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WO1993016178A2 - Sequences caracteristiques du produit de transcription des genes humains - Google Patents

Sequences caracteristiques du produit de transcription des genes humains Download PDF

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Publication number
WO1993016178A2
WO1993016178A2 PCT/US1993/001294 US9301294W WO9316178A2 WO 1993016178 A2 WO1993016178 A2 WO 1993016178A2 US 9301294 W US9301294 W US 9301294W WO 9316178 A2 WO9316178 A2 WO 9316178A2
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Prior art keywords
seq
polynucleotide
sequences
sequence
est
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PCT/US1993/001294
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WO1993016178A3 (fr
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Craig J. Venter
Mark D. Adams
Ruben F. Moreno
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO1993016178A2 publication Critical patent/WO1993016178A2/fr
Publication of WO1993016178A3 publication Critical patent/WO1993016178A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to newly identified polynucleotide sequences corresponding to transcription products of human genes, and to complete gene sequences associated therewith.
  • This invention relates to human genes. Identification and sequencing of human genes is a major goal of modern scientific research. The sequence of human genes is more than just a scientific curiosity. For example, by identifying genes and determining their sequences, scientists have been able to make large quantities of valuable human "gene products.” These include human insulin, interferon, Factor VIII, tumor necrosis factor, human growth hormone, tissue plasminogen activator, and numerous other compounds. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis). The present invention represents a quantum leap forward in civilization's knowledge of human gene sequences.
  • the present invention is based on identification and characterization of gene segments.
  • Genes are the basic units of inheritance. Each gene is a string of connected bases called nucleotides. Most genes are formed of deoxyribonucleic acid, DNA. (Some viruses contain genes of ribonucleic acid, RNA.) The genetic information resides in the particular sequence in which the bases are arranged. A short sequence of nucleotides is often called a polynucleotide or an oligonucleotide.
  • polypeptides are built from long strings of individual units. These units are amino acids.
  • the nucleotide sequence of a gene tells the cell the sequence in which to arrange the amino acids to make the polypeptide encoded by that gene.
  • chains of up to about 200 amino acids are called polypeptides, while proteins are larger molecules made up of polypeptide subunits; both types of molecules are referred to generally herein as polypeptides.
  • a triplet of nucleotides (codon) in DNA codes for each amino acid or signals the beginning or end of the message (anticodon).
  • the term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the original DNA sequence is transcribed.
  • RNA messenger RNA
  • mRNA messenger RNA
  • the mRNA in turn, can be translated into a polypeptide by the cell. This entire process is called gene expression, and the polypeptide is the gene product encoded by the gene.
  • cDNA complementary DNA
  • genes include those which code for polypeptides, those which are transcribed into RNA but are not translated into polypeptides, and those whose functional significance does not demand that they be transcribed at all.
  • Most genes are found on large molecules of DNA located in chromosomes. Double stranded cDNA carries all the information of a gene. Each base of the first strand is joined to a complementary base (hybridized) in the second strand.
  • the linear DNA molecules in chromosomes have thousands of genes distributed along their length. Chromosomes include both coding regions (coding for polypeptides) and noncoding regions; the coding regions. represent only about three percent of the total chromosome sequence.
  • An individual gene has regulatory regions that include a promoter which directs expression of the gene, a coding region which can code for a polypeptide, and a termination signal.
  • the regulatory DNA sequence is usually a noncoding region that determines if, where, when, and at what level a particular gene is expressed.
  • the coding regions of many genes are discontinuous, with coding sequences (exons) alternating with noncoding regions (introns).
  • the final mRNA copy of the gene does not include these introns (which can be much longer than the coding region itself), although it does contain certain untranslated regions that usually do not code for the polynucleotide gene product.
  • Untranslated sequences at the beginning and end of the mRNA are known as 5'- and 3'-untranslated regions, respectively. This nomenclature reflects the orientation of the nucleotide constituents of the mRNA.
  • a cDNA is a DNA copy of a messenger RNA, which contains all of the exons of a gene.
  • the cDNA can be thought of as having three parts: an untranslated 5' leader, an uninterrupted polypeptide-coding sequence, and a 3' untranslated region.
  • the untranslated leader and trailing sequences are important for initiation of translation, mRNA stability, and other functions.
  • the untranslated leader and trailing sequences are called 5'- and 3'-untranslated sequences, respectively.
  • the 3' untranslated sequence is usually longer than the 5' untranslated leader, and can be longer than the polypeptide-coding sequence.
  • the untranslated regions typically have many, randomly-distributed stop codons, and do not display the nonrandom base arrangements found in coding sequences.
  • the 5'-untranslated sequence is relatively short, generally between 20 and 200 bases.
  • the 3'-untranslated sequence is often many times longer, up to several thousand bases.
  • the translated or coding sequence begins with a translational start codon (AUG or GUG) and ends with a translational stop codon (UAA, UGA, or UAG).
  • translation begins at the first "start” codon on the mRNA and proceeds to the first "stop” codon. Coding sequences can be distinguished by their nonrandom distribution of bases; numerous computer algorithms have been developed to distinguish coding from noncoding regions in this way.
  • PCR polymerase chain reaction
  • oligonucleotide primers that hybridize to opposite strands.
  • Primer extension proceeds inward across the region between the two primers, and the product of DNA synthesis of one primer serves as a template for the other primer. Repeated cycles of DNA denaturation, annealing of primers, and extension result in an exponential increase in the number of copies of the region bounded by the primers.
  • a labeled segment of single-stranded DNA can be hybridized to a longer DNA sequence, such as a chromosome, to mark a specific location on the longer sequence.
  • a longer DNA sequence such as a chromosome
  • the Human Genome Project is an effort to sequence all human DNA (the human genome).
  • the human genome is estimated to comprise 50,000 - 100,000 genes, up to 30,000 of which might be expressed in the brain (Sutcliffe, Ann. Rev. Neurosci. 11:157 (1988)).
  • Once dedicated human chromosome sequencing begins in three to five years, it was expected that 12-15 years will be required to complete the sequence of the genome (Report of the Ad Hoc Program. Advisory Committee on Complex Genomes, Reston, Va., Feb. 1988, D. Baltimore Ed. (NIH, Bethesda, Md, 1988)).
  • the present invention can greatly accelerate the pace at which human genes can be identified and mapped.
  • GenBank listed the sequences of only a few thousand human genes and less than two hundred human brain mRNAs (GenBank Release 66.0, December, 1990).
  • Genomic sequencing proponents have argued the difficulty of finding every mR ⁇ A expressed in all tissues, cell types, and developmental states, and that much valuable information from intronic and intergenic regions, including control and regulatory sequences, will be missed by cD ⁇ A sequencing. (Report of the Committee on Mapping and Sequencing the Human Genome, National Research Council (National Academy Press, Washington, D.C. 1988)). Further, sequencing of transcribed regions of the genome using cDNA libraries has heretofore been considered impractical or unsatisfactory. Libraries of cDNA were believed to be dominated by repetitive elements, mitochondrial genes, ribosomal RNA genes, and other nuclear genes comprising common or housekeeping sequences.
  • cDNA libraries would provide few sequences corresponding to structural and regulatory polypeptides or peptides. See, for example, Putney, et al., Nature 302:718-721 (1983). Putney, et al. sequenced over 150 clones from a rabbit muscle cDNA library and identified clones for 13 of the 19 known muscle polypeptides, including one new isotype but no unknown coding sequences.
  • cDNA sequencing now provides a rapid method for obtaining enormous amounts of valuable genetic information and DNA products of great utility for the biotechnology and pharmaceutical industries. Not only can many distinct cDNAs be isolated and sequenced, even partial cDNAs can be used, with conventional, well-understood methods, to isolate entire genes, and to determine the chromosomal locations and biological functions of these genes. As is demonstrated here, fragments of only a few hundred bases are sufficient, in many cases, to identify the probable function of a new human gene if it is similar in structure to a gene from another animal, or from plants or bacteria.
  • fragments of untranslated regions of a cDNA can be used to: i) isolate the coding sequence of the cDNA; ii) isolate the complete gene; iii) determine the position of the gene on a human chromosome, and hence the potential of the gene to cause a human genetic disease; and iv) determine the function of the gene by means of experiments in which the function of the native gene is disrupted by the addition of a short DNA fragment to the cell, e.g., using triple helix or antisense probes.
  • coding regions comprise such a small portion of the human genome
  • identification and mapping of transcribed regions and coding regions of chromosomes is of significant interest.
  • human sequences are valuable for chromosome mapping, human identification, identification of tissue type and origin, forensic identification, and locating disease-associated genes (i.e., genes that are associated with an inherited human disease, whether through mutation, deletion, or faulty gene expression) on the chromosome.
  • sequences of the present invention were ascertained using a fast approach to cDNA characterization. This approach could facilitate the tagging of most expressed human genes within a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, provide new DNA-based therapeutics and diagnostics, and provide other valuable nucleotide reagents.
  • ESTs styled Expressed Sequence Tags
  • STSs random genomic DNA sequence tagged sites
  • aspects of the present invention thus include the individual ESTs, corresponding partial and complete cDNA, genomic DNA, mRNA, antisense strands, triple helix probes, PCR primers, coding regions, and constructs. Also, where one skilled in the art is enabled by this specification to prepare expression vectors and polypeptide expression products, they are also within the scope of the present invention, along with antibodies, especially monoclonal antibodies, to such expression products.
  • the single drawing Figure schematically illustrates the progression from chromosome to gene to mRNA to cDNA.
  • the sequences of the present invention were isolated from commercially available and custom made cDNA libraries using a rapid screening and sequencing technique.
  • the method comprises applying conventional automated DNA sequencing technology to screening clones, advantageously randomly selected clones, from a cDNA library.
  • the library is initially "enriched” through removal of ribosomal sequences and other common sequences prior to clone selection.
  • ESTs are generated from partial DNA sequencing of the selected clones.
  • the ESTs of the present invention were generated using low redundancy of sequencing, typically a single sequencing reaction. While single sequencing reactions may have an accuracy as low as 97%, this nevertheless provides sufficient fidelity for identification of the sequence and design of PCR primers.
  • transcripts of the gene will not be represented in cDNA libraries so the gene will not be identifiable by EST sequencing.
  • a new method called "exon amplification" can be used to isolate and identify transcripts of such genes.
  • Exon amplification works by artificially expressing part or all of a gene that is contained in a cloned fragment of genomic DNA such as a cosmid or yeast artificial chromosome (YAC).
  • the gene is cloned into a special vector, designed at MIT, that uses control elements from virus genes to express the protein-coding exons of the human gene of interest.
  • Exon trapping shows considerable promise as a general technique for identifying those genes in the human genome that cannot be found by cDNA cloning and EST sequencing.
  • Exon amplification will also be useful for identifying the genes in regions of genomic DNA to which disease genes have been mapped.
  • the exon amplification method can be used directly with the cosmid and YAC clones frown human chromosomes that are being obtained by both NIH and DOE supported human genome centers.
  • ESTs comprise DNA sequences corresponding to a portion of nuclear encoded messenger RNA.
  • An EST is of sufficient length to permit: (1) amplification of the specific sequence from a cDNA library, e.g., by polymerase chain reaction (PCR); (2) use of a synthetic polynucleotide corresponding to a partial or complete sequence of the EST as a hybridization probe of a cDNA library, generally having 30 - 50 base pairs; or (3) unique designation of the pure cDNA clone from which the EST was derived (the EST clone) for use as a hybridization probe of a cDNA library.
  • EST-derived primer pairs and sequences amplify or detectably hybridize to a sequence from a genomic library.
  • the ESTs disclosed herein are generally at least 150 base pairs in length.
  • the length of an EST is determined by the quality of sequencing data and the length of the cloned cDNA.
  • Raw data from the automated sequencers is edited to remove low quality sequence at the end of the sequencing run.
  • High quality sequences (usually a result of sequencing templates without excessive salt contamination) generally give about 400 bp of reliable sequence data; other sequences give fewer bases of reliable data.
  • a 150 bp EST is long enough to be translated into a 50 amino acid peptide sequence. This length is sufficient to observe similarities when they exist in a database search.
  • 150 bp is long enough to design PCR primers from each end of the sequence to amplify the complete EST. Sequences shorter than 150 bp are difficult to purify and use following PCR amplification. Furthermore, a 150 bp polynucleotide is likely to give a very strong signal with low background in a screen of a genomic library.
  • This problem can be circumvented by using the 3'-untranslated part of the cDNA alone as a probe for the chromosomal location or for the full -length cDNA or gene .
  • the 3'-untranslated region is more likely to be unique within gene families, since there is no evolutionary pressure to conserve a coding function of this region of the mRNA.
  • ESTs can be used to map the expressed sequence to a particular chromosome.
  • ESTs can be expanded to provide the full coding regions, as detailed below. In this manner, previously unknown genes can be identified.
  • cDNA libraries can be used to obtain ESTs
  • human brain cDNA libraries are exemplified and represent a preferred embodiment.
  • Suitable cDNA libraries can be freshly prepared or obtained commercially, e.g., as shown in Examples 1, 2, and 11.
  • the cDNA libraries from the desired tissue are preferably preprocessed by conventional techniques to reduce repeated sequencing of high and intermediate abundance clones and to maximize the chances of finding rare messages from specific cell populations.
  • preprocessing includes the use of defined composition prescreening probes, e.g., cDNA corresponding to mitochondria, abundant sequences, ribosomes, actins, myelin basic polypeptides, or any other known high abundance peptide; these prescreening probes used for preprocessing are generally derived from known ESTs.
  • Other useful preprocessing techniques include subtraction, which preferentially reduces the population of certain sequences in the library (e.g., see A. Swaroop et al., Nucl. Acids Res. 19, 1954 (1991)), and normalization, which results in all sequences being represented in approximately equal proportions in the library (Patanjali et al, Proc. Natl. Acad. Sci. USA 88:1943 (1991)).
  • the cDNA libraries used in the present method will ideally use directional cloning methods so that either the 5' end of the cDNA (likely to contain coding sequence) or the 3' end (likely to be a non-coding sequence) can be selectively obtained.”
  • Libraries of cDNA can also be generated from recombinant expression of genomic DNA. After they are amplified, ESTs can be obtained and sequenced, e.g., as illustrated in Example 11.
  • sequences of the present invention include the specific sequences set forth in the Sequence Listing and designated SEQ ID NO: 1 - SEQ ID NO: 2412.
  • the invention relates to those sequences of SEQ ID NOS: 1 - 2412 that comprise the cDNA coding sequences for polypeptides having less than 95% identity with known amino acid sequences (see Table 2) and more preferably less than 90% or 85% identity.
  • the invention relates to those sequences of SEQ ID NOS: 1 - 2412 that encode polypeptides having no similarity to known amino acid sequences (see Examples that follow). Precisely because they do not contain coding regions and are therefore more unique in their sequence structures, those sequences which meet neither of the preceding criteria can be most useful and are generally preferred for mapping.
  • the ESTs of the present invention generally represent relatively small coding regions or untranslated regions of human genes. Although most of these sequences do not code for a complete gene product, the ESTs of the present invention are highly specific markers for the corresponding complete coding regions.
  • the ESTs are of sufficient length that they will hybridize, under stringent conditions, only with DNA for that gene to which they correspond.
  • Suitably stringent conditions comprise conditions, for example, where at least 95%, preferably at least 97% or 98% identity (base pairing), is required for hybridization. This property permits use of the EST to isolate the entire coding region and even the entire sequence. Therefore, only routine laboratory work is necessary to parlay the unique EST sequence into the corresponding unique complete gene sequence.
  • each EST “corresponds" to a particular unique human gene. Knowledge of the EST sequence permits routine isolation and sequencing of the complete coding sequence of the corresponding gene. The complete coding sequence is present in a full-length cDNA clone as well as in the gene carried on genomic clones. Therefore, each EST "corresponds" to a cDNA (from which the EST was derived), a complete genomic gene sequence, a polypeptide coding region (which can be obtained either from the cDNA or genomic DNA) , and a polypeptide or amino acid sequence encoded by that region.
  • the first step in determining where an EST is located in the cDNA is to analyze the EST for the presence of coding sequence, e.g., as described in Example 14.
  • the CRM program predicts the extent and orientation of the coding region of a sequence. Based on this information, one can infer the presence of start or stop codons within a sequence and whether the sequence is completely coding or completely noncoding. If start or stop codons are present, then the EST can cover both part of the 5'-untranslated or 3'-untranslated part of the mRNA (respectively) as well as part of the coding sequence. If no coding sequence is present, it is likely that the EST is derived from the 3'-untranslated sequence due to its longer length and the fact that most cDNA library construction methods are biased toward the 3' end of the mRNA.
  • Radiolabel the isolated insert DNA e.g., with 32 P labels, preferably by nick translation or random primer labeling.
  • An EST is a specific tag for a messenger RNA molecule.
  • the complete sequence of that messenger RNA, in the form of cDNA, can be determined using the EST as a probe to identify a cDNA clone corresponding to a full-length transcript, followed by sequencing of that clone.
  • the EST or the full-length cDNA clone can also be used as a probe to identify a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons, and introns.
  • ESTs are used as probes to identify the cDNA clones from which an EST was derived.
  • ESTs, or portions thereof can be nick-translated or end-labelled with P 32 using polynucleotide kinase using labelling methods known to those with skill in the art (Basic Methods in Molecular Biology, L.G. Davis, M.D. Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986).
  • the lambda library can be directly screened with the labelled ESTs of interest or the library can be converted en masse to pBluescript (Stratagene, La Jolla, California) to facilitate bacterial colony screening. Both methods are well known in the art.
  • filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured and the DNA is fixed to the filters.
  • the filters are hybridized with the labelled probe using hybridization conditions described by Davis et al.
  • the ESTs, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification.
  • the resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque.
  • the colonies or plaques are selected, expanded and the DNA is isolated from the colonies for further analysis and sequencing.
  • the ESTs can additionally be used to screen Northern blots of mRNA obtained from various tissues or cell cultures, including the tissue of origin of the EST clone. Northern analysis will most often produce one to several positive bands. The bands can be selected for further study based on the predicted size of the mRNA.
  • Positive cDNA clones in phage lambda are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the EST and the other primer from the vector.
  • Clones with a larger vector-insert PCR product than the original EST clone are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size on a Northern blot.
  • the complete sequence of the clones can be determined.
  • the preferred method is to use exonuclease III digestion (McCombie, W.R, Kirkness, E., Fleming, J.T., Kerlavage, A.R., Iovannisci, D.M., and Martin-Gallardo, R., Methods: 3: 33-40, 1991).
  • a series of deletion clones is generated, each of which is sequenced.
  • the resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
  • a similar screening and clone selection approach can be applied to obtaining cosmid or lambda clones from a genomic DNA library that contains the complete gene from which the EST was derived (Kirkness, E.F., Kusiak, J.W., Menninger, J., Gocayne, J.D., Ward, D.C., and Venter, J.C. Genomics 10: 985-995 (1991). Although the process is much more laborious, these genomic clones can be sequenced in their entirety also.
  • a shotgun approach is preferred to sequencing clones with inserts longer than 10 kb (genomic cosmid and lambda clones). In shotgun sequencing, the clone is randomly broken into many small pieces, each of which is partially sequenced.
  • sequence fragments are then aligned to produce the final contiguous sequence with high redundancy.
  • An intermediate approach is to sequence just the promoter region and the intron-exon boundaries and to estimate the size of the introns by restriction endonuclease digestion (ibid.).
  • the polynucleotides of the present invention can be derived from natural sources or synthesized using known methods.
  • the sequences falling within the scope of the present invention are not limited to the specific sequences described, but include human allelic and species variations thereof and portions thereof of at least 15-18 bases. (Sequences of at least 15-18 bases can be used, for example, as PCR primers or as DNA probes.)
  • the invention includes the entire coding sequence associated with the specific polynucleotide sequence of bases described in the Sequence Listing, as well as portions of the entire coding sequence of at least 15-18 bases and allelic and species variations thereof.
  • the invention includes sequences coding for the same amino acid sequences as do the specific sequences disclosed herein.
  • sequences, constructs, vectors, clones, and other materials comprising the present invention can advantageously be in enriched or isolated form.
  • enriched means that the concentration of the material is at least about 2, 5, 10, 100, or 1000 times its natural concentration (for example), advantageously 0.01%, by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Further, removal of clones corresponding to ribosomal RNA and "housekeeping" genes and clones without human cDNA inserts results in a library that is "enriched" in the desired clones.
  • isolated requires that the material be removed from its original environment (e.g., the natural. environment if it is naturally occurring).
  • a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • sequences be in purified form.
  • purified does .not require absolute purity; rather, it is intended as a relative definition.
  • Individual EST clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity. The sequences obtained from these clones could not be obtained directly either from the library or from total human DNA.
  • the cDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • the conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection.
  • cDNA synthetic substance
  • cDNA pure individual cDNA clones can be isolated from the synthetic library by clonal selection.
  • creating a cDNA library from messenger RNA and subsequently isolating individual clones from that library results in an approximately 10°-fold purification of the native message.
  • Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • a cDNA library there are many species of mRNA represented. Each cDNA clone can be interesting in its own right, but must be isolated from the library before further experimentation can be completed. In order to sequence any specific cDNA, it must be removed and separated (i.e. isolated and purified) from all the other sequences. This can be accomplished by many techniques known to those of skill in the art. These procedures normally involve identification of a bacterial colony containing the cDNA of interest and further amplification of that bacteria. Once a cDNA is separated from the mixed clone library, it can be used as a template for further procedures such as nucleotide sequencing.
  • subgroupings of 50 ESTs are contemplated (e.g., SEQ ID NOS 1-50, 51-100, 101-150, etc.) as being within the scope of this invention, as are subgroupings of 5, 10, 25, 100, 200, and 500 ESTs and corresponding sequences.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a sense or antisense orientation.
  • the construct further comprises regulatory sequences, including for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial Bacterial: pBs, phagescript, ⁇ X174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukarvotic pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , and trc.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described construct.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a procaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • the constructs in host cells can be used in a conventional manner to produce the gene product coded by the recombinant sequence.
  • the encoded polypeptide can be synthetically produced by conventional peptide synthesizers.
  • ESTs have already been preliminarily categorized by analogy to related sequences in other organisms (see Table 2).
  • Table 10 of Example 10 categorizes particular ESTs broadly as metabolic, regulatory, and structural sequences where known. Constructs comprising genes or coding sequences corresponding to each of these categories are, therefore, specifically and individually contemplated.
  • Table 11 more particularly separates 127 new ESTs into
  • Each of the cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents.
  • the sequences can be used as diagnostic probes for the presence of a specific mRNA in a particular cell type.
  • these sequences can be used as diagnostic probes suitable for use in genetic linkage analysis (polymorphisms).
  • the sequences can be used as probes for locating gene regions associated with genetic disease, as explained in more detail below.
  • the EST and complete gene sequences of the present invention are also valuable for chromosome identification. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The present invention constitutes a major expansion of available chromosome markers. One hundred ESTS have already been mapped to chromosomes. Using the techniques described in Example 5 or 6, the remaining ESTs and the corresponding complete sequences can similarly be mapped to chromosomes. The mapping of ESTs and cDNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the ESTs. Computer analysis of the ESTs is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the EST will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular EST to a particular chromosome. Three or more clones can be assigned per day using a single thermal cycler. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map an EST to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific cDNA libraries. Results of mapping ESTs to chromosomal segments are listed in Tables 3 and 4.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • This technique can be used with cDNA as short as 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • FISH requires use of the clone from which the EST was derived, and the longer the better. 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time.
  • Reagents for chromosome mapping can be used individually (to mark a single chromosome or a single site on that chromosome) or as panels of reagents (for marking multiple sites and/or multiple chromosomes). Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping (see Tables 8 and 9).
  • a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb.)
  • Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individuals is required to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • sequences of the invention can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide sequence to DNA or RNA.
  • Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al, Nucl. Acids Res. 6: 3073 (1979); Cooney et al, Science 241: 456 (1988); and Dervan et al, Science 251: 1360 (1991)) or to the mRNA itself (antisense - Okano, J. Neurochem.
  • sequences of the present invention are also useful for identification of individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP.
  • RFLP is a pattern based technique, which does not directly focus on the actual DNA sequence of the individual.
  • the sequences of the present invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA.
  • Panels of corresponding DNA sequences from individuals can provide unique individual identifications, as each individual will have a unique set of such DNA sequences, due to allelic differences.
  • the sequences of the present invention can be used to particular advantage to obtain such identification sequences from individuals and from tissue, as explained in Examples 12 - 14.
  • the EST sequences from Examples 1 and 2 and the complete sequences from Example 13 uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the ESTs or complete coding sequences comprising a part of the present invention can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of Table 9 could comfortably provide positive individual identification with a panel of perhaps 100 to 1,000 primers which each yield a noncoding amplified sequence of 100 bp. If predicted coding sequences, such as those from Table 6, are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from ESTs or complete sequences of this invention is used to generate a unique ID database for an individual, those same reagents can later be used to identify tissue from that individual. Positive identification of that individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques are in forensic biology.
  • PCR technology can be used to amplify 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.
  • gene sequences are amplified at specific loci known to contain a large number of allelic variations, for example the DQ ⁇ class II HLA gene (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once this specific area of the genome is amplified, it is digested with one or more restriction enzymes to yield an identifying set of bands on a Southern blot probed with DNA corresponding to the DQ ⁇ class II HLA gene.
  • sequences of the present invention can be used to provide polynucleotide reagents specifically targeted to additional loci in the human genome, and can enhance the reliability of DNA-based forensic identifications. Those sequences targeted to noncoding regions (see, e.g., Tables 8 and 9) are particularly appropriate. As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Reagents for obtaining such sequence information are within the scope of the present invention. Such reagents can comprise complete ESTs or corresponding coding regions, or fragments of either of at least 15 bp, preferably at least 18 bp.
  • reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin.
  • Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the ESTs or complete sequences of the present invention. 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 culture for contamination.
  • each EST corresponds not only to a coding region, but also to a polypeptide.
  • the coding sequence is known, or the gene is cloned which encodes the polypeptide, conventional techniques in molecular biology can be used to obtain the polypeptide.
  • the amino acid sequence encoded by the polynucleotide sequence can be synthesized using commercially available peptide synthesizers. This is particularly useful in producing small peptides and fragments of larger polypeptides. (Fragments are useful, for example, in generating antibodies against the native polypeptide.)
  • the DNA encoding the desired polypeptide can be inserted into a host organism and expressed.
  • the organism can be a bacterium, yeast, cell line, or multicellular plant or animal.
  • the literature is replete with examples of suitable host organisms and expression techniques.
  • naked polynucleotide DNA or mRNA
  • This methodology can be used to deliver the polypeptide to the animal, or to generate an immune response against a foreign polypeptide.
  • the coding sequence can be inserted into a vector, which is then used to transfect a cell.
  • the cell which may or may not be part of a larger organism
  • Antibodies generated against the polypeptide corresponding to a sequence of the present invention can be obtained by direct injection of the naked polypeptide into an animal (as above) or by administering the polypeptide to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide. Moreover, a panel of such antibodies, specific to a large number of polypeptides, can be used to identify and differentiate such tissue.
  • lambda ZAP libraries were converted en masse to pBluescript plasmids, transfected into E. coli XL1-Blue cells, and plated on X-gal/IPTG/ampicillin plates.
  • a total of 1058 clones were picked at random from three human brain cDNA libraries: fetal brain, two-year-old hippocampus, and two-year-old temporal cortex (Stratagene catalog #936206, 936205, 935, respectively.
  • Stratagene 11099 N. Torrey Pines Rd., La Jolla, CA 92037).
  • the EST sequences from this Example 1 are identified as SEQ ID NOs 1-315.
  • cDNA libraries were used as sources of clones for sequencing.
  • Human hippocampus and fetal brain libraries, plasmid template preparation, sequencing reactions, and automated sequencing were performed as described (Adams, M.D., Kelley, J.M., Gocayne, J.D., Dubnick, M., Polymeropoulos, M.H., Xiao, H., Merril, C.R., Wu, A., Olde, B., Moreno, R.F., Kerlavage, A.R., McCombie, W.R., & Venter, J.C. Science, 252: 1651-56 (1991)).
  • a pooled probe consisting of inserts from 10 different EST clones with sequences that matched either mitochondrial genes or the 18S or 28S ribosomal RNAs was used to prescreen a gridded filter array of the hippocampus library; nonhybridizing clones are referred to as the "prescreened library”.
  • Another fetal brain library was constructed by and was a gift from Bento
  • BLAST Altschul, S. F., Gish, W., Miller, W., Myers, E.W., & Lipman, D. J. Mol. Biol. 215: 403-410 (1990)
  • BLAST output was parsed, and an interactive alignment editor was used to select which matches, if any, from each search to record in a relational EST database, which was developed to track sequencing, identification, tissue localization, physical mapping, and the public distribution of the clones, mapping and sequence data.
  • ESTs including SEQ ID NOs 1-315 were analyzed as follows. Initially, the EST sequences were examined for similarities in the GenBank nucleic acid database (GenBank Release 65.0), Protein Information Resource Release 26.0 (PIR), and ProSite (MacPattern from the EMBL data library, Fuchs R. Comput. Appl. Biosci. 7: 105 (1990) Release 5.0 were used). BLAST was used to search Genbank and the PIR (both maintained by the National Center for Biotechnology Information) ESTs without exact GenBank matches were translated in all six reading frames and each translation was compared with the protein sequence database PIR and the ProSite protein motif database. Comparisons with the ProSite motif database were done by means of the program MacPattern from the EMBL Data Library.
  • GenBank and PIR searches were conducted with the "basic local alignment search tool" programs for nucleotide (BLASTN) and peptide (BLASTX) comparisons (Altschul et al, J. Mol. Biol. 215: 403 (1990)). PIR searches were run on the National Center for Biotechnology Information BLAST network service.
  • the BLAST programs contain a very rapid database-searching algorithm that searches for local areas of similarity between two sequences and then extends the alignments on the basis of defined match and mismatch criteria. The algorithm does not consider the potential gaps to improve the alignment, thus sacrificing some sensitivity for a 6-80 fold increase in speed over other database-searching programs such as FASTA
  • ESTs matched previously sequenced human nuclear genes with more than 97% identity.
  • Four of these ESTs are from genes encoding enzymes involved in maintaining metabolic energy, including ADP/ATP translocase, aldolase C, hexokinase, and phosphoglycerate kinase.
  • Human homologs of genes for the bovine mitochondrial ATP synthase F o ß-subunit and porcine aconitase were also found (Table 2).
  • Brain-specific cDNAs included synaptophysin, glial fibrillary acidic protein (GFAP), and neurofilament light chain.
  • ESTs are from genes encoding proteins involved in signal transduction: 2',3'-cyclic nucleotide 3'-phosphodiesterase (2 ESTs), calmodulin, c-erbA- ⁇ -2, G s ⁇ , and Na + /K + ATPase ⁇ -subunit.
  • Other ESTs were matches to genes for ubiquitous structural proteins - - actins , tubulins , and fodrin (non-erythroid spectrin).
  • ESTs also document the presence in the hippocampus cDNA library of the ret protooncogene, the ras-related gene rhoB, and one of the chromosome 22 breakpoint cluster region transcripts.
  • ESTs are from genes known to be associated with genetic disorders (Online Mendelian Inheritance in Man). More than half of the human-matched ESTs from Example 1 have been mapped to chromosomes, indicating the bias of GenBank entries toward well-studied genes and proteins.
  • ESTs without significant GenBank matches were also compared to the ProSite database of recognized protein motifs. Not counting post-translational-modification signatures, fifty-four sequences contained motifs from the database. Some patterns, particularly the "leucine zipper", are found in scores or hundreds of proteins that do not share the functional property implied by the presence of the motif.
  • EST00257 shows strong nucleotide sequence similarity to the squid (67%) and Drosophila (70.4%) kinesin heavy chain. Kinesin was first described as a microtubule-associated motor protein involved in organelle transport in the squid giant axon (Vale et al, Cell 42: 39 (1985)). Six oncogene-related sequences were also among the cDNA clones sequenced.
  • EST00299 SEQ ID NO: 180
  • EST00283 SEQ ID NO: 271
  • EST00248 SEQ ID NO: 102
  • EST00248 SEQ ID NO: 102
  • EST00248 SEQ ID NO: 102
  • EST00248 SEQ ID NO: 102
  • EST00248 SEQ ID NO: 102
  • EST00248 SEQ ID NO: 102
  • EST00299 SEQ ID NO: 180
  • EST00283 SEQ ID NO: 271
  • Similarities with an S. cerevisiae RNA polymerase subunit and Torpedo electromotor neuron-associated protein were also observed.
  • Two ESTs may represent new members of known human gene families: EST00270 matched the three ß-tubulin genes with 88-91% identity and EST00271 (SEQ ID NO:248) matched ⁇ -actinin with 85% identity at the nucleotide level.
  • Enhancer of split protein interacts with a membrane protein that is the product of the Notch gene to convert a developmental signal into an altered pattern of gene expression (id. J. Mol. Biol. 215: 403 (1990)).
  • EST00256 (SEQ ID NO:188) matches near the 5' end of the Enhancer of split coding sequence, away from the mammalian G protein ⁇ subunit- and yeast cdc4-like elements (Hartley et al, Cell 55: 785 (1988); Klambt et al. EMBO J. 8: 203 (1989)).
  • Seven genes were represented by more than one EST.
  • Example 2 The ESTs of Example 2, including SEQ ID NOs 316-2407, were screened against known sequences listed in GenBank and other databases, as in Example 3. The results are reported in Table 2. The quality of the match is given as percent identity and length in base pairs for nucleotide matches and amino acid residues for peptide matches. In many cases ESTs match multiple domains on several related proteins; for example, EST00825 matches two transmembrane domains on both GABA and Norepinephrine transporters. Nucleotide databases are: GenBank (GB), and EMBL (E); peptide databases are: GenPept (GPU), Swiss-Prot (SP), and PIR.
  • Example 2 The great majority (83%) of the partial cDNA sequences reported in Example 2 are unrelated to any sequences previously described in the literature. Based on database matches to known genes from humans as well as from such evolutionarily distant organisms as E. coli , yeast, C. elegans, Drosophila , barley, AraJbidopsis, rice, and green algae, we have preliminarily identified the functional type of a number of the ESTs (Table 2). These include a novel gene similar to Notch/Tan- 1 (Adams et al., supra), a new neurotransmitter transporter gene, and a new member of the multi-drug resistance gene family.
  • MBP myelin basic protein genes
  • ESTs By matching ESTs to known database sequences, a phenotypic characterization of the tissue begins to emerge. Protein superfamilies matched by ESTs were grouped into three broad functional categories to assess the biological spectrum represented by these randomly selected cDNA clones. Structural and metabolic classes comprised about 30% of the ESTs with database matches. Twenty-five percent were involved in regulatory pathways and the remainder were not classifiable. Eleven of the eighteen enzymes of glycolysis and the citric acid cycle are represented by at least one subunit or isozyme.
  • osteopontin Young, M., Kerr, J., Termine, J., Wewer, U., Wang, M., McBride, W. & Fisher, L. Genomics 7:491-502
  • Oligonucleotide primer pairs were designed from EST sequences to minimize the chance of amplifying through an intron.
  • the oligonucleotides were 18-23 bp in length and designed for PCR amplification using the computer program INTRON (National Institutes of Mental Health, Bethesda, MD). The program is based on the assumptions that: 1) introns are genomic sequences that interrupt the coding and noncoding sequences of genes (Smith, J. Mol. Evol. 27:45-55 (1988)); 2) there are consensus sequences for splice junctions (Shapiro, et al., Nucl. Acids Res.
  • the program evaluates the likelihood that a given GG or CC dinucleotide represents a former exon-intron
  • every input strand is processed by the INTRON program twice, first evaluating the sense mRNA strand, and then processing the complementary or anti-sense strand.
  • the program evaluates each sequence by finding all GG or CC pairs (possible former splice sites), searching for STOP codons in all three reading frames, and analyzing the GG or CC pairs surrounded by stop codons. All regions of the EST that are unlikely to contain splice junctions based on CC content, GG content, and stop codon frequency are then marked by the program in uppercase.
  • PCR primers from known sequences are well known to those with skill in the art. For a review of PCR technology see Erlich, H.A., PCR Technology, Principles and Applications for DNA Amplification. 1992. W.H. Freeman and Co., New York. ESTs were examined for the presence of stop codons in each reading frame and for consensus splice junctions. The presence of stop codons and absence of splice junction sequences are more characteristic of 3' untranslated sequences than of introns. The untranslated sequences are unique to a given gene; thus, primers from these regions are less likely to prime other members of a gene family or pseudogenes.
  • PCR polymerase chain reactions
  • oligonucleotide primer 0.6 unit of Tag polymerase, and 1 uCu of a 32 P-labeled deoxycytidine triphosphate.
  • the PCR was performed in a microplate thermocycler (Techne) under the following conditions: 30 cycles of 94°C, 1.4 min; 55°C, 2 min; and 72°C, 2 min; with a final extension at 72°C for 10 min.
  • the amplified products were analyzed on a 6% polyacrylamide sequencing gel and visualized by
  • Somatic Cell Hybrid Mapping Panel Number 1 (NIGMS, Camden, NJ).
  • PCR was used to screen a series of somatic cell hybrid cell lines containing defined sets of human chromosomes for the presence of a given EST. DNA was isolated from the somatic hybrids and used as starting templates for PCR reactions using the primer pairs from EST sequences
  • the single human chromosome present in all cell hybrids that give rise to an amplified fragment represents the chromosome containing that EST.
  • the assignment of 100 ESTs and corresponding genes to chromosomes by PCR is shown in Table 3.
  • Somatic cell hybrids were prepared that contained defined subsets of chromosomes 6 and X. Methods for preparing and selecting somatic cell hybrids are known in the art. For a review of an exemplary procedure to generate somatic cell hybrids containing the short arm of human chromosome 6, see Zoghbi, et al., Genomics 9(4):713-720 (1991). For a general review of somatic cell hybridization see Ledbetter et al. (supra). The hybrids were processed to obtain DNA and analyzed by PCR and by fluorescence in situ
  • Example 5 The procedure of Example 5 is repeated for all of the ESTs from Examples 1 and 2 not previously mapped to human chromosomes. Data are generated corresponding to the data in Table 3 for all of the unmapped ESTs. As previously mentioned, virtually all of the ESTs will map to a unique chromosomal location. The inability of any ESTs to
  • This technique was used to map an EST to a particular location on a given chromosome.
  • Cell cultures, tissue, or whole blood were used to obtain chromosomes.
  • 0.5 ml. of whole blood was added to RPMI 1640 and incubated 96 hours in a 5%CO 2 /37°C incubator.
  • 0.05 ug/ml colcemide was added to the culture one hour before harvest.
  • Cells were collected and washed in PBS.
  • the suspension was incubated with a hypotonic solution of KC1 added dropwise to reach a final volume of 5 ml .
  • the cells were spun down and fixed by resuspending the cells in methanol and glacial acetic acid (3:1). The cell suspension was dropped onto glass slides and dried.
  • the slides were treated with RNase A and washed then dehydrated in a series of increasing concentrations of ethanol.
  • the EST to be localized was nick-translated using fluorescently labeled nucleotide (Korenberg, Jr., et al., Cell 53(3):391-400 (1988)). Following nick translation, unincorporated label was removed by spin dialysis through Sepharose. The probe was further extracted with phenolchloroform to remove additional protein. The chromosomes were denatured in formamide using techniques known in the art and the denatured probe was added to the slides. Following hybridization, the cells were washed. The slides were studied under a fluorescent microscope. In addition, the chromosomes can be stained for G-banding or Q-banding using techniques known in the art.
  • the resulting metaphase chromosomes had fluorescent tags localized to those regions of the chromosome that were homologous to the EST. Thus, a particular EST was localized to a particular region on a given chromosome.
  • SEQ ID NOs 396, 485, 506, 1880 and 1894 were mapped using fluorescent in situ hybridization to locations on chromosomes 17, 7, 10 and 1 respectively (See Table 4B below).
  • the ESTs of the present invention were statistically evaluated using the coding-region prediction program CRM via the GRAIL server (Uberbacher, E. & Mural, R. Proc.
  • the CRM program uses a neural network to combine results from several different coding regions by looking at different 6 bp sequences found in coding exons and in introns. The program additionally conducts reading frame searches and assesses randomness at the third position of codons. This, protocol categorizes sequences as having an excellent, good, marginal, or poor probability of containing coding regions. The results are reported in Tables 6-9. There were 219 ESTs categorized as "excellent” (Table 6); 120 categorized as "good” (Table 7); 113 categorized as

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Abstract

Séquences d'ADNc humain et géniques partielles et complètes correspondant à des marques séquencielles exprimées particulières. Les marques séquencielles exprimées qui sont des séquences d'ADNc dont la longueur varie généralement entre 150 et 500 paires de bases sont dérivées de banques d'ADNc du cerveau humain, correspondent à des gènes transcrits dans le cerveau humain et possèdent des séquences de base désignées ici par les no. d'identification 1-2421.
PCT/US1993/001294 1992-02-12 1993-02-12 Sequences caracteristiques du produit de transcription des genes humains WO1993016178A2 (fr)

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