
8 A method of claim 5, wherein the probe comprises at least 60 contiguous nucleotides
9 A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 1
10 A cell transformed with a recombinant polynucleotide of claim 9
11 A transgenic organism comprising a recombinant polynucleotide of claim 9
12 A method for producing a disease detection and treatment molecule polypeptide, the method compnsing a) cultuπng a cell under conditions suitable for expression of the disease detection and treatment molecule polypeptide, wherein said cell is transformed with a recombinant polynucleotide of claim 9, and b) recovering the disease detection and treatment molecule polypeptide so expressed
13 A purified disease detection and treatment molecule polypeptide (MDDT) encoded by at least one of the polynucleotides of claim 2
14 An isolated antibody which specifically binds to a disease detection and treatment molecule polypeptide of claim 13
15 A method of identifying a test compound which specifically binds to the disease detection and treatment molecule polypeptide of claim 13, the method compnsing the steps of a) providing a test compound, b) combining the disease detection and treatment molecule polypeptide with the test compound for a sufficient time and under suitable conditions for binding, and c) detecting binding of the disease detection and treatment molecule polypeptide to the test compound, thereby identifying the test compound which specifically binds the disease detection and treatment molecule polypeptide
84 16 A microarray wherein at least one element of the microarray is a polynucleotide of claim
17 A method for generating a transcript image of a sample which contains polynucleotides, the method compnsing the steps of a) labeling the polynucleotides of the sample, b) contacting the elements of the microanay of claim 16 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample
18 A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence of claim 1, the method compnsing a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide
19 A method of claim 6 for toxicity testing of a compound, further compnsing
(c) compaπng the presence, absence or amount of said target polynucleotide in a first biological sample and a second biological sample, wherein said first biological sample has been contacted with said compound, and said second sample is a control, whereby a change in presence, absence or amount of said target polynucleotide in said first sample, as compared with said second sample, is indicative of toxic response to said compound
85
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US14750199P 1999-08-05 1999-08-05
US147500P 1999-08-05
US147542P 1999-08-05
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US7611892B2 (en) 2000-03-24 2009-11-03 President And Fellows Of Harvard College Prostate-specific or testis-specific nucleic acid molecules, polypeptides, and diagnostic and therapeutic methods
US6664084B2 (en) * 2001-02-26 2003-12-16 Applera Corporation Isolated nucleic acid molecules encoding human glucuronyltransferase proteins, and related products and processes
JP2005508142A (en) * 2001-05-25 2005-03-31 インサイト・ゲノミックス・インコーポレイテッド Disease detection and therapeutic molecules

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US6458939B1 (en) * 1996-03-15 2002-10-01 Millennium Pharmaceuticals, Inc. Compositions and methods for the diagnosis, prevention, and treatment of neoplastic cell growth and proliferation
AU5796298A (en) * 1996-12-11 1998-07-03 Chiron Corporation Secreted human proteins
WO1998056951A1 (en) * 1997-06-11 1998-12-17 Abbott Laboratories Reagents and methods useful for detecting diseases of the lung
WO1999002724A2 (en) * 1997-07-11 1999-01-21 Mount Sinai Hospital Corporation Methods for identifying genes expressed in selected lineages, and a novel genes identified using the methods
EP1068307A1 (en) * 1998-03-31 2001-01-17 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them

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