Subscription based systems, methods and components for providing genomic and proteomic products and services

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is directed to systems and methods for providing research products and services (e.g., for industries involved in genomic and proteomic research), as well as research products supplied as part of the systems and methods.

[0003] 2. Background Art

[0004] Genomics relates to the study of genes and how they relate to the health, development, structure, and disease of an organism. The sequencing of the human genome has been a large focus of scientists over the past decade. Now that the task has been completed, life science research is shifting beyond sequencing to functional studies. This has given rise to the science of proteomics. Proteomics examines the role that proteins play with respect to both normal and abnormal biological (e.g., cellular) processes. Together, genomic and proteomic research are driving, for example, the race to mine the human genome to identify and exploit druggable targets.

[0005] A druggable target is a gene whose function can be modulated by a drug, such as an organic molecule with one or more pharmacological activities. The number of gene targets within the human genome that are of pharmaceutical relevance is limited. Presently, the pharmaceutical industry is focusing primarily on certain areas of high interest, such as CNS (central nervous systems) disorders, metabolic diseases, cardiovascular diseases, oncology, inflammation and infectious diseases. Within these areas, each pharmaceutical company has identified their own prioritized list of “druggable targets”.

[0006] Many currently available drugs were designed without the benefit of using clones encoding the intended druggable targets, and show undesirable, or sometimes unacceptable, side effects. It is generally believed that the poor side effect profiles of currently available drugs often stem from the interaction of these drugs with (sometimes multiple) family members of the target molecule. Each family member may be involved in a physiological function distinct from the other family members. More than one family member, however, may respond to a non-specific drug. As a consequence, a non-specific drug intended to exert its effects on one physiological function may in fact influence other physiological functions, thereby causing undesirable side effects. Therefore, the pharmaceutical industry is expressing an urgent need for access to complete sets of gene families.

[0007] Further, a major theme of pharmaceutical and biotechnology companies is to improve their lead compound selection process at the earliest stages of drug development. If these attempts are successful, those drug candidates that enter the clinic to treat human disease should possess much improved side effect and safety profiles. For example, drugs with undesirable or unacceptable side effects can be eliminated at the research stage, rather than at the clinical stage. Accordingly, there is a need to improve the lead compound selection process in order to reduce the costs associated with new drug development. Conducting research on open reading frame clones is one way of improving the identification of lead compounds. Thus, there is also a need to generate a representative open reading frame (ORF) clone collection for every human gene and/or gene family.

[0008] Pharmaceutical and biotechnology companies have invested significant resources in various genomics technologies developing, for example databases, gene expression platforms, etc. Further, a number of companies provide products and services related to these technologies. However, the offerings of these companies are generic, as opposed to customized, to the individual needs of the pharmaceutical and biotechnology companies. Heretofore, there has not been a single source upon which a pharmaceutical or biotechnology company could rely to meet most, if not all, of its needs for genomic and proteomic products and services. Thus, there is a need for an integrated system for providing customized genomic and proteomic products and services.

[0009] These needs and others are met by the present invention.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides subscription-based systems, methods, and components for providing research products and services (e.g., for use in industries involved in genomic and proteomic research and development). In addition, the present invention encompasses the products provided as well as methods of performing the services provided. The system includes a provider of research products and services and one or more customers desirous of obtaining one or more research products and/or services. Customers are identified as either subscribers or non-subscribers.

[0011] In some aspects, the system may comprise one or more databases. A database may comprise various types of information of interest to customers (e.g., individuals or organizations conducting research). For example, a database may contain information regarding products and/or services available (e.g., cloning services, expression services, expressed polypeptides, antibodies that bind expressed polypeptides, etc.), clones, sequences of clones, sequences of open reading frames (ORFs) contained in clones, physical characteristics of polypeptides expressed from open reading frames (e.g, molecular weight, amino acid composition, isoelectric point, etc.), activities (e.g., enzymatic, immunogenic, regulatory, etc.) of polypeptides expressed from ORFs, protein-protein interactions (e.g., identities of proteins that bind to/interact with polypeptides expressed from ORFs contained in clones), expression information (e.g., amount and/or activity of one or more polypeptides produced by one or more host cells containing one or more clones), functional regions (e.g., domains and/or sequences of polypeptides and/or nucleic acids having an activity and/or characteristic such as enzyme active sites, protein binding sites, promoter sequences, enhancer/repressor sequences, nucleic acid sequences bound by polypeptides, centromeres, telomers, etc.), and the like. A database may contain more than one type of information (e.g., two, three, four, five, six, seven, eight, nine, ten, etc. types of information) and a given type of information may be in more than one database. A database may contain private and/or public information. For example, a database may contain private information (e.g., trade secret and/or patentable information) regarding, for example, one or more clones (e.g., sequence of an ORF encoded by the clone, expression information, etc.) as well as public information (e.g., GenBank, EMBL, etc. sequences of related ORFs).

[0012] In one embodiment, one or more directories of available research products and services (e.g., genomic and proteomic research products and services) is maintained in a research products and services database. This database may be accessed by subscribers and non-subscribers (e.g., via an interface, such as a graphical user interface).

[0013] In one embodiment, the system may comprise one or more clone collection databases. Clone collection databases may be associated with the research products and services database or may be independent of the research products and services database. A clone collection database may comprise a private area that is only accessible by one or more subscribers and/or a public area that is accessible by both subscribers and non-subscribers. In one embodiment, the private area may be further sub-divided into private areas (e.g., for maintaining sub-categories of data and/or data accessible to specific subscribers). Such sub-divided portions of a private database may be accessible to one or more subscribers and inaccessible to others. A clone collection database may contain information identifying the characteristics of private and public clone collections available from the provider.

[0014] The system may further comprise one or more expression databases. An expression database may contain information identifying optimized expression systems for one or more clones in private and/or public clone collections. Such information may comprise one or more suitable host cells or cell types (e.g., mammalian cells, insect cells, etc.), as well as promoter information, enhancer information, repressor information, and the like. An expression database may comprise information regarding culture conditions suitable for a specific host cell type, isolation conditions for purifying a polypeptide encoded by a clone, and any other information related to expression of a polypeptide. An expression database may comprise information regarding an RNA expressed from a clone. The RNA may be translated or un-translated. The information may comprise information regarded 5′ and/or 3′ un-translated regions, RNA stability, etc. In some embodiments, an expression database may comprise information regarding suitable host cells for expression of a polypeptide having desired characteristics. For example, a database may contain information regarding post-translational modifications (e.g., glcosylation, acylation, etc.) that occur in a given host and information regarding the effects of such post-translational modification on one or more characteristics of the polypeptide (e.g., activity, immunogenicity, etc.).

[0015] In some embodiments, systems of the invention may be provided with one or more subscriber records. Such records may be use to, for example, manage subscriptions to the products and services of the provider. A subscriber record may include a subscription identification field, a subscription fee payment field, a clone purchase credit field, a clone purchase field, a subscriber site identification field, and/or combinations of any two or more of the above.

[0016] In one aspect, the present invention provides one or more compositions identified in one or more databases. The invention also encompasses reaction mixtures comprising such compositions and methods of making and using such reaction mixtures.

[0017] In one embodiment, the present invention provides the subscriber with access to the research products and services of the provider using a computer system and a graphical user interface. In addition to providing the subscriber with access to multiple databases, the present invention enables the subscriber to identify products and/or services, which may not have been previously available from the provider, that the subscriber desires to obtain. In one embodiment, clones to be built and added to the private or public clone collections of the provider may be identified by a subscriber. In some embodiments, the subscriber may be able to prioritize the order in which the identified clones are built and added to a clone collection. The present invention encompasses methods for preparing clone collections as well as clone collections prepared using the methods of the invention. Still further, the present invention provides research and development consulting services to one or more sites designated by the subscriber.

[0018] In some embodiments, the present invention provides clone collections. Clones making up a clone collection may contain any nucleic acids (e.g., two, three, five, ten, twenty, etc.) of interest, for example, nucleic acids that contain one or more open reading frames (ORFs), nucleic acids containing un-translated sequences, (e.g., 5′ and/or 3′ un-translated sequences, introns, etc.), which may be from cDNA and/or genomic DNA, nulceic acids containing promoter elements, and any other nucleic acid of interest to a customer. A clone collection may contain ORFs, which may be in vectors, representing all, substantially all, a majority, or a representative number of members of a class of polypeptides (e.g., all known polypeptides having a particular activity and/or characteristic of interest). A collection may comprise clones comprising ORFs encoding all, substantially all, a majority, or a representative number of polypeptides related to and/or affected by a particular activity. A collection may comprise clones comprising ORFs encoding all, substantially all, a majority, or a representative number of polypeptides involved in the metabolism (e.g., synthesis and degradation) of a metabolite of interest (e.g., a lipid, carbohydrate, peptide, etc.) as well as clones comprising one or more ORFs encoding polypeptides affected by the metabolite. One or more individual members of a clone collection may comprise ORFs flanked by recognition sites (e.g., recombination sites, topoisomerase sites, restriction enzyme sites, etc.). When a clone contains multiple recombination sites, such sites may or may not recombine with each other.

[0019] Clones of a collection may also contain one or more functional sequences (e.g., transcriptional regulatory sequences, sequences comprising stop codons, etc.). Such functional sequences may be operably linked to a sequence of interest (e.g., an ORF). Clones of a collection may also comprise one or more stop codons that may be repressible as well as one or more sequences encoding one or more tags (e.g., one or more C-terminal and/or N-terminal tags). One or members of a clone collection may comprise sequences other than ORFs. For example, one or more members of a clone might contain 5′-un-translated regions, regions of genomic nucleic acids, intron regions, promoter regions, enhancer regions, and the like.

[0020] The present invention also contemplates methods of making clones to be included in clone collections, methods of making clone collections, clones, and collections made by the methods of the invention, as well as reaction mixtures and compositions comprising one or more clones or collections.

[0021] Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the invention, are described in detail below with reference to the accompanying drawings. In the drawings, like reference numbers generally indicate identical, functionally similar and/or structurally similar elements. The drawing in which an element first appears is generally indicated by the leftmost digit(s) in the corresponding reference number.

BRIEF DESCRIPTION OF THE FIGURES

[0022] The present invention will be described with reference to the accompanying drawings, wherein:

[0023]
FIG. 1 is a block diagram of a system for providing genomic and proteomic products and services according to an embodiment of the present invention;

[0024]
FIG. 2A is a table describing exemplary genomic and proteomic products offered by a provider according to an embodiment of the present invention;

[0025]
FIG. 2B is a table describing exemplary genomic and proteomic services offered by a provider according to an embodiment of the present invention;

[0026]
FIG. 3 is a block diagram illustration of a subscriber record according to an embodiment of the present invention;

[0027]
FIG. 4 is a block diagram illustration depicting a client/server implementation according to an embodiment of the present invention;

[0028]
FIG. 5 is a block diagram illustration of an exemplary computer system embodiment of the client/server implementation of FIG. 4;

[0029]
FIG. 6 is a flow chart diagram of a method for providing genomic and roteomic products and services according to an embodiment of the present invention;

[0030]
FIG. 7 is a flow chart diagram of a method for providing genomic and roteomic products and services according to an embodiment of the present invention;

[0031]
FIG. 8 is a flow chart diagram of a method for providing clone construction and related genomic and proteomic products and services according to an embodiment of the present invention; and

[0032]
FIG. 9 is a flow chart diagram of a method for constructing a clone according to an embodiment of the present invention;

[0033]
FIG. 10 is a flow chart diagram of an exemplary implementation of an embodiment of the present invention;

[0034]
FIG. 11 is a schematic representation of some of the services that may be provided in conjunction with the present invention; and

[0035]
FIG. 12A-12F are schematic representations of configurations of vectors and sequences of interest that may be used in various embodiments of the invention.

TABLE OF CONTENTS

[0036] 1. Definitions

[0037] 2. Overview of the Invention

[0038] 3. Exemplary system embodiments

[0039] 3.1 Genomic and Proteomic Research Products and Services System

[0040] 3.1.1 Exemplary Products

[0041] 3.1.2 Exemplary Services

[0042] 3.1.3 Customers

[0043] b 3.2 Exemplary computer system embodiment

[0044] 3.2.1 Genomic and Proteomic Products and Services databases

[0045] 3.2.1.1 Subscriber database

[0046] 3.2.1.2 Clone collection database

[0047] 3.2.1.3 Expression Database

[0048] 3.2.2 Client/Server Architecture

[0049] 4. Exemplary operational embodiments

[0050] 4.1 Accessing Genomic and Proteomic Research Products and Services

[0051] 4.2 Providing Genomic and Proteomic Research Products and Services

[0052] 5. Detailed Description of Exemplary Products

[0053] 6. Detailed Description of Exemplary Services

[0054] 7. Conclusion

1. Definitions

[0055] In the description that follows, a number of terms used in recombinant nucleic acid technology are utilized extensively. In order to provide a clear and more consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0056] Genomic Products and Services: As used herein, the term genomic products and services refers to products and services that may be used to conduct research involving nucleic acids.

[0057] Proteomic Products and Services: As used herein, the term proteomic products and services refers to products and services that may be used to conduct research involving polypeptides.

[0058] Clone Collection: As used herein, “clone collection” refers to two or more nucleic acid molecules, each of which comprises one or more nucleic acid sequences of interest.

[0059] Customer: As used herein, the term customer refers to any individual, institution, corporation, university, or organization seeking to obtain genomic and proteomic products and services.

[0060] Provider: As used herein, the term provider refers to any individual, institution, corporation, university, or organization seeking to provide genomic and proteomic products and services.

[0061] Subscriber: As used herein, the term subscriber refers to any customer having an agreement with a provider to obtain public and private genomic and proteomic products and services at subscriber rates.

[0062] Non-subscriber: As used herein, the term non-subscriber refers to any customer who does not have an agreement with a provider to obtain public and private genomic and proteomic products and services at subscriber rates.

[0063] Host: As used herein, the term “host” refers to any prokaryotic or eukaryotic (e.g., mammalian, insect, yeast, plant, avian, animal, etc.) cell and/or organism that is a recipient of a replicable expression vector, cloning vector or any nucleic acid molecule. The nucleic acid molecule may contain, but is not limited to, a sequence of interest, a transcriptional regulatory sequence (such as a promoter, enhancer, repressor, and the like) and/or an origin of replication. As used herein, the terms “host,” “host cell,” “recombinant host” and “recombinant host cell” may be used interchangeably. For examples of such hosts, see Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[0064] Transcriptional Regulatory Sequence: As used herein, the phrase “transcriptional regulatory sequence” refers to a functional stretch of nucleotides contained on a nucleic acid molecule, in any configuration or geometry, that act to regulate the transcription of (1) one or more nucleic acid sequences that may comprise ORFs, (e.g., two, three, four, five, seven, ten, etc.) into messenger RNA or (2) one or more nucleic acid sequences into untranslated RNA. Examples of transcriptional regulatory sequences include, but are not limited to, promoters, enhancers, repressors, operators (e.g., the tet operator), and the like.

[0065] Promoter: As used herein, a promoter is an example of a transcriptional regulatory sequence, and is specifically a nucleic acid generally described as the 5′-region of a gene located proximal to the start codon or nucleic acid that encodes untranslated RNA. The transcription of an adjacent nucleic acid segment is initiated at or near the promoter. A repressible promoter's rate of transcription decreases in response to a repressing agent. An inducible promoter's rate of transcription increases in response to an inducing agent. A constitutive promoter's rate of transcription is not specifically regulated, though it can vary under the influence of general metabolic conditions.

[0066] Insert: As used herein, the term “insert” refers to a desired nucleic acid segment that is a part of a larger nucleic acid molecule. In many instances, the insert will be introduced into the larger nucleic acid molecule using techniques known to those of skill in the art, e.g., recombinational cloning, topoisomerase cloning or joining, ligation, etc.

[0067] Target Nucleic Acid Molecule: As used herein, the phrase “target nucleic acid molecule” refers to a nucleic acid molecule comprising at least one nucleic acid sequence of interest, preferably a nucleic acid molecule that is to be acted upon using the compounds and methods of the present invention. Such target nucleic acid molecules may contain one or more (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.) sequences of interest.

[0068] Recognition Sequence: As used herein, the phrase “recognition sequence” or “recognition site” refers to a particular sequence to which a protein, chemical compound, DNA, or RNA molecule (e.g., restriction endonuclease, a topoisomerase, a modification methylase, a recombinase, etc.) recognizes and binds. In the present invention, a recognition sequence may refer to a recombination site. For example, the recognition sequence for Cre recombinase is loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ Integrase. attB is an approximately 25 base pair sequence containing two 9 base pair core-type Int binding sites and a 7 base pair overlap region. attP is an approximately 240 base pair sequence containing core-type Int binding sites and arm-type Int binding sites as well as sites for auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)). Such sites may also be engineered according to the present invention to enhance production of products in the methods of the invention. For example, when such engineered sites lack the P1 or H1 domains to make the recombination reactions irreversible (e.g., attR or attP), such sites may be designated attR′ or attP′ to show that the domains of these sites have been modified in some way.

[0069] Recombination Proteins: As used herein, the phrase “recombination proteins” includes excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Examples of recombination proteins include Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0070] Recombinases: As used herein, the term “recombinases” is used to refer to the protein that catalyzes strand cleavage and re-ligation in a recombination reaction. Site-specific recombinases are proteins that are present in many organisms (e.g., viruses and bacteria) and have been characterized as having both endonuclease and ligase properties. These recombinases (along with associated proteins in some cases) recognize specific sequences of bases in a nucleic acid molecule and exchange the nucleic acid segments flanking those sequences. The recombinases and associated proteins are collectively referred to as “recombination proteins” (see, e.g., Landy, A., Current Opinion in Biotechnology 3:699-707 (1993)).

[0071] Numerous recombination systems from various organisms have been described. See, e.g., Hoess, et al., Nucleic Acids Research 14(6):2287 (1986); Abremski, et al., J. Biol. Chem. 261(1):391 (1986); Campbell, J. Bacteriol. 174(23):7495 (1992); Qian, et al., J. Biol. Chem. 267(11):7794 (1992); Araki, et al., J. Mol. Biol. 225(1):25 (1992); Maeser and Kahnmann, Mol. Gen. Genet. 230:170-176 (1991); Esposito, et al., Nucl. Acids Res. 25(18):3605 (1997). Many of these belong to the integrase family of recombinases (Argos, et al., EMBO J. 5:433-440 (1986); Voziyanov, et al., Nucl. Acids Res. 27:930 (1999)). Perhaps the best studied of these are the Integrase/att system from bacteriophage λ (Landy, A. Current Opinions in Genetics and Devel. 3:699-707 (1993)), the Cre/loxP system from bacteriophage P1 (Hoess and Abremski (1990) In Nucleic Acids and Molecular Biology, vol. 4. Eds,: Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT system from the Saccharomyces cerevisiae 2μ circle plasmid (Broach, et al., Cell 29:227-234 (1982)).

[0072] Recombination Site: A used herein, the phrase “recombination site” refers to a recognition sequence on a nucleic acid molecule that participates in an integration/recombination reaction by recombination proteins. Recombination sites are discrete sections or segments of nucleic acid on the participating nucleic acid molecules that are recognized and bound by a site-specific recombination protein during the initial stages of integration or recombination. For example, the recombination site for Cre recombinase is loxP, which is a 34 base pair sequence comprised of two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of Sauer, B., Curr. Opin. Biotech. 5:521-527 (1994)). Other examples of recombination sites include the attB, attP, attL, and attR sequences described in U.S. provisional patent applications Nos. 60/136,744, filed May 28, 1999, and 60/188,000, filed Mar. 9, 2000, and in co-pending U.S. patent applications Ser. Nos. 09/517,466 and 09/732,91—all of which are specifically incorporated herein by reference—and mutants, fragments, variants and derivatives thereof, which are recognized by the recombination protein λ Int and by the auxiliary proteins integration host factor (IHF), FIS and excisionase (Xis) (see Landy, Curr. Opin. Biotech. 3:699-707 (1993)).

[0073] Mutating specific residues in the core region of the att site can generate a large number of different att sites. As with the att1 and att2 sites utilized in GATEWAY™, each additional mutation potentially creates a novel att site with unique specificity that will recombine only with its cognate partner att site bearing the same mutation and will not cross-react with any other mutant or wild-type att site. Novel mutated att sites (e.g., attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described in previous patent application Ser. No. 09/517,466, filed Mar. 2, 2000, which is specifically incorporated herein by reference. Other recombination sites having unique specificity (i.e., a first site will recombine with its corresponding site and will not recombine or not substantially recombine with a second site having a different specificity) may be used to practice the present invention. Examples of suitable recombination sites include, but are not limited to, loxP sites; loxP site mutants, variants or derivatives such as loxP511 (see U.S. Pat. No. 5,851,808); frt sites; frt site mutants, variants or derivatives; dif sites; dif site mutants, variants or derivatives; psi sites; psi site mutants, variants or derivatives; cer sites; and cer site mutants, variants or derivatives.

[0074] Recombination sites may be added to molecules by any number of known methods. For example, recombination sites can be added to nucleic acid molecules by blunt end ligation, PCR performed with fully or partially random primers, or inserting the nucleic acid molecules into a vector using a restriction site flanked by recombination sites.

[0075] Recombinational Cloning: As used herein, the phrase “recombinational cloning” refers to a method whereby segments of nucleic acid molecules or populations of such molecules are exchanged, inserted, replaced, substituted or modified, in vitro or in vivo. Preferably, such cloning method is an in vitro method.

[0076] Suitable recombinational cloning systems that utilize recombination at defined recombination sites have been previously described in U.S. Pat. No. 5,888,732, U.S. Pat. No. 6,143,557, U.S. Pat. No. 6,171,861, U.S. Pat. No. 6,270,969, and U.S. Pat. No. 6,277,608, and in pending U.S. application Ser. No. 09/517,466, and in published U.S. application Ser. No. 20020007051, (each of which is fully incorporated herein by reference), all assigned to the Invitrogen Corporation, Carlsbad, Calif. In brief, the GATEWAY™ Cloning System described in these patents utilizes vectors that contain at least one recombination site to clone desired nucleic acid molecules in vivo or in vitro. In some embodiments, the system utilizes vectors that contain at least two different site-specific recombination sites that may be based on the bacteriophage lambda system (e.g., att1 and att2) that are mutated from the wild-type (att0) sites. Each mutated site has a unique specificity for its cognate partner att site (i.e., its binding partner recombination site) of the same type :(for example attB1 with attP1, or attL1 with att1) and will not cross-react with recombination sites of the other mutant type or with the wild-type att0 site. Different site specificities allow directional cloning or linkage of desired molecules thus providing desired orientation of the cloned molecules. Nucleic acid fragments flanked by recombination sites are cloned and subcloned using the GATEWAY™ system by replacing a selectable marker (for example, ccdB) flanked by att sites on the recipient plasmid molecule, sometimes termed the Destination Vector. Desired clones are then selected by transformation of a ccdB sensitive host strain and positive selection for a marker on the recipient molecule. Similar strategies for negative selection (e.g., use of toxic genes) can be used in other organisms such as thymidine kinase (TK) in mammals and insects.

[0077] Topoisomerase recognition site. As used herein, the term “topoisomerase recognition site” means a defined nucleotide sequence that is recognized and bound by a site specific topoisomerase. For example, the nucleotide sequence 5′-(C/T)CCTT-3′ is a topoisomerase recognition site that is bound specifically by most poxvirus topoisomerases, including vaccinia virus DNA topoisomerase I, which then can cleave the strand after the 3′-most thymidine of the recognition site to produce a nucleotide sequence comprising 5′-(C/T)CCTT-PO4-TOPO, i.e., a complex of the topoisomerase covalently bound to the 3′ phosphate through a tyrosine residue in the topoisomerase (see, Shuman, J. Biol. Chem. 266:11372-11379, 1991; Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; each of which is incorporated herein by reference; see, also, U.S. Pat. No. 5,766,891; PCT/US95/16099; and PCT/US98/12372). In comparison, the nucleotide sequence 5′-GCAACTT-3′ is the topoisomerase recognition site for type IA E. coli topoisomerase III.

[0078] Repression Cassette: As used herein, the phrase “repression cassette” refers to a nucleic acid segment that contains a repressor or a selectable marker present in the subcloning vector.

[0079] Selectable Marker: As used herein, the phrase “selectable marker” refers to a nucleic acid segment that allows one to select for or against a molecule (e.g., a replicon) or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. Examples of selectable markers include but are not limited to: (1) nucleic acid segments that encode products that provide resistance against otherwise toxic compounds (e.g., antibiotics); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that suppress the activity of a gene product; (4) nucleic acid segments that encode products that can be readily identified (e.g., phenotypic markers such as (β-galactosidase, green fluorescent protein (GFP), yellow flourescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell surface proteins); (5) nucleic acid segments that bind products that are otherwise detrimental to cell survival and/or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments described in Nos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acid segments that bind products that modify a substrate (e.g., restriction endonucleases); (8) nucleic acid segments that can be used to isolate or identify a desired molecule (e.g., specific protein binding sites); (9) nucleic acid segments that encode a specific nucleotide sequence that can be otherwise non-functional (e.g., for PCR amplification of subpopulations of molecules); (10) nucleic acid segments that, when absent, directly or indirectly confer resistance or sensitivity to particular compounds; and/or (11) nucleic acid segments that encode products that either are toxic (e.g., Diphtheria toxin) or convert a relatively non-toxic compound to a toxic compound (e.g., Herpes simplex thymidine kinase, cytosine deaminase) in recipient cells; (12) nucleic acid segments that inhibit replication, partition or heritability of nucleic acid molecules that contain them; and/or (13) nucleic acid segments that encode conditional replication functions, e.g., replication in certain hosts or host cell strains or under certain environmental conditions (e.g., temperature, nutritional conditions, etc.).

[0080] Site-Specific Recombinase: As used herein, the phrase “site-specific recombinase” refers to a type of recombinase that typically has at least the following four activities (or combinations thereof): (1) recognition of specific nucleic acid sequences; (2) cleavage of said sequence or sequences; (3) topoisomerase activity involved in strand exchange; and (4) ligase activity to reseal the cleaved strands of nucleic acid (see Sauer, B., Current Opinions in Biotechnology 5:521-527 (1994)). Conservative site-specific recombination is distinguished from homologous recombination and transposition by a high degree of sequence specificity for both partners. The strand exchange mechanism involves the cleavage and rejoining of specific nucleic acid sequences in the absence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem. 58:913-949).

[0081] Suppressor tRNAs. As used herein, the phrase “suppressor tRNA” refers to a molecule that mediates the incorporation of an amino acid in a polypeptide in a position corresponding to a stop codon in the mRNA being translated.

[0082] Homologous Recombination: As used herein, the phrase “homologous recombination” refers to the process in which nucleic acid molecules with similar nucleotide sequences associate and exchange nucleotide strands. A nucleotide sequence of a first nucleic acid molecule that is effective for engaging in homologous recombination at a predefined position of a second nucleic acid molecule will therefore have a nucleotide sequence that facilitates the exchange of nucleotide strands between the first nucleic acid molecule and a defined position of the second nucleic acid molecule. Thus, the first nucleic acid will generally have a nucleotide sequence that is sufficiently complementary to a portion of the second nucleic acid molecule to promote nucleotide base pairing.

[0083] Homologous recombination requires homologous sequences in the two recombining partner nucleic acids but does not require any specific sequences. As indicated above, site-specific recombination that occurs, for example, at recombination sites such as att sites, is not considered to be “homologous recombination,” as the phrase is used herein.

[0084] Vector: As used herein, the term “vector” refers to a nucleic acid molecule (preferably DNA) that provides a useful biological or biochemical property to an insert. Examples include plasmids, phages, viruses, autonomously replicating sequences (ARS), centromeres, and other sequences that are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. A vector can have one or more restriction endonuclease recognition sites (e.g., two, three, four, five, seven, ten, etc.) at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced in order to bring about its replication and cloning. Vectors can further provide primer sites (e.g., for PCR), transcriptional and/or translational initiation and/or regulation sites, recombinational signals, replicons, selectable markers, etc. Clearly, methods of inserting a desired nucleic acid fragment that do not require the use of recombination, transpositions or restriction enzymes (such as, but not limited to, uracil N-glycosylase (UDG) cloning of PCR fragments (U.S. Pat. Nos. 5,334,575 and 5,888,795, both of which are entirely incorporated herein by reference), T:A cloning, and the like) can also be applied to clone a fragment into a cloning vector to be used according to the present invention. The cloning vector can further contain one or more selectable markers (e.g., two, three, four, five, seven, ten, etc.) suitable for use in the identification of cells transformed with the cloning vector.

[0085] Subcloning Vector: As used herein, the phrase “subcloning vector” refers to a cloning vector comprising a circular or linear nucleic acid molecule that includes, preferably, an appropriate replicon. In the present invention, the subcloning vector can also contain functional and/or regulatory elements that are desired to be incorporated into the final product to act upon or with the cloned nucleic acid insert. The subcloning vector can also contain a selectable marker (preferably DNA).

[0086] Primer: As used herein, the term “primer” refers to a single stranded or double stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule (e.g., a DNA molecule). In one aspect, the primer may be a sequencing primer (for example, a universal sequencing primer). In another aspect, the primer may comprise a recombination site or portion thereof.

[0087] Adapter: As used herein, the term “adapter” refers to an oligonucleotide or nucleic acid fragment or segment (preferably DNA) that comprises one or more recombination sites (or portions of such recombination sites) that can be added to a circular or linear nucleic acid molecule as well as to other nucleic acid molecules described herein. When using portions of recombination sites, the missing portion may be provided by the nucleic acid molecule. Such adapters may be added at any location within a circular or linear molecule, although the adapters are preferably added at or near one or both termini of a linear molecule. Preferably, adapters are positioned to be located on both sides (flanking) a particular nucleic acid molecule of interest. In accordance with the invention, adapters may be added to nucleic acid molecules of interest by standard recombinant techniques (e.g., restriction digest and ligation). For example, adapters may be added to a circular molecule by first digesting the molecule with an appropriate restriction enzyme, adding the adapter at the cleavage site and reforming the circular molecule that contains the adapter(s) at the site of cleavage. In other aspects, adapters may be added by homologous recombination, by integration of RNA molecules, and the like. Alternatively, adapters may be ligated directly to one or more and preferably both termini of a linear molecule thereby resulting in linear molecule(s) having adapters at one or both termini. In one aspect of the invention, adapters may be added to a population of linear molecules, (e.g., a cDNA library or genomic DNA that has been cleaved or digested) to form a population of linear molecules containing adapters at one and preferably both termini of all or substantial portion of said population.

[0088] Adapter-Primer: As used herein, the phrase “adapter-primer” refers to a primer molecule that comprises one or more recombination sites (or portions of such recombination sites) that can be added to a circular or to a linear nucleic acid molecule described, herein. When using portions of recombination sites, the missing portion may be provided by a nucleic acid molecule (e.g., an adapter) of the invention. Such adapter-primers may be added at any location within a circular or linear molecule, although the adapter-primers are preferably added at or near one or both termini of a linear molecule. Such adapter-primers may be used to add one or more recombination sites or portions thereof to circular or linear nucleic acid molecules in a variety of contexts and by a variety of techniques, including but not limited to amplification (e.g., PCR), ligation (e.g., enzymatic or chemical/synthetic ligation), recombination (e.g., homologous or non-homologous (illegitimate) recombination) and the like.

[0089] Template: As used herein, the term “template” refers to a double stranded or single stranded nucleic acid molecule, all or a portion of which is to be amplified, synthesized, reverse transcribed, or sequenced. In the case of a double-stranded DNA molecule, denaturation of its strands to form a first and a second strand is preferably performed before these molecules may be amplified, synthesized or sequenced, or the double stranded molecule may be used directly as a template. For single stranded templates, a primer complementary to at least a portion of the template hybridizes under appropriate conditions and one or more polypeptides having polymerase activity (e.g., two, three, four, five, or seven DNA polymerases and/or reverse transcriptases) may then synthesize a molecule complementary to all or a portion of the template. Alternatively, for double stranded templates, one or more transcriptional regulatory sequences (e.g., two, three, four, five, seven or more promoters) may be used in combination with one or more polymerases to make nucleic acid molecules complementary to all or a portion of the template. The newly synthesized molecule, according to the invention, may be of equal or shorter length compared to the original template. Mismatch incorporation or strand slippage during the synthesis or extension of the newly synthesized molecule may result in one or a number of mismatched base pairs. Thus, the synthesized molecule need not be exactly complementary to the template. Additionally, a population of nucleic acid templates may be used during synthesis or amplification to produce a population of nucleic acid molecules typically representative of the original template population.

[0090] Incorporating: As used herein, the term “incorporating” means becoming a part of a nucleic acid (e.g., DNA) molecule or primer.

[0091] Library: As used herein, the term “library” refers to a collection of nucleic acid molecules (circular or linear). In one embodiment, a library may comprise a plurality of nucleic acid molecules (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, one hundred, two hundred, five hundred one thousand, five thousand, or more), that may or may not be from a common source organism, organ, tissue, or cell. In another embodiment, a library is representative of all or a portion or a significant portion of the nucleic acid content of an organism (a “genomic” library), or a set of nucleic acid molecules representative of all or a portion or a significant portion of the expressed nucleic acid molecules (a cDNA library or segments derived therefrom) in a cell, tissue, organ or organism. A library may also comprise nucleic acid molecules having random sequences made by de novo synthesis, mutagenesis of one or more nucleic acid molecules, and the like. Such libraries may or may not be contained in one or more vectors (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.). In some embodiments, a library may be “normalized” library (i.e., a library of cloned nucleic acid molecules from which each member nucleic acid molecule can be isolated with approximately equivalent probability).,

[0092] Normalized. As used herein, the term “normalized” or “normalized library” means a nucleic acid library that has been manipulated, preferably using the methods of the invention, to reduce the relative variation in abundance among member nucleic acid molecules in the library to a range of no greater than about 25-fold, no greater than about 20-fold, no greater than about 15-fold, no greater than about, 10-fold, no greater than about 7-fold, no greater than about 6-fold, no greater than about 5-fold, no greater than about 4-fold, no greater than about 3-fold or no greater than about 2-fold.

[0093] Amplification: As used herein, the term “amplification” refers to any in vitro method for increasing the number of copies of a nucleic acid molecule with the use of one or more polypeptides having polymerase activity (e.g., one, two, three, four or more nucleic acid polymerases or reverse transcriptases). Nucleic acid amplification results in the incorporation of nucleotides into a DNA and/or RNA molecule or primer thereby forming a new nucleic acid molecule complementary to a template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of nucleic acid replication. DNA amplification reactions include, for example, polymerase chain reaction (PCR). One PCR reaction may consist of 5 to 100 cycles of denaturation and synthesis of a DNA molecule.

[0094] Nucleotide: As used herein, the term “nucleotide” refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid molecule (DNA and RNA). The term nucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, [α-S]dATP, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the present invention, a “nucleotide” may be unlabeled or detectably labeled by well known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.

[0095] Nucleic Acid Molecule: As used herein, the phrase “nucleic acid molecule” refers to a sequence of contiguous nucleotides (riboNTPs, dNTPs, ddNTPs, or combinations thereof) of any length. A nucleic acid molecule may encode a full-length polypeptide or a fragment of any length thereof, or may be non-coding. As used herein, the terms “nucleic acid molecule” and “polynucleotide” may be used interchangeably and include both RNA and DNA.

[0096] Oligonucleotide: As used herein, the term “oligonucleotide” refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides that are joined by a phosphodiester bond between the 3′ position of the pentose of one nucleotide and the 5′ position of the pentose of the adjacent nucleotide.

[0097] Open Reading Frame (ORF): As used herein, an open reading frame or ORF refers to a sequence of nucleotides that codes for a contiguous sequence of amino acids. ORFs of the invention may be constructed to code for the amino acids of a polypeptide of interest from the N-termius of the polypeptide (typically a methionine encoded by a sequence that is transcribed as AUG) to the C-terminus of the polypeptide. ORFs of the invention include sequences that encode a contiguous sequence of amino acids with no intervening sequences (e.g., an ORF from a cDNA) as well as ORFs that comprise one or more intervening sequences (e.g., introns) that may be processed from an mRNA containing them (e.g., by splicing) when an mRNA containing the ORF is transcribed in a suitable host cell. ORFs of the invention also comprise splice variants of ORFs containing intervening sequences.

[0098] ORFs may optionally be provided with one or more sequences that function as stop codons (e.g., contain nucleotides that are transcribed as UAG, an amber stop codon, UGA, an opal stop codon, and/or UAA, an ochre stop codon). When present, a stop codon may be provided after the codon encoding the C-terminus of a polypeptide of interest (e.g., after the last amino acid of the polypeptide) and/or may be located within the coding sequence of the polypeptide of interest. When located after the C-terminus of the polypeptide of interest, a stop codon may be immediately adjacent to the codon encoding the last amino acid of the polypeptide or there may be one or more codons (e.g., one, two, three, four, five, ten, twenty, etc) between the codon encoding the last amino acid of the polypeptide of interest and the stop codon. A nucleic acid molecule containing an ORF may be provided with a stop codon upstream of the initiation codon (e.g., an AUG codon) of the ORF. When located upstream of the initiation codon of the polypeptide of interest, a stop codon may be immediately adjacent to the initiation codon or there may be one or more codons (e.g., one, two, three, four, five, ten, twenty, etc) between the initiation codon and the stop codon.

[0099] Polypeptide: As used herein, the term “polypeptide” refers to a sequence of contiguous amino acids of any length. The terms “peptide,” “oligopeptide,” or “protein” may be used interchangeably herein with the term “polypeptide.”

[0100] Hybridization: As used herein, the terms “hybridization” and “hybridizing” refer to base pairing of two complementary single-stranded nucleic acid molecules (RNA and/or DNA) to give a double stranded molecule. As used herein, two nucleic acid molecules may hybridize, although the base pairing is not completely complementary. Accordingly, mismatched bases do not prevent hybridization of two nucleic acid molecules provided that appropriate conditions, well known in the art, are used. In some aspects, hybridization is said to be under “stringent conditions.” By “stringent conditions,” as the phrase is used herein, is meant overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0101] Other terms used in the fields of recombinant nucleic acid technology and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable arts.

2. Overview

[0102] The present invention provides subscription-based and non-subscription based systems and methods for providing research products and services (e.g., for industries involved in genomic and proteomic research). A provider of genomic and proteomic research products and services provides such products and services to customers for a fee. In exchange for payment of a subscription fee, a customer may be designated a subscriber. Subscribers are charged subscriber fees for the genomic and proteomic research products and services they request. In one embodiment, the subscriber fees are less than the fees charged to non-subscribers.

[0103] Users of the system are provided access to one or more clone collections of the provider. The users may also be given access to databases that contain data describing the attributes of the clones represented in the clone collections. In addition to providing the subscriber with access to multiple databases, the present invention enables the subscriber to identify clones to be built and added to the clone collections of the provider. Access to these clones may or may not be provided to non-subscribers and/or to other subscribers. Further, the subscriber is able to prioritize the order in which the identified clones are to be built and added to the clone collection. In this way, the clone collection can be customized and prioritized according to the research needs of the subscriber. Still further, the present invention provides research and development consulting services to one or more sites designated by the subscriber.

3. Exemplary System Embodiments

[0104] 3.1 Genomics and Proteomics Research Products and Services System

[0105]
FIG. 1 is a block diagram illustration of a system 100 for providing genomic and proteomic products and services according to an embodiment of the present invention. In FIG. 1, a provider 105 provides genomic and proteomic products 103 and services 107 to customers.

[0106] 3.1.1 Exemplary Products

[0107]
FIG. 2A provides an exemplary list of the types of products offered by the provider 105. Such products may comprise clone collections, individual clones, compositions comprising one or more clones and/or collections of clones, reaction mixtures comprising one or more clones and/or collections of clones, polypeptides, antibodies, libraries (e.g., cDNA libraries, genomic libraries, etc.), and kits, as well as individual clones. Additional details of these exemplary products are provided below. Further, these exemplary products are provided for example only and are not intended to limit the present invention.

[0108] 3.1.2 Exemplary Services

[0109]
FIG. 2B provides an exemplary list of the types of services offered by the provider 105. Such services include clone construction services, protein expression services, antibody production services, library (e.g., cDNA library, genomic library, etc.) construction services, and research and development consulting services. In some embodiments, library construction services may comprise construction of a library having specified characteristics (e.g., full-length, normalized, etc.). Library construction services may be performed using tissues and/or organisms of any source. In some embodiments, libraries may be constructed from human, mouse, dog, rat, and/or other mammalian tissues. Libraries may be constructed from more than one tissue source within an organism, for example, from brain, liver, kidney, pancreas, lung, heart, etc. Libraries may be normalized, full-length and/or both normalized and full-length libraries. Thus, the present invention contemplates cDNA library construction (e.g., full-length and/or normalized) for human, mouse, dog, rat, and other organisms. The invention also contemplates normalization of standard cDNA libraries (e.g., for organisms other than human, mouse, dog, or rat). Additional details of these exemplary products and services, as well as other products and services, are provided below. Further, these exemplary services are provided for example only and are not intended to limit the present invention.

[0110] 3.1.3 Customers

[0111] Referring again to FIG. 1, in an embodiment of the present invention, the exemplary products and services set out in FIGS. 2A and 2B are provided to the customers in exchange for the payment of fees associated with the products or services requested. In one embodiment of the present invention, the customers can elect to pay a subscription fee in order to be designated as a subscriber. Accordingly, the customers in FIG. 1 are shown as subscribers 112 and non-subscribers 110. In another embodiment of the present invention, subscribers 112 are able to obtain subscriber benefits offered by the provider 105.

[0112] One example subscriber benefit is the ability to purchase the products and services of the provider 115 at subscriber rates. In one embodiment of the present invention, subscriber rates are less than non-subscriber rates. An additional subscriber benefit includes the ability to access private clone collections (i.e., clone collections only made available to all or some subscribers). Another subscriber benefit includes the ability to identify clones to be built and added to the clone collections maintained by the provider 105. The ability to prioritize the order in which clones are built and added to the clone collections maintained by the provider 105 is an additional subscriber benefit. In some embodiments, a subscriber may have the ability to specify the size of a clone collection (e.g., one, ten, fifty, one hundred, five hundred, one thousand, etc.) and may also have the ability to specify when one or more specific clones are made and supplied (e.g., the clones will be made and supplied within 2 to 8, 3 to 20, 2 to 20, 4 to 20, 6 to 20, 6 to 15, etc. weeks). Yet another subscriber benefit is the ability to designate one or more sites to receive research and development consulting services from the provider 105. In one embodiment, research and development consulting services include providing the subscriber designated sites with information relating to new products and services being developed by the provider. In another embodiment, the research and development consulting services also include provider evaluation of new products and services being developed by the subscriber. In other embodiments, the number of sites that the subscriber can designate is one, two, three, four, five or six. However, the subscriber may designate more sites (e.g., eight, ten, twenty, etc.) by paying an additional fee for each additional site designated.

[0113] Referring to FIG. 3, for each customer who chooses to become a subscriber, a subscriber record 300 may-be maintained. The subscriber record may be used to maintain information identifying each subscriber 112 and for tracking the products and services provided to each of the subscribers. In one embodiment, the subscriber record comprises a subscriber identification field 305, a subscription fee field 310, a clone purchase credit field 315, a clone total order field 320, and a subscriber site identification field 325. In this embodiment, the subscriber identification field may be used to record a unique subscriber identification number for each subscriber 112. The subscription fee field is used to record the subscription fee paid by the subscriber 112. The clone purchase credit field 315 may be used to record the amount of funds the subscriber 112 has credited toward the purchase of clones. The clone total order field may be used to record the number of clones the subscriber 112 has ordered during a designated accounting period. For example, the provider 112 could track the number of clones ordered during a month, quarter or year. The subscriber site identification field 325 may be used to record unique identifiers for one or more sites designated by the subscriber 112. In an embodiment of the present invention, the designated sites receive research and development consulting services from the provider 105. Additional subscriber record fields will be apparent to a person skilled in the relevant arts based at least on the teachings contained herein.

[0114] 3.2 Exemplary Computer System Embodiment

[0115] In one embodiment of the present invention, system 100 is implemented in part using one or more computer systems. FIG. 4 is a block diagram of a client/server system 400 for providing genomic and proteomic products and services according to an embodiment of the present invention.

[0116] 3.2.1 Databases

[0117] In one embodiment, one or more databases are used to store data related to the genomic and proteomic products and services. In one embodiment, the databases may be organized by fields, records, and files. A field may represent a single piece of information. A record may represent one complete set of fields. Finally, a collection of records may be organized into a file. In FIG. 4, system 400 includes a subscriber database 425, a clone collection database 430, and an expression database 435.

[0118]

3
.2.1.1 Subscriber Database

[0119] Subscriber database 425 contains a subscriber record, such as subscriber record 300 of FIG. 3, for each subscriber of genomic and proteomic products and services.

[0120] 3.2.1.2 Clone Collection Database

[0121] The clone collection database 430 is configured to store data describing the attributes of the clones available in one or more clone collections (e.g., public and/or private clone collections). Examples of attributes that may be stored in a clone collection database include, but are not limited to, the nucleotide sequence of an ORF in a clone, the source of the template used to construct the ORF, the sequences of known allelic variants of the ORF, sequences of splice variants, sites of known polymorphisms and/or mutations in the ORF (e.g., single nucleotide polymorphisms, etc.), post-translational modifications (e.g., glycosylation, protein splicing, etc.) that are known to occur to the polypeptide expressed from the ORF, sites at which such post-translational modifications occur, and other similar information. Clone collection databases may comprise attributes of the polypeptides expressed from one or more clones. Attributes of a polypeptide that a clone collection database may comprise include, but are not limited to, the amino acid sequence, amino acid residues known to be involved in one or more activity (e.g., active site residues, epitopes, etc.), locations of structural and/or functional domains, molecular weight, isoelectric point, catalytic activities, number and kind of post-translational modifications, amino acids theat are post-translationrally modified, the amino acid sequence of structurally related polypeptides, and the like.

[0122] Clone collection databases may be searchable (e.g., with a nucleotide and/or polypeptide sequence). In some embodiments, it may be possible to search a clone collection database with all or a portion of the amino acid sequence of a polypeptide in order to identify clones encoding all or a portion of the polypeptide or encoding all or a portion of one or more related polypeptides. In some embodiments, the amino acid sequence of a portion of a polypeptide (e.g., a structural and/or functional domain, an amino acid motif, etc.) may be used to search a clone collection database to identify one or more clones encoding polypeptides that have an amino acid sequence similar to the search sequence (e.g., have a similar domain and/or motif).

[0123] In some embodiments, a clone collection database may contain sequence information. Such sequence information may or may not be of any particular clone present in the collection. For example, a clone collection database may have sequence information concerning one or more nucleic acids, which may encode one or more polypeptides, that are not present in a clone collection. In some embodiments, a subscriber may request that a clone be prepared from all or a part of such a sequence.

[0124] In one embodiment of the present invention, the clone collection database 430 includes a private area and a public area. The private area of clone collection database 430 maintains information describing clones that are only available to one or more subscribers. The public area of the clone collection database 430 maintains information describing the clones from the provider's clone collections that are available to everyone (i.e., all customers).

[0125] 3.2.1.3 Expression Database

[0126] The expression database 435 is configured to store data describing the results of protein expression analyses performed for the clones in the clone collections. In this way, optimized protein expression systems identifying the best vector and host for a particular clone are readily accessible.

[0127] In addition to vector and host systems, a protein expression database may comprise information related to codon usage in one or more hosts. The optimum codon usage based on any particular host may be identified. Clones employing the optimum codon usage may be constructed and added to a clone collection in order to optimize the expression of one or more polypeptides in one or more hosts. In some embodiments, clones in a clone collection may encode polypeptides using optimized codons for a particular organism (e.g., E. coli, yeast, insect cells, mammalian cells, etc.). A clone collection may comprise multiple sequences encoding the same polypeptide but employing different codons in order to optimize the expression of the polypeptide in a variety of host cells.

[0128] In addition, protein expression databases may comprise other information including, but not limited to, information regarding the characteristics of a polypeptide expressed from an ORF in the clone collection. Characteristics that might be included include the molecular weight of the expressed polypeptide, the site, extent and nature of post-translational modification undergone by the polypeptide in its native organism, the specific activity of the polypeptide, known stimulators and/or inhibitors of an activity of the polypeptide, physiological role of the polypeptide in its native organism, and similar information.

[0129] 3.2.1.4 Client/Server Architecture

[0130] A provider server 420 provides access to subscriber database 425, clone collection database 430, and expression database 435. Customer computer systems 410 are connected to provider server 420 via a communications network 415 (such as a local area network, a wide area network, point-to-point links, the Internet, etc., or combinations thereof). Users may access and traverse the functions provided by the provider server 420 in any number of ways via interaction with menus or icons provided by a user interface. Other ways of accessing system 400 will be apparent to persons skilled in the relevant arts based at least on the teachings contained herein.

[0131] In an embodiment, the provider server 420 and the customer systems 410 are implemented using a computer system 500 such as that shown in FIG. 5.

[0132] Referring to FIG. 5, the computer system 500 includes one or more processors 502. Processor 502 is connected to a communication bus 504. The computer system 500 also includes a main memory 506. Main memory 506 is preferably random access memory (RAM). Computer system 500 further includes secondary memory 508. Secondary memory 508 includes, for example, hard disk drive 510 and/or removable storage drive 512. Removable storage drive 512 could be, for example, a floppy disk drive, a magnetic tape drive, a compact disk drive, a program cartridge and cartridge interface, or a removable memory chip. Removable storage drive 512 reads from and writes to a removable storage unit 514. Removable storage unit 514, also called a program storage device or computer program product, represents a floppy disk, magnetic tape, compact disk, or other data storage device. Computer programs or computer control logic are stored in main memory 506 and/or secondary memory 508 and/or removable storage unit 514. When executed, these computer programs enable the provider server 420 and customer systems 410 to perform various functions of the present invention as discussed herein. In particular, the computer programs enable the processor 502 to perform some of the functions of the present invention. Accordingly, such computer programs represent controllers of the system 400. Computer system 500 further includes a communications interface 516. Communications interface 516 facilitates communications between computer system 500 and local or remote external devices 518. External devices 518 could be, for example, personal computers, displays, databases, and additional computer systems 500. In particular, communications interface 516 enables computer system 500 to send and receive software and data to/from external devices 518 via signals, which are also herein referred to as computer program products. Examples of communications interface 516 include a modem, a network interface, and a communications port.

4. Exemplary Operational Embodiments

[0133] Exemplary methods for providing genomic and proteomic products and services in accordance with embodiments of the present invention will now be described with reference to FIG. 1, FIG. 4, and the steps described in FIGS. 6-8 and 10.

[0134]

4
.1 Accessing Genomic and Proteomic Research Products and Services

[0135] Referring to FIG. 6, in a step 605, a determination may be made as to whether a customer is a subscriber or not. The results of this determination will often dictate the nature, extent, configuration, and other details of products and services to which the customer is provided access.

[0136] Next, if the customer is a subscriber, then the customer may be presented with means for enabling the selection of public and private genomic and proteomic products and services from the provider 105 (step 610). In one embodiment, a listing of available products and services is provided to the customer on a display associated with a customer computer system such as customer system 410 illustrated in FIG. 4. The user is then able to select products and services from the list using an input device such as a keyboard or mouse.

[0137] Once a product or service has been selected, in a step 615, the provider 105 responds by providing the selected product or service at an established subscriber rate.

[0138] Alternatively, where the customer is not a subscriber, in a step 620, the customer may be, for example, presented with means for enabling the selection of public genomic and proteomic products and services from the provider 105. The products and services available to a non-subscriber may be the same or different from those available to a subscriber. In some embodiments, more products and services may be available to a subscriber than are available to a non-subscriber.

[0139] Once a product or service has been selected, in a step 625, the provider 105 responds by providing the selected product or service at an established non-subscriber rate.

[0140] Steps 610 or 620 provide the subscribers and non-subscribers with multiple products and services from which to choose. Accordingly, in steps 615 or 625, a variety of operational flows could be executed; such operational flows are within the scope and spirit of the invention. Further, as a consequence of providing a particular product or service, the need for additional products or services may arise. Accordingly, in an embodiment of the present invention, the need for additional products and services is anticipated.

[0141] An exemplary method for providing additional products and services related to an initial product or service provided to the subscribers and non-subscribers in now provided with reference to FIG. 7.

[0142] In step 705, a determination is made as to whether a customer is a subscriber or not. The results of this determination will dictate the nature, extent, configuration, and other details of products and services to which the customer is provided access.

[0143] Next, if the customer is a subscriber, then the customer is presented with means for enabling the selection of public and private genomic and proteomic products and services from the provider 105 (step 710).

[0144] Alternatively, where the customer is not a subscriber, in a step 715, the customer is presented with means for enabling the selection of public genomic and proteomic products and services from the provider 105.

[0145] In one embodiment, a listing of available products and services is provided to the customer on a display associated with a customer computer system such as customer system 410 illustrated in FIG. 4. The user is then able to select products and services from the list using an input device such as a keyboard or mouse.

[0146] Once an initial selection of products or services has been made, in a step 720, the provider 105 responds by providing the selected initial product or service. In one embodiment, the customer will be charged a subscriber rate or a non-subscriber rate for the selected product or service.

[0147] In a step 725, products or services that are related to the initial products or services provided are identified. For example, an initial product may be a clone from a clone collection, related products would include, but not be limited to, a polypeptide encoded by the clone, an expression system (e.g., a vector comprising the ORF for the polypeptide and a suitable host cell) for expressing the polypeptide, antibodies that specifically bind to the polypeptide, reagents for assaying an activity of the polypeptide and the like. Related services may include the production of any related product, for example, expression and purification of the polypeptide, production of antibodies specific to the polypeptide, and the like.

[0148] Next, the customer is presented with means for enabling the selection of the identified products or services that are related to the initially provided product or service (step 730).

[0149] If the customer elects to obtain a related product or service (step 735), the provider 105 responds by providing the related product or service (step 740).

[0150] If the customer does not wish to obtain the related product or service, in a step 745, he or she can elect to request new products or services. In this case, the customer is again presented with the option of selecting initial genomic and proteomic products and services (steps 710 or 715).

[0151] 4.2 Providing Genomic and Proteomic Research Products and Services

[0152] Requesting clone construction is one service that can be requested by both subscribers and non-subscribers and is likely to lead to the need for additional products or services. FIGS. 8 and 9 will now be used to describe an exemplary method for providing clone construction and activities related thereto in accordance with one embodiment of the present invention.

[0153] In a step 805, the provider constructs one or more clones in response to a customer's selection of this service. An exemplary method for constructing clones is described with reference to the steps shown in FIG. 9.

[0154] In a step 905, target templates are identified. A target template may be a nucleic acid molecule that contains a nucleic acid sequence of interest that a customer desires to be included in a clone. In an embodiment of the present invention, all or a portion of a nucleic acid sequence of interest may be compared (e.g., BLASTed) against a number of available public and/or private clone databases in order to identify potential templates from which to amplify corresponding sequence of interest (e.g., ORF).

[0155] Next, in a step 910, clones corresponding to the identified potential templates are processed: The desired template is isolated and a clone comprising the desired nucleic acid sequence is prepared from the template using standard techniques (e.g., PCR cloning, recombinational cloning, restriction digest and ligation cloning, topoisomerase-mediated cloning, etc.). For example, the desired nucleic acid sequence of interest may be amplified form a template using PCR primers that flank the desired sequence. PCR primers may contain sequences corresponding to one or more recognition sites. For example, a PCR primer may contain the sequence of all or a portion of a recombination site, all or a portion of a topoisomerase site, all or a portion of a restriction enzyme site, or combinations of the above. After amplification, the, amplification product may be inserted into one or more vectors making use of one or more of the recognition sites. For example, after PCR, an amplification product comprising recombination sites may be contacted with one or more vectors comprising compatible recombination sites and one or more recombination proteins under conditions causing the amplification product to be inserted in the vector.

[0156] A clone comprises a nucleic acid sequence of interest. A nucleic acid sequence of interest may be any nucleic acid sequence. For example, a nucleic acid sequence of interest may comprise an ORF. The ORF may correspond to all or a portion of a polypeptide (e.g., may be a full-length ORF or a partial ORF). A sequence comprising an ORF may further comprise one or more stop codons, one or more promoters, one or more enhancers, one or more polyadenylation sites, one or more splice sites or other sequences known to those skilled in the art. A nucleic acid sequence of interest may comprise a sequence of an un-translated RNA molecule. For example, a sequence of interest may comprise the sequence of a tRNA, a ribozyme, an RNAi, an anti-sense molecule and the like.

[0157] In one embodiment, full-length clones that correspond to the targets are inoculated into 96-well Bio-Blocks for subsequent mini-preps. In parallel, PCR primers, which flank each ORF including the stop codon, are designed. In an embodiment, primers include the full attB1 and attB2 sites. In this way, subsequent cloning of the amplicons into a Gateway-compatible donor vector (e.g. pDONR221) can be performed. Primers may be synthesized at a 50 nmol scale, desalted purity, in the same format as the arrayed clones (96-well) in order to facilitate set-up of the amplification reactions. For those targets which are deemed vital to the collection but are not present within the clone collections, the provider utilizes its collection of >50 full-length and normalized full-length human cDNA libraries as potential templates from which to amplify the ORF. Primer design and synthesis proceeds as described earlier. Amplification of the ORF proceeds using a DNA polymerase, preferably one with proofreading activity (e.g. Platinum Pfx), under conditions which will minimize the potential for PCR-induced nucleotide mutations (e.g. base changes, insertions, deletions). Immediately following amplification, products are run out on a 1% agarose gel containing ethidium bromide (0.25 μg/ml) and visualized on a gel documentation system in order to confirm amplification of the correct product. Products are then purified in a 96-well format using a commercially available filter plate to remove excess primer and unincorporated nucleotides. Purified PCR products are then reacted with pDONR221 in a BxP Gateway™ cloning reaction in a 96-well format to produce entry clones. Upon termination of the BxP reaction with proteinase K, DNA is transformed, for example, into MultiShot™ TOP10 chemically competent E. coli and selected on solid medium containing kanamycin (50 μg/ml). One or two individual antibiotic-resistant colonies are then selected per clone and subjected to diagnostic PCR using vector-specific primers in order to confirm presence of the ORF insert within the entry vector.

[0158] Next, in a step 915, the entry clones produced in step 910 are confirmed. In one embodiment, confirmation is achieved via agarose gel electrophoresis and subsequent visualization on a gel documentation system.

[0159] Processing of the entry clones continues in step 920. In one embodiment, confirmed entry clones from step 915 are inoculated into liquid media containing kanamycin (50 μg/ml) and cultured overnight for the purpose of producing glycerol stocks of each of the entry clones. Full-length nucleotide sequence verification of the glycerol stocks is then completed. The confirmed entry clones are then prepped and initially subjected to 5′ and 3′ end sequencing using the universal sequencing sites within the vector. Full-length sequencing proceeds via primer walking and results in 2× coverage of the ORFs.

[0160]

[0161] Finally, in step 925, once the sequence data is annotated and confirmed, the entry clones are entered into the clone collection. In one embodiment, the clone is added to either the public clone collection or the private clone collection.

[0162] In accordance with an embodiment of the present invention, the customer is able to identify the clones that are built and added to the clone collection. Further, the subscriber may stipulate the order in which clones are built and added to the clone collection. In this way, the populating of the clone collection is prioritized to meet the research needs of the subscriber.

[0163] Returning to FIG. 8, once the clones have been constructed and added to the clone collection, in a step 810, the clone collection database may be updated with information describing the attributes of the newly added entry clones.

[0164] In a step 815, where the customer is a subscriber, the subscriber record for the customer may be updated. Accordingly, the amount of funds credited for clone purchases may be reduced by an amount equal to the subscriber fee for this service. Additionally, the total number of clones ordered is incremented by an amount equal to the number of clones ordered.

[0165] In a step 820, the provider identifies optimized protein expression systems for one or more of the clones in the clone collection. In one embodiment, data describing the characteristics of the optimized protein expression systems is maintained in the expression database 435. Optimized protein expression systems may identify the vector and host shown to yield protein of a particular type or quantity. An optimized protein expression system may identify codons to be used for one or more amino acids that result in improved expression in one or more host cells. One or more clones may be constructed that use one or more of the optimized codons to encode the polypeptide to be expressed. By taking advantage of this service, the customer can avoid the time and expense involved with identifying optimized protein expression systems on their own.

[0166] In a step 825, the provider determines if the customer would like to obtain protein produced by any of the clones in the clone collection. If protein is desired, then in step 830, the purified protein products are produced and/or provided to the customer.

[0167] In a step 835, the provider determines if the customer would like to obtain antibodies produced by any of the clones in the clone collection. If antibodies are desired, then in step 840, antibody products are provided to the customer.

[0168] In accordance with the above described system and methods, a customer is able to obtain customized genomic and proteomic products and services. In this way, a single resource for assisting with the efficient identification of pharmacologically accessible targets is realized.

[0169]
FIG. 10 illustrates yet another exemplary method for iteratively providing genomic and proteomic products and services in accordance with one embodiment of the present invention.

[0170] In a step 1005, customers are given access to one or more databases by the provider.

[0171] In a step 1010, customers may request a product or service, such as requesting reagents, for example.

[0172] In response, in a step 1015, the provider supplies the requested reagents.

[0173] Next, in a step 1020, customers may request additional reagents related to the originally requested product or service. For example, customers may request protein antibodies, etc.

[0174] In response, in a step 1025, the provider supplies-the related reagents requested by the customers.

[0175] The steps described herein are presented for explanation only and are not intended to limit the present invention. Based at least on the teachings described herein, a person skilled in the relevant arts will recognize that one or more steps could be added or removed without departing from the spirit and scope of the present invention. Further details of the products and services available in accordance with embodiments of the present invention will now be described.

5. Detailed Exemplary Products Description

[0176] Clone Collections

[0177] In some embodiments of the invention, a collection of clones (e.g., clones comprising an ORF or other sequence of interest) may be constructed. A collection of clones may be constructed in response to a request from a subscriber and may comprise one or more sequences identified by a subscriber. A clone collection may comprise clones comprising any sequences that are of interest to a subscriber. A clone collection may contain sequences representing all, substantially all, a majority, or a representative number of all known members of a class of polypeptides. For example, a collection may contain clones comprising ORFs of all known polypeptides having a particular activity and/or characteristic of interest (e.g., all human polypeptides having a particular enzymatic activity of interest).

[0178] Collections may comprise clones comprising ORFs encoding all, substantially all, a majority, or a representative number of polypeptides related to and/or affected by a particular activity. For example, a collection may comprise clones comprising ORFs relating to or affected by a particular ligand. Clones in a collection of this type might comprise ORFs encoding signal transduction polypeptides (e.g., receptors), related signaling polypeptides (e.g., polypeptides involved in signaling pathways), and polypeptides affected by the ligand (e.g., polypeptides induced, repressed, activated, in-activated, etc.).

[0179] Collections may comprise clones comprising ORFs encoding all, substantially all, a majority, or a representative number of polypeptides involved in the metabolism (e.g., synthesis and degradation) of a metabolite of interest (e.g., a lipid, carbohydrate, peptide, etc.) as well as clones comprising ORFs encoding the polypeptides affected by the metabolite. For example, a collection may contain clones comprising ORFs encoding the enzymes of the biosynthetic pathway that results in the production of a metabolite of interest, those involved in the degradative pathway of the metabolite as well as those affected by the presence or absence of the metabolite. Representative metabolites include, but are not limited to, lipids (e.g., eicosanoids, prostaglandins, prostacyclins, thromboxanes, leukotrienes, steroid hormones, etc.) carbohydrates (e.g., inositol phosphate), peptides (e.g., cytokines, chemokines, interleukins, growth factors) and the like.

[0180] Examples of collections that may be prepared include, but are not limited to, those in Tables 1-15 or subsets thereof. Tables 1-15 contain the GenBank accession numbers of sequences relating to various molecules of interest (e.g., polypeptides, hormones, small molecules, etc.). Sequences relating to a molecule of interest may comprise sequences of the molecules of interest (e.g., when the molecule of interest is a polypeptide or nucleic acid), sequences of polypeptides involved in the metabolism (e.g., synthesis and/or degradation) of the molecule of interest, sequences of polypeptides that are affected by the molecule of interest (directly or indirectly), and/or polypeptides involved in signaling or other processes mediated by the molecule of interest. The accession numbers of the sequences listed in the tables, as well as the underlying full GenBank record of each accession number (e.g., sequences and references cited) are specifically incorporated herein by reference.

[0181] Nucleic acid sequences of interest to be included in a clone collection of the invention (e.g., ORFs, tRNAs, ribozymes, RNAis, 5′-un-translated regions, promoters, enhancers, etc.) may be provided in any suitable vector for inclusion in a collection. In some instances, it may be desirable to position a nucleic acid sequence of interest (e.g., an ORF or other nucleic acid of interest) in the vector such that the orientation of the nucleic acid sequence of interest with respect to the vector is controlled. This may be accomplished by equipping nucleic acid sequence of interest with one or more adapter sequences prior to inserting the nucleic acid into the vector. Adapter sequences may comprise one or more functional sites such as one or more recognition sites (e.g., restriction enzyme recognition sites, one or more recombination sites and/or one or more topoisomerase recognition sites). Suitable adapter sequences may be attached to a nucleic acid sequence of interest using techniques well known in the art, for example, by ligating an adapter to the nucleic acid or by amplifying the nucleic acid with a primer containing the adapter sequences.

[0182] Clone collections of the invention may contain two or more clones (e.g., a plurality of individual clones each comprising a vector and a nucleic acid sequence of interest or insert). In many instances, the nucleic acid inserts will reside in a vector such that the insert is not normally transcribed. In such instances, the vectors of the clone collection may be used to propagate and/or transfer the inserts to other nucleic acid molecules (e.g., vectors, chromosomes, etc.). In other instances, clone collections of the invention will be designed so that nucleic acid insert is operably linked to an expression control element (e.g., a promoter). Regardless of whether the nucleic acid insert resides in a vector in an expressible format, the insert may be linked to nucleic acid which is co-transcribed with the insert under appropriate conditions. As an example, when the nucleic acid insert is an ORF, the ORF may be linked to nucleic acid which encodes an amino acid sequence which is not normally associated with the expression product of the ORF. Thus, upon transcription and translation, a fusion protein is produced.

[0183] As explained elsewhere herein, fusion proteins may be produced when stop codon suppression is employed. In other words, a stop codon may be located between the ORF and the nucleic acid which encodes the other amino acid sequence and stop codon suppression can be used to geneate a fusion product. Of course, expression of the ORF in the absence of stop codon suppression will yield the product of the ORF without the other amino acid sequence.

[0184] As noted above, clone collections of the invention may contain essentially any number of clones. Further, these clones may encode RNA and/or polypeptide fusion products. Clone collections of the invention may contain from about 2 to about 100,000 clones, from about 2 to about 50,000 clones, from about 2 to about 40,000 clones, from about 2 to about 30,000 clones, from about 2 to about 20,000 clones, from about 2 to about 10,000 clones, from about 2 to about 5,000 clones, from about 2 to about 2,000 clones, from about 20 to about 100,000 clones, from about 20 to about 50,000 clones, from about 20 to about 30,000 clones, from about 20 to about 20,000 clones, from about 20 to about 10,000 clones, from about 20 to about 5,000 clones, from about 50 to about 100,000 clones, from about 50 to about 50,000 clones, from about 50 to about 40,000 clones, from about 50 to about 30,000 clones, from about 50 to about 20,000 clones, from about 50 to about 10,000 clones, from about 50 to about 5,000 clones, from about 50 to about, 3,000 clones, from about 50 to about 1,000 clones, from about 100 to about 100,000 clones, from about 100 to about 50,000 clones, from about 100 to about 40,000 clones, from about 100 to about 30,000 clones, from about 100 to about 20,000 clones, from about 100 to about 10,000 clones, from about 100 to about 5,000 clones, from about 100 to about 3,000 clones, from about 200 to about 100,000 clones, from about 200 to about 50,000 clones, from about 200 to about 40,000 clones, from about 200 to about 30,000 clones, from about 200 to about 20,000 clones, from about 200 to about 10,000 clones, from about 200 to about 5,000 clones, from about 200 to about 4,000 clones, from about 200 to about 3,000 clones, from about 200 to about 2,000 clones, from about 200 to about 1,000 clones, from about 300 to about 100,000 clones, from about 300 to about 50,000 clones, from about 30 to about 30,000 clones, from about 300 to about 20,000 clones, from about 300 to about 10,000 clones, from about 300 to about 5,000 clones, from about 300 to about 3,000 clones, from about 300 to about 2,000 clones, from about 300 to about 1,000 clones, from about 400 to about 100,000 clones, from about 400 to about 50,000 clones, from about 400 to about 30,000 clones, from about 400 to about 10,000 clones, from about 400 to about 5,000 clones, from about 400 to about 3,000 clones, from about 400 to about 2,000 clones, from about 400 to about 1,000 clones, from about 500 to about 100,000 clones, from about 500 to about 50,000 clones, from about 500 to about 25,000 clones, from about 500 to about 10,000 clones, from about 500 to about 5,000 clones, from about 500 to about 3,000 clones, from about 500 to about 2,000 clones, from about 500 to about 1,000 clones, from about 750 to about 100,000 clones, from about 750 to about 50,000 clones, from about 750 to about 30,000 clones, from about 750 to about 10,000 clones, from about 750 to about 5,000 clones, from about 750 to about 3,000 clones, from about 750 to about 2,000 clones, from about 750 to about 1,000 clones, from about 1,00 to about 100,000 clones, from about 1,000 to about 50,000 clones, from about 1,000 to about 30,000 clones, from about 1,000 to about 10,000 clones, from about 1,000 to about 5,000 clones, from about 1,000 to about 3,000 clones, from about 2,000 to about 100,000 clones, from about 2,000 to about 50,000 clones, from about 2,000 to about 30,000 clones, from about 2,000 to about 10,000 clones, from about 2,000 to about 5,000 clones, from about 2,000 to about 150,000 clones, from about 2,000 to about 200,000 clones, from about 2,000 to about 300,000 clones, from about 2,000 to about 400,000 clones, from about 2,000 to about 500,000 clones, from about 2,000 to about 600,000 clones, from about 2,000 to about 800,000 clones, from about 2,000 to about 1,000,000 clones, from about 5,000 to about 1,000,000 clones, from about 5,000 to about 500,000 clones, from about 5,000 to about 250,000 clones, from about 5,000 to about 100,000 clones, from about 5,000 to about 50,000 clones, from about 5,000 to about 25,000 clones, from about 5,000 to about 10,000 clones, from about 10,000 to about 100,000 clones, from about 10,000 to about 250,000 clones, from about 10,000 to about 500,000 clones, from about 10,000 to about 750,000 clones, from about 10,000 to about 1,000,000 clones, from about 10,000 to about 50,000 clones, from about 10,000 to about 25,000 clones, from about 20,000 to about 100,000 clones, from about 20,000 to about 250,000 clones, from about 20,000 to about 500,000 clones, from about 20,000 to about 1,000,000 clones, from about 20,000 to about 50,000 clones, from about 20,000 to about 40,000 clones, from about 40,000 to about 100,000 clones, from about 40,000 to about 250,000 clones, from about 40,000 to about 500,000 clones, from about 40,000 to about 1,000,000 clones, from about 40,000 to about 75,000 clones, from about 60,000 to about 80,000 clones, from about 60,000 to about 100,000 clones, from about 60,000 to about 250,000 clones, from about 60,000 to about 500,000 clones, or from about 60,000 to about 1,000,000 clones.

[0185] A clone collection may comprise clones containing any nucleic acid sequences of interest. As examples, collections of clones which encode proteins involved in the same or related biological processes (see Tables 1-15); clones with inserts from a particular/individual organism (e.g., a human), clones with inserts from a particular species of organism, and clones with inserts from a particular strain of an organism (e.g., E. coli K12). In some embodiments, a clone collection may comprise nucleic acid sequences of interest that are derived from human, mouse, dog, rat, and/or other mammalian tissues. Clone collections may be constructed from more than one tissue source within an organism, for example, from brain, liver, kidney, pancreas, lung, heart, etc.

[0186] Nucleic acid segments used to prepare clones of collections of the invention may or may not contain one or more recombination sites, and/or one or more topoisomerase recognition site. Further, in some collections, some clones may contain one or more recombination sites and/or one or more topoisomerase recognition site while other clones may not contain any such sites.

[0187] In some instances, a clone to be included in a clone collection may comprise a vector containing an ORF. A vector may be provided with one or more functional sequences. Functional sequences on the vector may be used to control the expression of a polypeptide of interest from an ORF and to influence the characteristics of the expressed polypeptide. Such sequences may be located anywhere in the vector that allows them to exert their function. For example, a vector may comprise a variety of sequences including, but not limited to, sequences suitable for use as primer sites (e.g., sequences to which a primer, such as a sequencing primer or amplification primer may hybridize to initiate nucleic acid synthesis, amplification or sequencing), transcription or translation signals or regulatory sequences such as promoters and/or enhancers, ribosomal binding sites, Kozak sequences, start codons, termination signals such as stop codons, origins of replication, recombination sites (or portions thereof), selectable markers, and ORFs or portions of ORFs to create protein fusions (e.g., N-terminal or C-terminal) such as GST, GUS, GFP, YFP, CFP, maltose binding protein, 6 histidines (HIS6), epitopes, haptens and the like and combinations thereof. In some embodiments, any one or more of the functional sequences discussed above may be operably linked to an ORF to form a nucleic acid sequence of interest comprising the ORF and one or more functional sequences. Thus functional sequences may be provided on a vector and/or as part of a nucleic acid sequence of interest.

[0188] An ORF may be cloned from a known sequence (e.g., all or a part of a sequence having a GenBank accession number) using standard techniques (see, Sambrook, et al., supra). For example, PCR amplification may be conducted using a template nucleic acid comprising the ORF. In some embodiments, primers for amplification may comprise all or a portion of one or more recognition sequences (e.g., restriction sites, topoisomerase recognition sites, and/or recombination sites). The amplification product may be inserted into a nucleic acid molecule (e.g., a vector) using techniques known in the art. In some preferred embodiments, primers for amplification of an ORF may comprise a recombination site and the amplification product may be inserted into a vector using GATEWAY™ recombinational cloning techniques available from Invitrogen Corporation, Carlsbad, Calif.

[0189] After cloning an ORF into a vector, the entire ORF may be sequenced to ensure that the cloned ORF has the desired sequence. Sequencing may be accomplished using standard techniques (e.g., dideoxy sequencing).

[0190] In some embodiments, ORFs of the invention and/or vectors comprising the ORFs of the invention may be provided with one or more recombination sites. Recombination sites for use in the invention may be any nucleic acid that can serve as a substrate in a recombination reaction. Such recombination sites may be wild-type or naturally occurring recombination sites, or modified, variant, derivative, or mutant recombination sites. Examples of recombination sites for use in the invention include, but are not limited to, phage-lambda recombination sites (such as attP, attB, attL, and attR and mutants or derivatives thereof) and recombination sites from other bacteriophages such as phi80, P22, P2, 186, P4 and P1 (including lox sites such as loxP and loxP511).

[0191] Recombination proteins and mutant, modified, variant, or derivative recombination sites for use in the invention include those described in U.S. Pat. Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, and 6,277,608 and in U.S. application Ser. No. 09/438,358 (filed Nov. 12, 1999), based upon U.S. provisional application No. 60/108,324 (filed Nov. 13, 1998). Mutated att sites (e.g., attB 1-10, attP 1-10, attR 1-10 and attL 1-10) are described in U.S. provisional patent application Nos. 60/122,389, filed Mar. 2, 1999, 60/126,049, filed Mar. 23, 1999, 60/136,744, filed May 28, 1999, 60/169,983, filed Dec. 10, 1999, and 60/188,000, filed Mar. 9, 2000, and in U.S. application Ser. Nos 09/517,466, filed Mar. 2, 2000, and 09/732,914, filed Dec. 11, 2000 (published as 20020007051-A1) the disclosures of which are specifically incorporated herein by reference in their entirety. Other suitable recombination sites and proteins are those associated with the GATEWAY™ Cloning Technology available from Invitrogen Corp., Carlsbad, Calif., and described in the product literature of the GATEWAY™ Cloning Technology, the entire disclosures of all of which are specifically incorporated herein by reference in their entireties.

[0192] Sites that may be used in the present invention include att sites. The 15 bp core region of the wild-type att site (GCTTTTTTAT ACTAA (SEQ ID NO:)), which is identical in all wild-type att sites, may be mutated in one or more positions. Other att sites that specifically recombine with other att sites can be constructed by altering nucleotides in and near the 7 base pair overlap region, bases 6-12 of the core region. Thus, recombination sites suitable for use in the methods, molecules, compositions, and vectors of the invention include, but are not limited to, those with insertions, deletions or substitutions of one, two, three, four, or more nucleotide bases within the 15 base pair core region (see U.S. application Ser. Nos. 08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732) and 09/177,387, filed Oct. 23, 1998, which describes the core region in further detail, and the disclosures of which are incorporated herein by reference in their entireties). Recombination sites suitable for use in the methods, compositions, and vectors of the invention also include those with insertions, deletions or substitutions of one, two, three, four, or more nucleotide bases within the 15 base pair core region that are at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to this 15 base pair core region.

[0193] Analogously, the core regions in attB1, attP1, attL1 and attR1 are identical to one another, as are the core regions in attB2, attP2, attL2 and attR2. Nucleic acid molecules suitable for use with the invention also include those comprising insertions, deletions or substitutions of one, two, three, four, or more nucleotides within the seven base pair overlap region (TTTATAC, bases 6-12 in the core region). The overlap region is defined by the cut sites for the integrase protein and is the region where strand exchange takes place. Examples of such mutants, fragments, variants and derivatives include, but are not limited to, nucleic acid molecules in which (1) the thymine at position 1 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (2) the thymine at position 2 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (3) the thymine at position 3 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (4) the adenine at position 4 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or thymine; (5) the thymine at position 5 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or adenine; (6) the adenine at position 6 of the seven bp overlap region has been deleted or substituted with a guanine, cytosine, or thymine; and (7) the cytosine at position 7 of the seven bp overlap region has been deleted or substituted with a guanine, thymine, or adenine; or any combination of one or more (e.g., two, three, four, five, etc.) such deletions and/or substitutions within this seven bp overlap region. The nucleotide sequences of representative seven base pair core regions are set out below.

[0194] Altered att sites have been constructed that demonstrate that (1) substitutions made within the first three positions of the seven base pair overlap (TTTATAC) strongly affect the specificity of recombination, (2) substitutions made in the last four positions (TTTATAC) only partially alter recombination specificity, and (3) nucleotide substitutions outside of the seven bp overlap, but elsewhere within the 15 base pair core region, do not affect specificity of recombination but do influence the efficiency of recombination. Thus, nucleic acid molecules and methods of the invention include those comprising or employing one, two, three, four, five, six, eight, ten, or more recombination sites which affect recombination specificity, particularly one or more (e.g., one, two, three, four, five, six, eight, ten, twenty, thirty, forty, fifty, etc.) different recombination sites that may correspond substantially to the seven base pair overlap within the 15 base pair core region, having one or more mutations that affect recombination specificity. Particularly preferred such molecules may comprise a consensus sequence such as NNNATAC wherein “N” refers to any nucleotide (i.e., may be A, G, T/U or C). Preferably, if one of the first three nucleotides in the consensus sequence is a T/U, then at least one of the other two of the first three nucleotides is not a T/U.

[0195] The core sequence of each att site (attB, attP, attL and attR) can be divided into functional units consisting of integrase binding sites, integrase cleavage sites and sequences that determine specificity. Specificity determinants are defined by the first three positions following the integrase top strand cleavage site. These three positions are shown with underlining in the following reference sequence: CAACTTTTTTATAC AAAGTTG (SEQ ID NO: ). Modification of these three positions (64 possible combinations, Table 16) can be used to generate att sites that recombine with high specificity with other att sites having the same sequence for the first three nucleotides of the seven base pair overlap region. The possible combinations of first three nucleotides of the overlap region are shown in Table 16.

[0196] Representative examples of seven base pair att site overlap regions suitable for in methods, compositions and vectors of the invention are shown in Table 17. The invention further includes nucleic acid molecules comprising one or more (e.g., one, two, three, four, five, six, eight, ten, twenty, thirty, forty, fifty, etc.) nucleotides sequences set out in Table 17. Thus, for example, in one aspect, the invention provides nucleic acid molecules comprising the nucleotide sequence GAAATAC, GATATAC, ACAATAC, or TGCATAC.

[0197] As noted above, alterations of nucleotides located 3′ to the three base pair region discussed above can also affect recombination specificity. For example, alterations within the last four positions of the seven base pair overlap can also affect recombination specificity.

[0198] For example, mutated att sites that may be used in the practice of the present invention include attB1 (AGCCTGCTTT TTTGTACAAA CTTGT (SEQ ID NO: )), attP1 (TACAGGTCAC TAATACCATC TAAGTAGTTG ATTCATAGTG ACTGGATATG TTGTGTTTTA CAGTATTATG TAGTCTGTTT TTTATGCAAA ATCTAATTTA ATATATTGAT ATTTATATCA TTTTACGTTT CTCGTTCAGC TTTTTTGTAC AAAGTTGGCA TTATAAAAAA GCATTGCTCA TCAATTTGTT GCAACGAACA GGTCACTATC AGTCAAAATA AAATCATTAT TTG (SEQ ID NO: )), attL1 (CAAATAATGA TTTTATTTTG ACTGATAGTG ACCTGTTCGT TGCAACAAAT TGATAAGCAA TGCTTTTTTA TAATGCCAAC TTTGTACAAA AAAGCAGGCT (SEQ ID NO: )), and attR1 (ACAAGTTTGT ACAAAAAAGC TGAACGAGAA ACGTAAAATG ATATAAATAT CAATATATTA AATTAGATTT TGCATAAAAA ACAGACTACA TAATACTGTA AAACACAACA TATCCAGTCA CTATG (SEQ ID NO: )). Table 18 provides the sequences of the regions surrounding the core region for the wild type att sites (attB0, P0, R0, and L0) as well as a variety of other suitable recombination sites. Those skilled in the art will appreciated that the remainder of the site may be the same as the corresponding site (B, P, L, or R) listed above.

[0199] Other recombination sites having unique specificity (i.e., a first site will recombine with its corresponding site and will not substantially recombine with a second site having a different specificity) are known to those skilled in the art and may be used to practice the present invention. Corresponding recombination proteins for these systems may be used in accordance with the invention with the indicated recombination sites. Other systems providing recombination sites and recombination proteins for use in the invention include the FLP/FRT system from Saccharomyces cerevisiae, the resolvase family (e.g., γδ, TndX, TnpX, Tn3 resolvase, Hin, Hjc, Gin, SpCCE1, ParA, and Cin), and IS231 and other Bacillus thuringiensis transposable elements. Other suitable recombination systems for use in the present invention include the XerC and XerD recombinases and the psi, dif and cer recombination sites in E. coli. Other suitable recombination sites may be found in U.S. Pat. No. 5,851,808 issued to Elledge and Liu which is specifically incorporated herein by reference.

[0200] The materials and methods of the invention may further encompass the use of “single use” recombination sites which undergo recombination one time and then either undergo recombination with low frequency (e.g., have at least five fold, at least ten fold, at least fifty fold, at least one hundred fold, or at least one thousand fold lower recombination activity in subsequent recombination reactions) or are essentially incapable of undergo recombination. The invention also provides methods for making and using nucleic acid molecules which contain such single use recombination sites and molecules which contain these sites. Examples of methods which can be used to generate and identify such single use recombination sites are set out in PCT/US00/21623, published as WO 01/11058, which claims priority to U.S. provisional patent application No. 60/147,892, filed Aug. 9, 1999, both of which are specifically incorporated herein by reference.

[0201] Single use recombination sites are especially useful for either decreasing the frequency of or preventing recombination when either large number of nucleic acid segments are attached to each other or multiple recombination reactions are performed. Thus, the invention further includes nucleic acid molecules which contain single use recombination sites, as well as methods for performing recombination using these sites.

[0202] Recombination sites used with the invention may also have embedded functions or properties. An embedded functionality is a function or property conferred by a nucleotide sequence in a recombination site that is not directly associated with recombination efficiency or specificity. For example, recombination sites may contain protein coding sequences (e.g., intein coding sequences), intron/exon splice sites, origins of replication, and/or stop codons. Further, recombination sites that have more than one (e.g., two, three, four, five, etc.) embedded functions or properties may also be prepared.

[0203] In some instances it will be advantageous to remove either RNA corresponding to recombination sites from RNA transcripts or amino acid residues encoded by recombination sites from polypeptides translated from such RNAs. Removal of such sequences can be performed in several ways and can occur at either the RNA or protein level. One instance where it may be advantageous to remove RNA transcribed from a recombination site will be when constructing a fusion polypeptide between a polypeptide of interest and a coding sequence present on the vector. The presence of an intervening recombination site between the ORF of the polypeptide of interest and the vector coding sequences may result in the recombination site (1) contributing codons to the mRNA that result in the inclusion of additional amino acid residues in the expression product, (2) contributing a stop codon to the mRNA that prevents the production of the desired fusion protein, and/or (3) shifting the reading frame of the mRNA such that the two protein are not fused “in-frame.”

[0204] In one aspect, the invention provides methods for removing nucleotide sequences encoded by recombination sites from RNA molecules. One example of such a method employs the use of intron/exon splice sites to remove RNA encoded by recombination sites from RNA transcripts. Nucleotide sequences that encode intron/exon splice sites may be fully or partially embedded in the recombination sites used in the present invention and/or may encoded by adjacent nucleic acid sequence. Sequences to be excised from RNA molecules may be flanked by splice sites that are appropriately located in the sequence of interest and/or on the vector. For example, one intron/exon splice site may be encoded by a recombination site and another intron/exon splice site may be encoded by other nucleotide sequences (e.g., nucleic acid sequences of the vector or a nucleic acid of interest). Nucleic acid splicing is well known to those skilled in the art and is discussed in the following publications: R. Reed, Curr. Opin. Genet. Devel. 6:215-220 (1996); S. Mount, Nucl. Acids. Res. 10:459-472, (1982); P. Sharp, Cell 77:805-815, (1994); K. Nelson and M. Green, Genes and Devel. 23:319-329 (1988); and T. Cooper and W. Mattox, Am. J. Hum. Genet. 61:259-266 (1997).

[0205] Splice sites can be suitably positioned in a number of locations. For example, a vector designed to express an inserted ORF with an N-terminal fusion—for example, with a detectable marker—the first splice site could be encoded by vector sequences located 3′ to the detectable marker coding sequences and the second splice site could be partially embedded in the recombination site that separates the detectable marker coding sequences from the coding sequences of the ORF. Further, the second splice site either could abut the 3′ end of the recombination site or could be positioned a short distance (e.g., 2, 4, 8, 10, 20 nucleotides) 3′ to the recombination site. In addition, depending on the length of the recombination site, the second splice site could be fully embedded in the recombination site.

[0206] A modification of the method described above involves the connection of multiple (i.e., two or more) nucleic acid segments such that, upon expression, a fusion protein is produced. In one specific example, one nucleic acid segment encodes a detectable marker—for example, a vector comprising the GFP coding sequence—and another nucleic acid segment encodes an ORF of interest. Each of these segments may contain one or more recombination sites at one or both ends. In addition, the nucleic acid segment that encodes the detectable marker may contain an intron/exon splice site near its 3′ terminus and the nucleic acid segment that contains the ORF of interest may also contain an intron/exon splice site near its 5′ terminus. Upon recombination, the nucleic acid segment that encodes the detectable marker is positioned 5′ to the nucleic acid segment that encodes the ORF of interest. Further, these two nucleic acid segments are separated by a recombination site that is flanked by intron/exon splice sites. Excision of the intervening recombination site thus occurs after transcription of the fusion mRNA. Thus, in one aspect, the invention is directed to methods for removing RNA transcribed from recombination sites from transcripts generated from nucleic acids described herein. In many embodiments, the processed RNA will encode an ORF of interest which upon expression results in the production of a fusion protein.

[0207] Splice sites may be introduced into nucleic acid molecules to be used in the present invention in a variety of ways. One method that could be used to introduce intron/exon splice sites into nucleic acid segments is PCR. For example, primers could be used to generate nucleic acid segments corresponding to an ORF of interest and containing both a recombination site and an intron/exon splice site.

[0208] The above methods can also be used to remove RNA corresponding to recombination sites when the nucleic acid segment that is recombined with another nucleic acid segment encodes RNA that is not produced in a translatable format. One example of such an instance is where a nucleic acid segment is inserted into a vector in a manner that results in the production of antisense RNA. This antisense RNA may be fused, for example, with RNA that encodes a ribozyme. Thus, the invention also provides methods for removing RNA corresponding to recombination sites from such molecules.

[0209] The invention further provides methods for removing one or more amino acid sequences from protein expression products by protein splicing. Nucleotide sequences that encode protein splice sites may be fully or partially embedded in the sequence of the protein expression product and/or protein splice sites may be encoded by adjacent nucleotide sequences. In some embodiments, the invention provides methods of removing tag sequences by protein splicing. Suitable splice sites are encoded in the sequence of interest and/or in vector sequences and a tag sequence may be removed by splicing after translation. In some embodiments, the invention provides methods for removing amino acid sequences encoded by functional sequences (e.g., recombination sites) from protein expression products by protein splicing. Nucleotide sequences that encode protein splice sites may be fully or partially embedded in the recombination sites that encode amino acid sequences excised from proteins or protein splice sites may be encoded by adjacent nucleotide sequences. Similarly, one protein splice site may be encoded by a recombination site and another protein splice site may be encoded by other nucleotide sequences (e.g., nucleic acid sequences of the vector or a nucleic acid of interest).

[0210] It has been shown that protein splicing can occur by excision of an intein from a protein molecule and ligation of flanking segments (see, e.g., Derbyshire et al., Proc. Natl. Acad. Sci. (USA) 95:1356-1357 (1998)). In brief, inteins are amino acid segments that are post-translationally excised from proteins by a self-catalytic splicing process. A considerable number of intein consensus sequences have been identified (see, e.g., Perler, Nucleic Acids Res. 27:346-347 (1999)). Thus, inteins can be used, for example, to separate tags from proteins encoded by ORFs of interest.

[0211] Similar to intron/exon splicing, N- and C-terminal intein motifs have been shown to be involved in protein splicing. Thus, the invention further provides compositions and methods for removing one or more amino acid sequences from protein expression products by protein splicing. Nucleotide sequences that encode protein splice sites may be fully or partially embedded in the sequence of the protein expression product and/or protein splice sites may be encoded by adjacent nucleotide sequences. In some embodiments, the invention provides compositions and methods for removing amino acid residues encoded by functional sequences (e.g., recombination sites) from protein expression products by protein splicing. In a particular embodiment, this aspect of the invention is related to the positioning of nucleic acid sequences that encode intein splice sites on both the 5′ and 3′ end of recombination sites positioned between two coding regions. Thus, when the protein expression product is incubated under suitable conditions, amino acid residues encoded by these recombination sites will be excised. In another particular embodiment, this aspect of the invention is related to the positioning of nucleic acid sequences that encode intein splice sites on both the 5′ and 3′ end of amino acid tag sequences, which may be on the N-terminal, C-terminal and/or interior of the expression product. Thus, when the protein expression product is incubated under suitable conditions, amino acid residues of the tag sequence will be excised.

[0212] Protein splicing may be used to remove all or part of the amino acid sequences encoded by one or more recombination sites or amino acids sequences of one or more tags. Nucleic acid sequence that encode inteins may be, for example, fully or partially embedded in recombination sites or may adjacent to such sites. In certain circumstances, it may be desirable to remove a considerable number of amino acid residues. For example, an expression product may comprise a tag sequence and amino acids encoded by a recombination site. Such amino acids may extend beyond the N- and/or C-terminal ends of a polypeptide of interest. In such instances, intein coding sequence may be located a distance (e.g., 30, 50, 75, 100, etc. nucleotides) 5′ and/or 3′ of the sequences to be removed (e.g., the sequences encoded by the recombination site and the tag sequence).

[0213] While conditions suitable for intein excision will vary with the particular intein, as well as the protein that contains this intein, Chong et al., Gene 192:271-281 (1997), have demonstrated that a modified Saccharomyces cerevisiae intein, referred to as Sce VMA intein, can be induced to undergo self-cleavage by a number of agents including 1,4-dithiothreitol (DTT), β-mercaptoethanol, and cysteine. For example, intein excision/splicing can be induced by incubation in the presence of 30 mM DTT, at 4° C. for 16 hours.

[0214] Polypeptides

[0215] In some embodiments, the present invention provides polypeptides expressed from clones containing ORFs. The polypeptides may be expressed as native polypeptides, i.e., without any modifications to the primary sequence. Polypeptides may also be expressed as fusion proteins (e.g., N-terminal and/or C-terminal) and/or may be post-translationally modified (e.g., glycosylated, etc.).

[0216] In some embodiments, the polypeptides expressed from cloned ORFs of the present invention may be modified to contain a tag (e.g., an affinity tag) in order to facilitate the purification of the polypeptide. Suitable tags are well known to those skilled in the art and include, but are not limited to, repeated sequences of amino acids such as six histidines, epitopes such as the hemagglutinin epitope, the V5 epitope, and the myc epitope, and other amino acid sequences that permit the simplified purification of the polypeptide.

[0217] The invention further relates to fusion proteins comprising (1) a polypeptide, or fragment thereof, having one or more desired characteristics and/or activities and (2) a tag (e.g., an affinity tag), as well as nucleic acid molecules and collections of nucleic acid molecules which encode such fusion proteins. In particular embodiments, the invention includes a polypeptide described herein having one or more (e.g., one, two, three, four, five, six, seven, eight, etc.) tags. These tags may be located, for example, (1) at the N-terminus, (2) at the C-terminus, or (3) at both the N-terminus and C-terminus of the protein, or a fragment thereof having one or more desired characteristic and/or activity. A tag may also be located internally (e.g., between regions of amino acid sequence derived from a polypeptide encoded by a cloned ORF). The invention further includes collections of RNA (e.g., mRNA) and polypeptide expression products (e.g., fusion proteins, non-fusion proteins etc.) encoded by clone collections described herein.

[0218] Tags used in the invention may vary in length but will typically be from about 5 to about 100, from about 10 to about 100, from about 15 to about 100, from about 20 to about 100, from about 25 to about 100, from about 30 to about 100 from about 35 to about 100, from about 40 to about 100, from about 45 to about 100, from about 50 to about 100, from about 55 to about 100, from about 60 to about 100, from about 65 to about 100, from about 70 to about 100, from about 75 to about 100, from about 80 to about 100, from about 85 to about 100, from about 90 to about 100, from about 95 to about 100, from about 5 to about 80, from about 10 to about 80, from about 20 to about 80, from about 30 to about 80, from about 40 to about 80, from about 50 to about 80, from about 60 to about 80, from about 70 to about 80, from about 5 to about 60, from about 10 to about 60, from about 20 to about 60, from about 30 to about 60, from about 40 to about 60, from about 50 to about 60, from about 5 to about 40, from about 10 to about 40, from about 20 to about 40, from about 30 to about 40, from about 5 to about 30, from about 10 to about 30, from about 20 to about 30, from about 5 to about 25, from about 10 to about 25, or from about 15 to about 25 amino acid residues in length.

[0219] Tags used in the practice of the invention may serve any number of purposes. For example, such tags may (1) contribute to protein-protein interactions both internally within a protein (e.g., between a tag sequence and a polypeptide sequence to which the tag has been attached) and with other protein molecules, (2) make the polypeptide amenable to particular purification methods (e.g., affinity purification), (3) enable one to identify whether the polypeptide is present in a composition (e.g. ELISA, Western blot, etc.), and/or (4) stabilize or destabilize intra-protein interactions with the protein to which the tag has been added (e.g., increase or decrease thermostability of the protein).

[0220] Examples of tags which may be used in the practice of the invention include metal binding domains (e.g., a poly-histidine segments such as a three, four, five, six, or seven histidine region), immunoglobulin binding domains (e.g., (1) Protein A; (2) Protein G; (3) T cell, B cell, and/or Fc receptors;

[0221] and/or (4) complement protein antibody-binding domain); sugar binding domains (e.g., a maltose binding domain); and detectable domains (e.g., at least a portion of β-galactosidase). Fusion proteins may contain one or more tags such as those described above. Typically, fusion proteins that contain more than one tag will contain these tags at one terminus or both termini (i.e., the N-terminus and the C-terminus) of the polypeptide, although one or more tags may be located internally in addition to those present at the termini. Further, more than one tag may be present at one terminus, internally and/or at both termini of the polypeptide. For example, three consecutive tags could be linked end-to-end at the N-terminus of the polypeptide. The invention further includes compositions and reaction mixture that contain the above fusion proteins, as well as methods for preparing these fusion proteins, nucleic acid molecules (e.g., vectors) which encode these fusion proteins and recombinant host cells that contain these nucleic acid molecules. The invention also includes methods for using these fusion proteins as described elsewhere herein.

[0222] Tags that enable one to identify whether the fusion protein is present in a composition include, for example, tags that can be used to identify the protein in an electrophoretic gel. A number of such tags are known in the art and include epitopes and antibody binding domains, which can be used for Western blots.

[0223] The amino acid composition of the tags for use in the present invention may vary. In some embodiments, a tag may contain from about 1% to about 5% amino acids that have a positive charge at physiological pH, e.g., lysine, arginine, and histidine, or from about 5% to about 10% amino acids that have a positive charge at physiological pH, or from about 10% to about 20% amino acids that have a positive charge at physiological pH, or from about 10% to about 30% amino acids that have a positive charge at physiological pH, or from about 10% to about 50% amino acids that have a positive charge at physiological pH, or from about 10% to about 75% amino acids that have a positive charge at physiological pH. In some embodiments, a tag may contain from about 1% to about 5% amino acids that have a negative charge at physiological pH, e.g., aspartic acid and glutamic acid, or from about 5% to about 10% amino acids that have a negative charge at physiological pH, or from about 10% to about 20% amino acids that have a negative charge at physiological pH, or from about 10% to about 30% amino acids that have a negative charge at physiological pH, or from about 10% to about 50% amino acids that have a negative charge at physiological pH, or from about 10% to about 75% amino acids that have a negative charge at physiological pH. In some embodiments, a tag may comprise a sequence of amino acids that contains two or more contiguous charged amino acids that may be the same or different and may be of the same or different charge. For example, a tag may contain a series (e.g., two, three, four, five, six, ten etc.) of positively charged amino acids that may be the same or different. A tag may contain a series (e.g., two, three, four, five, six, ten etc.) of negatively charged amino acids that may be the same or different. In some embodiments, a tag may contain a series (e.g., two, three, four, five, six, ten etc.) of alternating positively charged and negatively charged amino acids that may be the same or different (e.g., positive, negative, positive, negative, etc.). Any of the above-described series of amino acids (e.g., positively charged, negatively charged or alternating charge) may comprise one or more neutral polar or non-polar amino acids (e.g., two, three, four, five, six, ten etc.) spaced between the charged amino acids. Such neutral amino acids may be evenly distributed through out the series of charged amino acids (e.g., charged, neutral, charged, neutral) or may be unevenly distributed throughout the series (e.g., charged, a plurality of neutral, charged, neutral, a plurality of charged, etc.).

[0224] In some embodiments, tags to be attached to the polypeptides of the invention may have an overall charge at physiological pH (e.g., positive charge or negative charge). The size of the overall charge may vary, for example, the tag may contain a net plus one, two, three, four, five, etc. or may possess a net negative one, two, three, four, five, etc.

[0225] In some embodiments, it may be desirable to remove all or a portion of a tag sequence from a fusion protein comprising a tag sequence and a polypeptide sequence encoded by a cloned ORF of the invention. In embodiments of this type, one or more amino acids forming a cleavage site, e.g., for a protease enzyme, may be incorporated into the primary sequence of the fusion protein. The cleavage site may be located such that cleavage at the site may remove all or a portion of the tag sequence from the fusion protein. In some embodiments, the cleavage site may be located between the tag sequence and the sequence of the polypeptide such that all of the tag sequence is removed by cleavage with a protease enzyme that recognizes the cleavage site. Examples of suitable cleavage sites include, but are not limited to, the Factor Xa cleavage site having the sequence Ile-Glu-Gly-Arg (SEQ ID NO: ), which is recognized and cleaved by blood coagulation factor Xa, and the thrombin cleavage site having the sequence Leu-Val-Pro-Arg (SEQ ID NO:), which is recognized and cleaved by thrombin. Other suitable cleavage sites are known to those skilled in the art and may be used in conjunction with the present invention.

[0226] Polypeptides of the invention may be post-translationally modified, for example, may be glycosylated, acylated, etc. Various eukaryotic expression systems may used to produce glycosylated polypeptides (e.g., baculovirus, vaccinia virus, yeast, etc.). Those skilled in the art will appreciate that the number and character of glycosyl chains that may be added to the polypeptides of the invention by post-translational modification may vary depending upon the expression system used (e.g., expression vector and host cell). The invention thus includes collections of vectors, which allow for the expression of glycosylated polypeptides, as well as vectors (e.g., an entry vector) that can be used to prepare such expression vectors.

[0227] Antibodies

[0228] Antibodies may be prepared that are specific to one or more of the polypeptides encoded by the cloned ORFs of a collection. Antibodies may be polyclonal and/or monoclonal. They may be prepared against an entire polypeptide or against a fragment of the polypeptide.

[0229] In some instances, antibodies are prepared that recognize all, substantially all, or a representative number of the polypeptides encoded by the ORFs of a collection. In other instances, antibodies may be prepared that are specific to a single polypeptide. In some embodiments, antibodies may be prepared that specifically bind to a subset of the polypeptides encoded by the ORFs of a collection. Thus, the invention also includes collections of antibodies that bind to proteins encoded by one or more ORFs of a collection.

[0230] Antibodies may be used for the detection of the polypeptides in an immunoassay, such as ELISA, Western blot, radioimmunoassay, enzyme immunoassay, and may be used in immunocytochemistry. In some embodiments, an anti-polypeptide antibody may be in solution and the polypeptide to be recognized may be in solution (e.g., an immunopreciptitation) or may be on or attached to a solid surface (e.g., a Western blot). In other embodiments, the antibody may be attached to a solid surface and the polypeptide may be in solution (e.g., affinity chromatography).

[0231] Antibodies to the polypeptides encoded by the ORFs of a collection may be used to determine the presence, absence or amount of one or more of the polypeptides in a sample (e.g., a patient-derived sample). The amount of specifically bound polypeptide may be determined using an antibody to which is attached a label or other marker, such as a radioactive, a fluorescent, or an enzymatic label. Alternatively, a labeled secondary antibody (e.g., an antibody that recognizes the antibody that is specific to the polypeptide) may be used to detect a polypeptide-antibody complex between the specific antibody and the polypeptide.

[0232] cDNA and cDNA Libraries

[0233] In some embodiments, the present invention provides cDNA molecules and/or cDNA libraries.

[0234] In some embodiments, the present invention provides a collection of clones comprising all, substantially all, a majority, or a representative number of clones of a cDNA library. Clones of a cDNA library may be provided as full length clones, i.e., as DNA copies of the mRNAs, or may only contain the sequence corresponding to the ORF, i.e., from the start codon to the stop codon. As discussed above, clones containing an ORF may be provided with or without a stop codon and with or without one or more tag sequences.

[0235] cDNA and/or cDNA libraries can be prepared from any prokaryotic or eukaryotic cells, tissues and/or organs. The cells, tissues and/or organs may be normal, diseased, transformed, established, progenitors, precursors, fetal or embryonic. Diseased cells may, for example, include those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) or parasites), in genetic or biochemical pathologies (e.g., cystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy or multiple sclerosis) or in cancerous processes. Transformed or established animal cell lines may include, for example, COS cells, CHO cells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293 cells, L929 cells, F9 cells, and the like.

[0236] cDNA libraries of the invention may be normalized. A normalized library is a library that has been produced such that all or substantially all of the members of the library can be isolated with approximately equal probability. Suitable examples of normalized libraries and method of making such libraries may be found in U.S. Pat. No. 6,399,334, which is specifically incorporated herein by reference.

[0237] Kits

[0238] In another aspect, the invention provides kits that may be used in conjunction with the invention. Kits according to this aspect of the invention may comprise one or more containers, which may contain one or more components selected from the group consisting of one or more nucleic acid molecules (e.g., one or more vectors comprising a selectable marker, one or more vectors comprising one or more recombination sites and/or functional sequences, and the like) and/or clones comprising nucleic acid sequences of interest (e.g., sequences encoding ORFs, RNAi, ribozymes, etc.), one or more primers, one or more polymerases, one or more reverse transcriptases, one or more recombination proteins (or other enzymes for carrying out the methods of the invention), one or more buffers, one or more detergents, one or more restriction endonucleases, one or more nucleotides, one or more terminating agents (e.g., ddNTPs), one or more transfection reagents, pyrophosphatase, and the like. In some embodiments, kits of the invention may comprise a plurality of clones of the invention wherein each clone is in a different container. In some embodiments of this type, a kit may comprise a plurality of clones, each of which is separately contained in a well of a 96-well plate.

[0239] A wide variety of nucleic acid molecules and/or clones comprising nucleic acid sequences of interest (e.g., sequences encoding ORFs, RNAi, ribozymes, etc.) can be used with the invention. Further, when nucleic acid sequences of interest are provided with flanking recombination sites, these sequences can be combined with a wide range of other nucleic acid molecules comprising recombination sites (e.g., vectors, genomic DNA, etc) in wide range of ways. Examples of nucleic acid molecules that can be supplied in kits of the invention include those that contain functional sequences such as promoters, signal peptides, enhancers, repressors, selection markers, transcription signals, translation signals, primer hybridization sites (e.g., for sequencing or PCR), recombination sites, restriction sites and polylinkers, sites that suppress the termination of translation in the presence of a suppressor tRNA, suppressor tRNA coding sequences, sequences that encode domains and/or regions (e.g., 6 His tag) for the preparation of fusion proteins, origins of replication, telomeres, centromeres, and the like.

[0240] Similarly, collections and/or libraries can be supplied in kits of the invention. These collections and/or libraries may be in the form of replicable nucleic acid molecules or they may comprise nucleic acid molecules that are not associated with an origin of replication. As one skilled in the art would recognize, the nucleic acid molecules of libraries, as well as other nucleic acid molecules that are not associated with an origin of replication, either could be inserted into other nucleic acid molecules that have an origin of replication or would be an expendable kit components.

[0241] Further, in some embodiments, collections and/or libraries supplied in kits of the invention may comprise two components: (1) the nucleic acid molecules of these collections and/or libraries and (2) 5′ and/or 3′ recombination sites and/or topoisomerase recognition sites. In some embodiments, when the nucleic acid molecules of a collection and/or library are supplied with 5′ and/or 3′ recombination sites, it will be possible to insert these molecules into nucleic acid molecules comprising one or more compatible recombination sites, which also may be supplied as a kit component, using recombination reactions. In other embodiments, recombination sites can be attached to the nucleic acid molecules of the collections and/or libraries before use (e.g., by the use of a ligase, which may also be supplied with the kit). In such cases, nucleic acid molecules that contain recombinatiori sites or primers that can be used to generate recombination sites may be supplied with the kits.

[0242] Nucleic acid molecules to be supplied in kits of the invention (e.g., vectors, clones comprising ORFs, etc.) can vary greatly. In some instances, these molecules will contain an origin of replication, at least one selectable marker, and at least one recombination site. For example, molecules supplied in kits of the invention can have four separate recombination sites that allow for insertion of sequence of interest at two different locations. Other attributes of vectors supplied in kits of the invention are described elsewhere herein.

[0243] In some embodiments, the kits of the invention may comprise a plurality of containers, each container comprising one or more nucleic acid segments comprising a nucleic acid sequence of interest (e.g., sequence encoding an ORF, RNAi, ribozyme, etc.) and/or recombination sites. Segments may be provided with recombination sites such that a series of segments (e.g., two, three, four, five six, seven, eight, nine, ten, etc.) may be combined in order to construct a nucleic acid comprising multiple sequences of interest, which may be the same or different. Segments may be combined in reactions involving two or more segments (e.g., three, four, five, six, seven, eight, nine, ten, etc.). Each segment may be from about 100 bp to about 35 kb in length, or from about 100 bp to about 20 kb in length, or from about 100 bp to about 10 kb in length, or from about 100 bp to about 5 kb in length, or from about 100 bp to about 2.5 kb in length, or from about 100 bp to about 1 kb in length, or from about 100 bp to about 500 bp in length.

[0244] A kit of the present invention may comprise a container containing a nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest (e.g., sequence encoding an ORF, RNAi, ribozyme, etc.) and comprising two recombination sites that do not recombine with each other. The recombination sites may flank a selectable marker that allows selection for or against the presence of the nucleic acid molecule in a host cell or identification of a host cell containing or not containing the nucleic acid. A nucleic acid molecule to be included in a kit may comprise more than two recombination sites, for example, a nucleic acid molecule may comprise multiple pairs of recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten, etc.) where members of a pair of recombination sites do not recombine or substantially recombine with each other. In some embodiments, members of one pair of recombination sites do not recombine with members of another pair present in the same nucleic acid molecule.

[0245] Kits of the invention may comprise containers containing one or more recombination proteins. Suitable recombination proteins have been disclosed above and include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, Cin, Tn3 resolvase, ΦC31, TndX, XerC, and XerD.

[0246] Kits of the invention may also comprise one or more topoisomerase proteins and/or one or more nucleic acids comprising one or more topoisomerase recognition sequence. Suitable topoisomerases include Type IA topoisomerases, Type IB topoisomerases and/or Type II topoisomerases. Suitable topoisomerases include, but are not limited to, poxvirus topoisomerases, including vaccinia virus DNA topoisomerase I, E. coli topoisomerase III, E. coli topoisomerase I, topoisomerase III, eukaryotic topoisomerase II, archeal reverse gyrase, yeast topoisomerase III, Drosophila topoisomerase III, human topoisomerase III, Streptococcus pneumoniae topoisomerase III, bacterial gyrase, bacterial DNA topoisorerase IV, eukaryotic DNA topoisomerase II, and T-even phage encoded DNA topoisomerases, and the like. Suitable recognition sequences have been described above.

[0247] In use, a nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest, which may be provided in a kit of the invention, may be combined with a nucleic acid molecule comprising a functional sequence (e.g., using recombinational cloning, topoisomerase-mediated cloning, etc.). The nucleic acid molecule comprising all or a nucleic acid sequence of interest may be provided, for example, with two recombination sites that do not recombine with each other. The nucleic acid molecule comprising a functional sequence may also be provided with two recombination sites, each of which is capable of recombining with one of the two sites present on the a nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest. In the presence of the appropriate recombination proteins, the nucleic acid molecule comprising a functional sequence recombines the nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest in order to form a recombinant nucleic acid molecule containing the functional sequence and all or a portion of a nucleic acid sequence of interest. In embodiments of this type, the functional sequence may become operably linked to the nucleic acid sequence of interest as a result of the recombination reaction. When the nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest comprises multiple pairs of recombination sites, multiple nucleic acid molecules comprising functional sequences and/or other sequences of interest, which may be the same or different, may be combined with the nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest in order to form a nucleic acid molecule comprising all or a portion of a nucleic acid sequence of interest and also comprising multiple functional sequences and/or multiple sequences of interest. In such embodiments, some or all of the functional sequences and/or other sequences of interest may be operably linked to one or more nucleic acid sequences of interest or portion thereof.

[0248] Kits of the invention can also be supplied with primers. These primers will generally be designed to anneal to molecules having specific nucleotide sequences. For example, these primers can be designed for use in PCR to amplify a particular nucleic acid molecule. Further, primers supplied with kits of the invention can be sequencing primers designed to hybridize to vector sequences. Thus, such primers will generally be supplied as part of a kit for sequencing nucleic acid molecules that have been inserted into a vector.

[0249] One or more buffers (e.g., one, two, three, four, five, eight, ten, fifteen) may be supplied in kits of the invention. These buffers may be supplied at a working concentrations or may be supplied in concentrated form and then diluted to the working concentrations. These buffers will often contain salt, metal ions, co-factors, metal ion chelating agents, etc. for the enhancement of activities of the stabilization of either the buffer itself or molecules in the buffer. Further, these buffers may be supplied in dried or aqueous forms. When buffers are supplied in a dried form, they will generally be dissolved in water prior to use.

[0250] Kits of the invention may contain virtually any combination of the components set out above or described elsewhere herein. As one skilled in the art would recognize, the components supplied with kits of the invention will vary with the intended use for the kits. Thus, kits may be designed to perform various functions set out in this application and the components of such kits will vary accordingly.

[0251] Kits of the invention may comprise one or more pages of written instructions for carrying out the methods of the invention. For example, instructions may comprise methods steps necessary to carryout recombinational cloning of an ORF provided with recombination sites and a vector also comprising recombination sites and optionally further comprising one or more functional sequences.

6. Detailed Exemplary Services Description

[0252] The present invention provides numerous services of value to business in the biotechnology and pharmaceutical fields. With reference to FIG. 11, a clone (e.g., an entry clone) may be prepared. A clone may comprise a nucleic acid sequence of interest to a subscriber, which sequence may be optionally flanked by one or more recognition sites (e.g., recombination sites, topoisomerase sites, etc.). Using recombinational cloning, the nucleic acid sequence of interest may be transferred to a plurality of expression vectors and tested in a plurality of expression systems to identify a suitable system or systems. Factors that may be considered in determining the expression system(s) of choice may include amount and/or activity of the polypeptide, cost per unit of polypeptide produced, and/or length of time required to produce a desired amount of polypeptide.

[0253] After a suitable expression system has been selected, the present invention also provides the service of producing and purifying the polypeptide of interest. This can be done using techniques known in the art including, but not limited to, chromatography, electrophoresis, differential precipitation and the like.

[0254] Purified polypeptide may be used for a variety of purposes. Purified polypeptide may be characterized by any number of methods. For example, crystals may be grown of the polypeptide and the crystal structure determined. This may be useful to identify an active site of a polypeptide, which may then be further used to model compounds to identify those that modulate polypeptide activity. Purified polypeptide may be used directly, for example in assays. Polypeptides also may be used to generate antibodies.

[0255] In some embodiments, clones (e.g., entry clones) containing nucleic acid sequences of interest may be further manipulated to produce vectors that may be used in gene targeting applications. For example, an ORF (with or without additional sequences) may be introduced into a cell and/or organism to produce a recombinant cell and/or organism that expresses the polypeptide encoded by the ORF.

[0256] Construction of Clones and Clone Collections

[0257] Suitable nucleic acid sequences to be cloned and included in a collection may be identified using techniques known in the art. For example, a collection may comprise clones of members of a family of proteins. A collection of clones may comprise nucleic acids that do not encode proteins (e.g., ribozymes, tRNAs, RNAis, etc).

[0258] Suitable sequences (e.g., protein-encoding or otherwise) to be included in a collection may be identified by percentage sequence identity with, for example, a reference sequence. For example, a family may be a set of sequences having a sequence that is at least a specified percentage (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, etc.) identical to a reference sequence.

[0259] By a sequence of interest (e.g., amino acid or nucleotide) at least, for example, 70% “identical” to a reference sequence, it is intended that the sequence of interest is identical to the reference sequence except that the sequence of interest may include up to 30 alterations per each 100 positions (e.g., amino acids or nucleotides) of the reference sequence.

[0260] In other words, to obtain a protein having an amino acid sequence at least 70% identical to a reference amino acid sequence, up to 30% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 30% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino (N-) and/or carboxy (C-) terminal positions of the reference amino acid sequence and/or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence and/or in one or more contiguous groups within the reference sequence. As a practical matter, whether a given amino acid sequence is, for example, at least 70% identical to the amino acid sequence of a reference protein can be determined conventionally using known computer programs such as the CLUSTAL W program (Thompson, J. D., et al., Nucleic Acids Res. 22:4673-4680 (1994)).

[0261] To obtain a nucleic acid sequence at least 70% identical to a reference nucleic acid sequence, up to 30% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 30% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the 5′-terminal, 3′-terminal and/or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence and/or in one or more contiguous groups within the reference sequence. Percent sequence identity may be determined using a computer program as discussed herein.

[0262] Sequence identity may be determined by comparing a reference sequence or a subsequence of the reference sequence to a test sequence. The reference sequence and the test sequence are optimally aligned over an arbitrary number of residues termed a comparison window. In order to obtain optimal alignment, additions or deletions, such as gaps, may be introduced into the test sequence. The percent sequence identity is determined by determining the number of positions at which the same residue is present in both sequences and dividing the number of matching positions by the total length of the sequences in the comparison window and multiplying by 100 to give the percentage. In addition to the number of matching positions, the number and size of gaps is also considered in calculating the percentage sequence identity.

[0263] Sequence identity is typically determined using computer programs. A representative program is the BLAST (Basic Local Alignment Search Tool) program publicly accessible at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/). This program compares segments in a test sequence to sequences in a database to determine the statistical significance of the matches, then identifies and reports only those matches that are more significant than a threshold level. A suitable version of the BLAST program is one that allows gaps, for example, version 2.X (Altschul, et al., Nucleic Acids Res. 25(17):3389-402, 1997). Standard BLAST programs for searching nucleotide sequences (blastn) or protein (blastp) may be used. Translated query searches in which the query sequence is translated, i.e., from nucleotide sequence to protein (blastx) or from protein to nucleic acid sequence (tbblastn) may also be used as well as queries in which a nucleotide query sequence is translated into protein sequences in all 6 reading frames and then compared to an NCBI nucleotide database which has been translated in all six reading frames (tbblastx).

[0264] Additional suitable programs for identifying ORFs to be included in a collection of a,family of proteins include, but are not limited to, PHI-BLAST (Pattern Hit Initiated BLAST, Zhang, et al., Nucleic Acids Res. 26(17):3986-90, 1998) and PSI-BLAST (Position-Specific Iterated BLAST, Altschul, et al, Nucleic Acids Res. 25(17):3389-402, 1997).

[0265] Programs may be used with default searching parameters. Alternatively, one or more search parameter may be adjusted. Selecting suitable search parameter values is within the abilities of one of ordinary skill in the art.

[0266] Once a suitable nucleic acid molecule comprising the nucleic acid sequence of interest has been identified, the nucleic acid sequence of interest (e.g., ORF) may be prepared from the nucleic acid molecule. In some embodiments, the sequence of interest may be amplified by PCR using primers constructed to contain a sequence corresponding to all or a portion of a recombination site. After amplification, the amplification product may be contacted with one or more recombination proteins and one or more vectors comprising recombination sites to effect insertion of the amplification product into the vector.

[0267] With reference to FIG. 12, a vector used to prepare a clone of the invention may or may not provide one or more sequences that may be operably linked to the sequence of interest. In FIG. 12A, a sequence of interest (Insert) is cloned into a vector. The vector contains an origin of replication and a selectable marker and does not contain any sequences that are operably linked to the Insert. FIG. 12B shows the case where the sequence of interest is cloned into a vector containing one or more transcriptional regulatory sequences (e.g., promoters). Such transcriptional regulatory sequences may be operably linked to the sequence of interest (Insert). The promoter can be used to produce RNA corresponding to the sequence of interest, which may or may not be translated into a polypeptide. FIG. 12C shows the situation where the vector comprises a tag sequence located at the 3′ end of the sequence of interest. The tag sequence is separated from the sequence of interest by a suppressible stop codon. The tag is also followed by a stop codon. Transcription and translation in the absence of a suppressor tRNA results in the expression of a polypeptide having a native C-terminal. Expression of a suppressor tRNA that suppresses the suppressible stop codon results in the expression of a polypeptide containing a C-terminal tag. FIG. 12D shows the case where the vector contains a promoter followed by a tag sequence and an internal ribosome entry site (IRES) operably linked to a sequence of interest (Insert). Transcription from the promoter and translation of the resultant mRNA results in the production of two different polypeptides. Translation starting at the ATG of the tag sequence results in the production of a polypeptide having an N-terminal tag. Translation starting at an ATG in the context of an IRES results in a polypeptide not containing an N-terminal tag sequence. FIG. 12E shows the case where the vector contains the promoter, tag, and IRES structure of FIG. 12D in combination with the suppressible stop codon and tag sequence of FIG. 12C. A tag at the N-terminal (Tag1) may be the same or different as a tag at the C-terminal (Tag2). A construct of this sort permits the expression of native polypeptide when translation is initiated at the IRES and terminated at the suppressible stop codon, an N-terminal tagged protein when translation begins at the ATG of the Tag1 sequence and terminates at the suppressible stop codon, an N- and C-terminal tagged polypeptide when translation begins at the ATG of the Tag1 sequence and termination at the suppressible stop codon is suppressed by the presence of the appropriate suppressor tRNA, and a C-terminal tagged polypeptide when translation is initiated at the IRES and termination at the suppressible stop codon is suppressed by the presence of the appropriate suppressor tRNA. FIG. 12E shows the case when the vector provides a tag sequence that may be operably linked to the sequence of interest. In embodiments of this type, the sequence of interest may or may not contain a promoter.

[0268] Recognition sites (e.g., recombination sites, topoisomerase recognition sites, restriction enzyme recognition sites, etc.) may be provided at one or both ends of any one or more of the segments of the vectors identified in FIGS. 12A-F (e.g., promoter, Insert, Tag1, Tag2, ori, IRES, and/or suppressible stop codon). When more than one recombination sites are provided, they may have the same or different specificities. Vectors used to prepare clones and/or collections of clones may be any vector that can be used for molecular cloning and/or expression, including, but not limited to, plasmids, cosmids, phagemids, BACs, YACS, baculoviruses, adenovirus, and the like

[0269] In some embodiments, the present invention provides the service of constructing a clone comprising the entire coding sequence of an open reading frame. A customer may have a portion of a sequence of interest, for example, may have the sequence of a proteolytic fragment of a polypeptide of interest. Using the sequence information provided by the customer, a sequence corresponding to the full-length coding sequence can be obtained and used to construct a clone of the invention.

[0270] In some embodiments, the present invention provides the service of constructing a clone comprising a sequence corresponding to the full-length of an mRNA molecule. For example, an mRNA molecule may be identified by a customer, for example, by providing a sequence of the polypeptide encoded by the mRNA. Using techniques known in the art, for example, 5′-RACE, a cDNA molecule corresponding to the full-length of the mRNA (including 5′ and/or 3′-un-translated regions) may be obtained and used to construct a clone of the invention. Any method known in the art may be used to construct the full length clones of the invention.

Protein Expression Services

[0271] Expression of Polypeptides

[0272] In some embodiments, the present invention provides the service of optimizing the expression of a polypeptide for a subscriber. In addition, the invention contemplates the construction of a panel of expression vectors comprising the ORF of a polypeptide.

[0273] To optimize expression of the polypeptides of the present invention, inducible or constitutive promoters may be used to express high levels of a polypeptide in a recombinant host. Similarly, high copy number vectors, well known in the art, may be used to achieve high levels of expression. Vectors having an inducible high copy number may also be useful to enhance expression of the polypeptides of the invention in a recombinant host.

[0274] To express the desired polypeptide in a prokaryotic cell (such as, E. coli, B. subtilis, Pseudomonas, etc.), it is necessary to operably link the ORF encoding the polypeptide to a functional prokaryotic promoter. Such promoters may be used to enhance expression and may either be constitutive or regulatable (i.e., inducible or derepressible) promoters. Examples of constitutive promoters include the int promoter of bacteriophage λ, and the bla promoter of the β-lactamase gene of pBR322. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (PR and PL), trp, recA, lacZ, lacI, tet, gal, trc, and tac promoters of E. coli. The B. subtilis promoters include α-amylase (Ulmanen, et al., J Bacteriol 162:176-182 (1985)) and Bacillus bacteriophage promoters (Gryczan, T., In: The Molecular Biology Of Bacilli, Academic Press, New York (1982)). Streptomyces promoters are described by Ward, et al., Mol. Gen. Genet. 203:468478 (1986)). Prokaryotic promoters are also reviewed by Glick, J. Ind. Microbiol. 1:277-282 (1987); Cenatiempto, Y., Biochimie 68:505-516 (1986); and Gottesman, Ann. Rev. Genet. 18:415-442 (1984). Expression in a prokaryotic cell also requires the presence of a ribosomal binding site upstream of the gene-encoding sequence. Such ribosomal binding sites are disclosed, for example, by Gold, et al., Ann. Rev. Microbiol. 35:365404 (1981).

[0275] To enhance the expression of polypeptides of the invention in a eukaryotic cell, well known eukaryotic promoters and hosts may be used. Suitable promoters include, for example, the cytomegalovirus promoter, the gal 10 promoter and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter.

[0276] Examples of eukaryotic hosts suitable for use with the present invention include fungal cells (e.g., Saccharomyces cerevisiae cells, Pichia pastoris cells, etc.), plant cells, and animal (e.g., insect and mammalian) cells (e.g., Drosophila melanogaster cells, Spodoptera frugiperda Sf9 and Sf21 cells, Trichoplusa High-Five cells, C. elegans cells, Xenopus laevis cells, CHO cells, COS cells, VERO cells, BHK cells, Hela cells, 293 cells, etc.).

[0277] Those skilled in the art will appreciate that each organism has preferred codons for each amino acid. Thus, the present invention contemplates optimizing the codon usage to comport with the host cell type chosen. A nucleic acid encoding the polypeptide of interest can be constructed so as to contain the codons most commonly used by a particular organism in order to optimize the expression of the polypeptide in the particular organism.

[0278] A polypeptide encoded by a cloned ORF of the present invention is preferably produced by growth in culture of the recombinant host containing and expressing the desired polypeptide. Fragments of a polypeptide encoded by an ORF of the invention are also included in the present invention. Such fragments include proteolytic fragments and fragments having a desired characteristic and/or activity (e.g., antigenic fragments, enzymatically active fragments, etc.).

[0279] Any nutrient that can be assimilated by a host containing a clone comprising an ORF may be added to the culture medium. Optimal culture conditions should be selected case by case according to the strain used and the composition of the culture medium. Antibiotics may also be added to the growth media to insure maintenance of vector DNA containing the desired ORF to be expressed. Media formulations have been described in DSM or ATCC Catalogs and Sambrook et al., In: Molecular Cloning, a Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0280] Recombinant host cells producing polypeptide expressed from a cloned ORF of the invention can be separated from liquid culture, for example, by centrifugation. In general, the collected cells (e.g., eukaryotic or prokaryotic) are dispersed in a suitable buffer, and then broken open by well known procedures (eg., hypotionic lysis, detergent treatment, enzyme treatment, french press, sonication, and the like) to allow extraction of the polypeptide by the buffer solution. After removal of cell debris by ultracentrifugation or centrifugation, the polypeptide can be purified by standard protein purification techniques such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis or the like. Assays to detect the presence of the polypeptide during purification are well known in the art and can be used during conventional biochemical purification methods to determine the presence of the polypeptide.

[0281] The invention also relates to host cells comprising one or more of the vectors and/or nucleic acids molecules of the invention containing one or more nucleic acids of interest (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), particularly those vectors described in detail herein. Representative host cells that may be used according to this aspect of the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Preferred bacterial host cells include Escherichia spp. cells (particularly E. coli cells and most particularly E. coli strains DH10B, Stb12, DH5α, DB3, DB3.1 (preferably E. coli LIBRARY EFFICIENCY® DB3.1™ Competent Cells; Invitrogen Corp., Carlsbad, Calif.), DB4 and DB5 (see U.S. application Ser. No. 09/518,188, filed on Mar. 2, 2000, and U.S. Provisional Application No. 60/122,392, filed on Mar. 2, 1999, the disclosures of which are incorporated by reference herein in their entireties), Bacillus spp. cells (particularly B. subtilis and B. megaterium cells), Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells (particularly S. marcessans cells), Pseudomonas spp. cells (particularly P. aeruginosa cells), and Salmonella spp. cells (particularly S. typhimurium and S. typhi cells). Preferred animal host cells include insect cells (most particularly Drosophila melanogaster cells, Spodoptera frugiperda Sf9 and SJ21 cells and Trichoplusa High-Five,cells), nematode cells (particularly C. elegans cells), avian cells, amphibian cells (particularly Xenopus laevis cells), reptilian cells, and mammalian cells (most particularly NIH3T3, 293, CHO, COS, VERO, BHK and human cells). Preferred yeast host cells include Saccharomyces cerevisiae cells and Pichia pastoris cells. These and other suitable host cells are available commercially, for example, from Invitrogen Corp., (Carlsbad, Calif.), American Type Culture Collection (Manassas, Va.), and Agricultural Research Culture Collection (NRRL; Peoria, Ill.).

[0282] Methods for introducing the vectors and/or nucleic acids molecules of the invention into the host cells described herein, to produce host cells comprising one or more of the vectors and/or nucleic acids molecules of the invention, will be familiar to those of ordinary skill in the art. For instance, the nucleic acid molecules and/or vectors of the invention may be introduced into host cells using well known techniques of infection, transduction, electroporation, transfection, and transformation. The nucleic acid molecules and/or vectors of the invention may be introduced alone or in conjunction with other nucleic acid molecules and/or vectors and/or proteins, peptides or RNAs. Alternatively, the nucleic acid molecules and/or vectors of the invention may be introduced into host cells as a precipitate, such as a calcium phosphate precipitate, or in a complex with a lipid. Electroporation also may be used to introduce the nucleic acid molecules and/or vectors of the invention into a host. Likewise, such molecules may be introduced into chemically competent cells such as E. coli. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells. Thus nucleic acid molecules of the invention may contain and/or encode one or more packaging signal (e.g., viral packaging signals that direct the packaging of viral nucleic acid molecules). Hence, a wide variety of techniques suitable for introducing the nucleic acid molecules and/or vectors of the invention into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art. Such techniques are reviewed at length, for example, in Sambrook, J., et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 16.30-16.55 (1989), Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W. H. Freeman and Co., pp. 213-234 (1992), and Winnacker, E.-L., From Genes to Clone, New York: VCH Publishers (1987), which are illustrative of the many laboratory manuals that detail these techniques and which are incorporated by reference herein in their entireties for their relevant disclosures.

[0283] The present invention also provides the option of producing a polypeptide with a tag sequence from the same clone used to produce the un-tagged polypeptide by suppressing one or more stop codons present in the clone. Mutant tRNA molecules that recognize what are ordinarily stop codons suppress the termination of translation of an mRNA molecule and are termed suppressor tRNAs. Three codons are used by both eukaryotes and prokaryotes to signal the end of gene. When transcribed into mRNA, the codons have the following sequences: UAG (amber), UGA (opal) and UAA (ochre). Under most circumstances, the cell does not contain any tRNA molecules that recognize these codons. Thus, when a ribosome translating an mRNA reaches one of these codons, the ribosome stalls and falls off the RNA, terminating translation of the mRNA. The release of the ribosome from the mRNA is mediated by specific factors (see S. Mottagui-Tabar, Nucleic Acids Research 26(11), 2789, 1998). A gene with an in-frame stop codon (TAA, TAG, or TGA) will ordinarily encode a protein with a native carboxy terminus. However, suppressor tRNAs, can result in the insertion of amino acids and continuation of translation past stop codons.

[0284] A number of such suppressor tRNAs have been found. Examples include, but are not limited to, the supE, supP, supD, supF and supZ suppressors, which suppress the termination of translation of the amber stop codon, supb, gIT, supL, supN, supC and supM suppressors, which suppress the function of the ochre stop codon and glyT, trpT and Su-9 suppressors, which suppress the function of the opal stop codon. In general, suppressor tRNAs contain one or more mutations in the anti-codon loop of the tRNA that allows the tRNA to base pair with a codon that ordinarily functions as a stop codon. The mutant tRNA is charged with its cognate amino acid residue and the cognate amino acid residue is inserted into the translating polypeptide when the stop codon is encountered. For a more detailed discussion of suppressor tRNAs, the reader may consult Eggertsson, et al., (1988) Microbiological Review 52(3):354-374, and Engleerg-Kukla, et al. (1996) in Escherichia coli and Salmonella Cellular and Molecular Biology, Chapter 60, pps 909-921, Neidhardt, et al. eds., ASM Press, Washington, D.C.

[0285] Mutations that enhance the efficiency of termination suppressors, i.e., increase the read through of the stop codon, have been identified. These include, but are not limited to, mutations in the uar gene (also known as the prfA gene), mutations in the ups gene, mutations in the sueA, sueB and sueC genes, mutations in the rpsD (ramA) and rpsE (spcA) genes and mutations in the rplL gene. Suppression in some organisms (e.g., E. coli) may be improved when the stop codon is followed immediately by the nucleotide adenosine. Thus, the present invention contemplates nucleic acid sequences comprising stop codons followed by adenosine (e.g., comprising the sequences TAGA, TAAA and/or TGAA).

[0286] Under ordinary circumstances, host cells would not be expected to be healthy if suppression of stop codons is too efficient. This is because of the thousands or tens of thousands of genes in a genome, a significant fraction will naturally have one of the three stop codons; complete read-through of these would result in a large number of aberrant proteins containing additional amino acids at their carboxy termini. If some level of suppressing tRNA is present, there is a race between the incorporation of the amino acid and the release of the ribosome. Higher levels of tRNA may lead to more read-through although other factors, such as the codon context, can influence the efficiency of suppression.

[0287] Organisms ordinarily have multiple genes for tRNAs. Combined with the redundancy of the genetic code (multiple codons for many of the amino acids), mutation of one tRNA gene to a suppressor tRNA status does not lead to high levels of suppression. The TAA stop codon is the strongest, and most difficult to suppress. The TGA is the weakest, and naturally (in E. coli) leaks to the extent of 3%. The TAG (amber) codon is relatively tight, with a read-through of ˜1% without suppression. In addition, the amber codon can be suppressed with efficiencies on the order of 50% with naturally occurring suppressor mutants.

[0288] Suppression has been studied for decades in bacteria and bacteriophages. In addition, suppression is known in yeast, flies, plants and other eukaryotic cells including mammalian cells. For example, Capone, et al. (Molecular and Cellular Biology 6(9):3059-3067, 1986) demonstrated that suppressor tRNAs derived from mammalian tRNAs could be used to suppress a stop codon in mammalian cells. A copy of the E. coli chloramphenicol acetyltransferase (cat) gene having a stop codon in place of the codon for serine 27 was transfected into mammalian cells along with a gene encoding a human serine tRNA that had been mutated to form an amber, ochre, or opal suppressor derivative of the gene. Successful expression of the cat gene was observed. An inducible mammalian amber suppressor has been used to suppress a mutation in the replicase gene of polio virus and cell lines expressing the suppressor were successfully used to propagate the mutated virus (Sedivy, et al., Cell 50: 379-389 (1987)). The context effects on the efficiency of suppression of stop codons by suppressor tRNAs has been shown to be different in mammalian cells as compared to E. coli (Phillips-Jones, et al., Molecular and Cellular Biology 15(12): 6593-6600 (1995), Martin, et al., Biochemical Society Transactions 21: (1993)) Since some human diseases are caused by nonsense mutations in essential genes, the potential of suppression for gene therapy has long been recognized (see Temple, et al., Nature 296(5857):537-40 (1982)). The suppression of single and double nonsense mutations introduced into the diphtheria toxin A-gene has been used as the basis of a binary system for toxin gene therapy (Robinson, et al., Human Gene Therapy 6:137-143 (1995)).

[0289] The present invention contemplates fusion polypeptides wherein a portion of the fusion protein is translated from an mRNA sequence that is 3′-to at least one stop codon. In general terms, a gene may be expressed in four forms: native at both amino and carboxy termini, modified at either end, or modified at both ends. A construct containing an ORF of interest may include the N-terminal methionine ATG codon, and a stop codon at the carboxy end, of the open reading frame, or ORF, thus ATG-ORF-stop. Frequently, a gene construct will include translation initiation sequences, tis, that may be located upstream of the ATG that allow expression of the ORF, thus tis-ATG-ORF-stop. Constructs of this sort allow expression of a gene as a protein that contains the same amino and carboxy amino acids as in the native, uncloned, protein. When such a construct is fused in-frame with an amino-terminal protein tag, e.g., GST, the tag will have its own tis, thus tis-ATG-tag-tis-ATG-ORF-stop, and the bases comprising the tis of the ORF will be translated into amino acids between the tag and the ORF. In addition, some level of translation initiation may be expected in the interior of the mRNA (i.e., at the ORF's ATG and not the tag's ATG) resulting in a certain amount of native protein expression contaminating the desired protein.

[0290] DNA (lower case): tis1-atg-tag-tis2-atg-orf-stop

[0291] RNA (lower case, italics): tis1-atg-tag-tis2-atg-orf-stop

[0292] Protein (upper case): ATG-TAG-TIS2-ATG-ORF (tis1 and stop are not translated)+contaminating ATG-ORF (translation of ORF beginning at tis2).

[0293] Using one or more of the cloning techniques described herein (e.g., recombinational cloning, topoisomerase-mediated cloning, etc.) it is a simple matter for those skilled in the art to construct a vector containing a tag adjacent to a recombination site permitting the in frame fusion of a tag to the C- and/or N-terminus of the ORF of interest.

[0294] Given the ability to rapidly create a number of clones in a variety of vectors, there is a need in the art to maximize the number of ways a single cloned ORF can be expressed without the need to manipulate the ORF-containing clone itself. The present invention meets this need by providing materials and methods for the controlled expression of a C- and/or N-terminal fusion to a target ORF using one or more suppressor tRNAs to suppress the termination of translation at a stop codon. Thus, the present invention provides materials and methods in which an ORF-containing clone is prepared such that the ORF is flanked with recombination sites.

[0295] The construct may be prepared with a sequence coding for a stop codon preferably at the C-terminus of the ORF of interest. In some embodiments, a stop codon can be located adjacent to the ORF, for example, within a recombination site flanking the ORF or at or near the 3′ end of the sequence of the ORF before a recombination site. The ORF construct can be transferred through recombination to various vectors that can provide various C-terminal or N-terminal tags (e.g., GFP, GST, His Tag, GUS, etc.) to the ORF of interest. When the stop codon is located at the carboxy terminus of the ORF, expression of the corresponding polypeptide with a “native” carboxy end amino acid sequence occurs under non-suppressing conditions (i.e., when the suppressor tRNA is not expressed) while expression of the polypeptide as a carboxy fusion protein occurs under suppressing conditions. Those skilled in the art will recognize that any suppressors and any stop codons could be used in the practice of the present invention.

[0296] In some embodiments, the gene coding for the suppressing tRNA may be incorporated into the vector from which the ORF of interest is to be expressed. In other embodiments, the gene for the suppressor tRNA may be in the genome of the host cell. In still other embodiments, the gene for the suppressor may be located on a separate other vector—i.e., plasmid, cosmid, virus, etc.—and provided in trans.

[0297] More than one copy of a gene encoding a suppressor tRNA may be provided in all of the embodiments described herein. For example, a host cell may be provided that contains multiple copies of a gene encoding the suppressor tRNA. Alternatively, multiple gene copies of the suppressor tRNA under the same or different promoters may be provided in the same vector background as the target gene of interest. In some embodiments, multiple copies of a suppressor tRNA may be provided in a different vector than the one containing the target gene of interest. In other embodiments, one or more copies of the suppressor tRNA gene may be provided on the vector containing the ORF of the polypeptide of interest and/or on another vector and/or in the genome of the host cell or in combinations of the above. When more than one copy of a suppressor tRNA gene is provided, the genes may be expressed from the same or different promoters that may be the same or different as the promoter used to express the ORF encoding the polypeptide of interest.

[0298] In some embodiments, two or more different suppressor tRNA genes may be provided. In embodiments of this type one or more of the individual suppressors may be provided in multiple copies and the number of copies of a particular suppressor tRNA gene may be the same or different as the number of copies of another suppressor tRNA gene. Each suppressor tRNA gene, independently of any other suppressor tRNA gene, may be provided on the vector used to express the ORF of interest and/or on a different vector and/or in the genome of the host cell. A given tRNA gene may be provided in more than one place in some embodiments. For example, a copy of the suppressor tRNA may be provided on the vector containing the ORF of interest while one or more additional copies may be provided on an additional vector and/or in the genome of the host cell. When more than one copy of a suppressor tRNA gene is provided, the genes may be expressed from the same or different promoters that may be the same or different as the promoter used to express the gene encoding the protein of interest and may be the same or different as a promoter used to express a different tRNA gene.

[0299] In some embodiments of the present invention, the ORF of interest and the gene expressing the suppressor tRNA may be controlled by the same promoter. In other embodiments, the ORF of interest may be expressed from a different promoter than the suppressor tRNA. Those skilled in the art will appreciate that, under certain circumstances, it may be desirable to control the expression of the suppressor tRNA and/or the ORF of interest using a regulatable promoter. For example, either the ORF of interest and/or the gene expressing the suppressor tRNA may be controlled by a promoter such as the lac promoter or derivatives thereof such as the tac promoter. In some embodiments, both the ORF of interest and the suppressor tRNA gene are expressed from the T7 RNA polymerase promoter and, optionally, are expressed as part of one RNA molecule. In embodiments of this type, the portion of the RNA corresponding to the suppressor tRNA is processed from the originally transcribed RNA molecule by cellular factors.

[0300] In some embodiments, the expression of the suppressor tRNA gene may be under the control of a different promoter from that of the ORF of interest. In some embodiments, it may be possible to express the suppressor gene before the expression of the ORF. This would allow levels of suppressor to build up to a high level, before they are needed to allow expression of a fusion protein by suppression of a the stop codon. For example, in embodiments of the invention where the suppressor gene is controlled by a promoter inducible with IPTG, the ORF may be controlled by the T7 RNA polymerase promoter and the expression of the T7 RNA polymerase may controlled by a promoter inducible with an inducing signal other than IPTG, e.g., NaCl, one could turn on expression of the suppressor tRNA gene with IPTG prior to the induction of the T7 RNA polymerase gene and subsequent expression of the ORF of interest. In some embodiments, the expression of the suppressor tRNA might be induced about 15 minutes to about one hour before the induction of the T7 RNA polymerase gene. In one embodiment, the expression of the suppressor tRNA may be induced from about 15 minutes to about 30 minutes before induction of the T7 RNA polymerase gene. In some embodiments, the expression of the T7 RNA polymerase gene is under the control of an inducible promoter.

[0301] In additional embodiments, the expression of the ORF of interest and the suppressor tRNA can be arranged in the form of a feedback loop. For example, the ORF of interest may be placed under the control of the T7 RNA polymerase promoter while the suppressor gene is under the control of both the T7 promoter and the lac promoter. The T7 RNA polymerase gene itself is also under the control of both the T7 promoter and the lac promoter. In addition, the T7 RNA polymerase gene has an amber stop mutation replacing a normal tyrosine codon, e.g., the 28th codon (out of 883). No active T7 RNA polyymerase can be made before levels of suppressor are high enough to give significant suppression. Then expression of the polymerase rapidly rises, because the T7 polymerase expresses the suppressor gene as well as itself. In other preferred embodiments, only the suppressor gene is expressed from the T7 RNA polymerase promoter. Embodiments of this type would give a high level of suppressor without producing an excess amount of T7 RNA polymerase. In other preferred embodiments, the T7 RNA polymerase gene has more than one amber stop mutation. This will require higher levels of suppressor before active T7 RNA polymerase is produced.

[0302] In some embodiments of the present invention it may be desirable to have more than one stop codon suppressible by more than one suppressor tRNA. A recombinant vector may be constructed so as to permit the regulatable expression of N- and/or C-terminal fusions of a polypeptide expressed from an ORF of interest from the same construct. A vector may comprise a first tag sequence expressed from a promoter and may include a first stop codon in the same reading frame as the tag. The stop codon may be located anywhere in the tag sequence and is preferably located at or near the C-terminal of the tag sequence. The stop codon may also be located in a recombination site or in an internal ribosome entry sequence (IRES). The vector may also include an ORF of interest that includes a second stop codon. The first tag and the ORF of interest are preferably in the same reading frame although inclusion of a sequence that causes frame shifting to bring the first tag into the same reading frame as the ORF of interest is within the scope of the present invention. The second stop codon is preferably in the same reading frame as the ORF of interest and is preferably located at or near the end of the coding sequence of the ORF. The second stop codon may optionally be located within a recombination site located 3′ to the ORF of interest. The construct may also include a second tag sequence in the same reading frame as the ORF of interest and the second tag sequence may optionally include a third stop codon in the same reading frame as the second tag. A transcription terminator and/or a polyadenylation sequence may be included in the construct after the coding sequence of the second tag. The first, second and third stop codons may be the same or different. In some embodiments, all three stop codons are different. In embodiments where the first and the second stop codons are different, the same construct may be used to express an N-terminal fusion, a C-terminal fusion and the native protein by varying the expression of the appropriate suppressor tRNA. For example, to express the native protein, no suppressor tRNAs are expressed and protein translation is controlled by an appropriately located IRES. When an N-terminal fusion is desired, a suppressor tRNA that suppresses the first stop codon is expressed while a suppressor tRNA that suppresses the second stop codon is expressed in-order to produce a C-terminal fusion. In some instances it may be desirable to express a doubly tagged protein of interest in which case suppressor tRNAs that suppress both the first and the second stop codons may be expressed.

[0303] Antibody Production Services

[0304] One or more of the polypeptides encoded by the ORFs of a collection may be used as immunogens to prepare polyclonal an/or monoclonal antibodies capable of binding the polypeptides using techniques well known in the art (Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988). In brief, antibodies are prepared by immunization of suitable subjects (e.g., mice, rats, rabbits, goats, etc.) with all or a part of the polypeptides of the invention. If the polypeptide or fragment thereof is sufficiently immunogenic, it may be used to immunize the subject. If necessary or desired to increase immunogenicity, the polypeptide or fragment may be conjugated to a suitable carrier molecule (e.g., BSA, KLH, and the like). Polypeptides of the invention or fragments thereof may be conjugated to carriers using techniques well known in the art. For example, they may be directly conjugated to a carrier using, for example, carbodiimide reagents. Other suitable linking reagents are commercially available from, for example, Pierce Chemical Co., Rockford, Ill.

[0305] Suitably prepared polypeptides of the invention or fragments thereof may be administered by injection over a suitable time period. They may be administered with or without the use of an adjuvant (e.g., Freunds). They may be administered one or more times until antibody titers reach a desired level.

[0306] In some embodiments, it may be desirable to produce monoclonal antibodies to the, polypeptides of the invention or fragments thereof. Immortalized cell lines that produce the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or other techniques well known in the art. Cells producing the desired monoclonal antibody can be cultured either in vitro or by production in ascites fluid.

[0307] In some embodiments, it may be desirable to use a fragment of an antibody that is capable of binding a polypeptide of the invention or fragment thereof. For example, Fab, Fab′, of F(ab′)2 fragments may be produced using techniques well known in the art.

[0308] Construction of cDNA Libraries

[0309] In some embodiments, the present invention provides the service of preparing cDNA molecules and cDNA libraries for a subscriber. Such cDNAs and cDNA libraries may be prepared for any cell or tissue source.

[0310] In accordance with the invention, cDNA molecules (single-stranded or double-stranded) may be prepared, from a variety of nucleic acid template molecules. Preferred nucleic acid molecules for use in the present invention include single-stranded or double-stranded DNA and RNA molecules, as well as double-stranded DNA:RNA hybrids. More preferred nucleic acid molecules include messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, although mRNA molecules are the preferred template according to the invention.

[0311] The nucleic acid molecules that are used to prepare cDNA molecules according to the methods of the present invention may be prepared synthetically, according to standard organic chemical synthesis methods that will be familiar to one of ordinary skill. More preferably, the nucleic acid molecules may be obtained from natural sources, such as a variety of cells, tissues, organs or organisms. Cells that may be used as sources of nucleic acid molecules may be prokaryotic (bacterial cells, including but not limited to those of species of the genera Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium, Xanthomonas and Streptomyces) or eukaryotic (including fungi (especially yeasts), plants, protozoans and other parasites, and animals including insects (particularly Drosophila spp. cells), nematodes (particularly Caenorhabditis elegans cells), and mammals (particularly human cells)).

[0312] Mammalian somatic cells that may be used as sources of nucleic acids include blood cells (reticulocytes and leukocytes), endothelial cells, epithelial cells, neuronal cells (from the central or peripheral nervous systems), muscle cells (including myocytes and myoblasts from skeletal, smooth or cardiac muscle), connective tissue cells (including fibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes and osteoblasts) and other stromal cells (e.g., macrophages, dendritic cells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes) may also be used as sources of nucleic acids for use in the invention, as may the progenitors, precursors and stem cells that give rise to the above somatic and germ cells. Also suitable for use as nucleic acid sources are mammalian tissues or organs such as those derived from brain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous, skin, genitourinary, circulatory, lymphoid, gastrointestinal and connective tissue sources, as well as those derived from a mammalian (including human) embryo or fetus.

[0313] Any of the above prokaryotic or eukaryotic cells, tissues and organs may be normal, diseased, transformed, established, progenitors, precursors, fetal or embryonic. Diseased cells may, for example, include those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) or parasites), in genetic or biochemical pathologies (e.g., cystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy or/multiple sclerosis) or in cancerous processes. Transformed or established animal cell lines may include, for example, COS cells, CHO cells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293 cells, L929 cells, F9 cells, and the like. Other cells, cell lines, tissues, organs and organisms suitable as sources of nucleic acids for use in the present invention will be apparent to one of ordinary skill in the art.

[0314] Once the starting cells, tissues, organs or other samples are obtained, nucleic acid molecules (such as mRNA) may be isolated therefrom by methods that are well-known in the art (See, e.g., Maniatis, T., et al., Cell 15:687-701 (1978); Okayama, H., and Berg, P., Mol. Cell. Biol. 2:161-170 (1982); Gubler, U., and Hoffman, B. J., Gene 25:263-269 (1983)). The nucleic acid molecules thus isolated may then be used to prepare cDNA molecules and cDNA libraries in accordance with the present invention.

[0315] In the practice of the invention, cDNA molecules or cDNA libraries are produced by mixing one or more nucleic acid molecules obtained as described above, which is preferably one or more mRNA molecules such as a population of mRNA molecules, with a reverse transcriptase and/or a DNA polymerase under conditions favoring the reverse transcription of the nucleic acid molecule to form a cDNA molecule (single-stranded or double-stranded). Methods of preparing cDNA and cDNA libraries are well known in the art (see, e.g., Gubler, U., and Hoffman, B. J., Gene 25:263-269 (1983); Krug, M. S., and Berger, S. L., Meth. Enzymol. 152:316-325 (1987); Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 8.60-8.63 (1989); WO 99/15702; WO 98/47912; and WO 98/51699). Other methods of cDNA synthesis which may advantageously use the present invention will be readily apparent to one of ordinary skill in the art.

[0316] Methods for generating full-length cDNA molecules are known in the art. For example, U.S. Pat. No. 6,197,554 issued to Lin, et al., discloses a method for preparing a full-length cDNA library from a single cell or a small number of cells suing repeated reverse transcription and amplification steps. U.S. Pat. No. 6,187,544, issued to Bergsma, et al., discloses a method for high throughput cloning of full length cDNA sequences using a plurality of clone arrays prepared from cDNA libraries which have been preferably enriched for 5′ mRNA sequences and size fractionated into several discrete ranges (sub-libraries). U.S. Pat. No. 6,174,669, issued to Hayashizaki, et al., relates to a method for making full-length cDNAs having a length corresponding to full-length mRNAs by binding a tag molecule to a diol structure present in the cap of mRNAs, reverse transcribing the mRNA to make a RNA-DNA hybrid and isolating the RNA-DNA hybrids using the tag molecule.

[0317] In some embodiments, the libraries constructed according to the present invention may be normalized. As discussed above, a normalized library is one that has been constructed so as to reduce the relative variation in abundance among member nucleic acid molecules in the library. In brief, a library may be normalized by reducing the abundance of molecules that are represented at a high level in the library.

[0318] The present invention encompasses methods of preparing normalized libraries and the normalized libraries (i.e., libraries of cloned nucleic acid molecules from which each member nucleic acid molecule can be isolated with approximately equivalent probability) prepared by such methods, clones comprising such members of such libraries, and compositions comprising such clones and/or libraries.

[0319] A normalized library may be produced by synthesizing one or more nucleic acid molecules complementary to all or a portion of the nucleic acid molecules of the library, wherein the synthesized nucleic acid molecules comprise at least one hapten, thereby producing haptenylated nucleic acid molecules (which may be RNA molecules or DNA molecules); incubating a nucleic acid library to be normalized with the haptenylated nucleic acid molecules (e.g. also referred to as driver) under conditions favoring the hybridization of the more highly abundant molecules of the library with the haptenylated nucleic acid molecules; and removing the hybridized molecules, thereby producing a normalized library.

[0320] In some embodiments, the relative concentration of all members of the normalized library are within one to two orders of magnitude. In another aspect, contaminating nucleic acid molecules (e.g., vectors without inserts) are removed from the normalized library. In this manner, all or a substantial portion of the normalized library will comprise vectors containing inserted nucleic acid molecules of the library.

[0321] In some embodiments, a population of mRNA is incubated under conditions sufficient to produce a population of cDNA molecules complementary to all or a portion of said mRNA molecules. Conditions may comprise mixing the population of mRNA molecules with one or more polypeptides having reverse transcriptase activity and incubating the mixture under conditions sufficient to produce a population of single stranded cDNA molecules complementary to all or a portion of the mRNA molecules. The single stranded cDNA molecules may then be used to make double stranded cDNA molecules by incubating the mixture under appropriate conditions in the presence of one or more DNA polymerases. The resulting population of double-stranded or single-stranded cDNA molecules makes up a library that may be normalized using the methods of the invention. Such cDNA libraries may be inserted into one or more vectors prior to normalization. Alternatively, the cDNA libraries may be normalized prior to insertion within one or more vectors, and after normalization may be cloned into one or more vectors.

[0322] The library to be normalized may be contained in (inserted in) one or more vectors, which may be a plasmid, a cosmid, a phagemid, a virus and the like. Such vectors preferably comprise one or more promoters that allow the synthesis of at least one RNA molecule from all or a portion of the nucleic acid molecules (preferably cDNA molecules) inserted in the vector. Thus, by use of the promoters, haptenylated RNA molecules complementary to all or a portion of the nucleic acid molecules of the library may be made and used to normalize the library in accordance with the invention. Such synthesized RNA molecules (which have been haptenylated) will be complementary to all or a portion of the vector inserts of the library. More highly abundant molecules in the library may then be preferentially removed by hybridizing the haptenylated RNA molecules to the library, thereby producing the normalized library of the invention. Without being limited, the synthesized RNA molecules are thought to be representative of the library; that is, more highly abundant species in the library result in more highly abundant haptenylated RNA using the above method. The relative abundance of the molecules within the library, and therefore, within the haptenylated RNA determines the rate of removal of particular species of the library; if a particular species abundance is high, such highly abundant species will be removed more readily while low abundant species will be removed less readily from the population. Normalization by this process thus allows one to substantially equalize the level of each species within the library.

[0323] In another aspect of the invention, the library to be normalized need not be inserted in one or more vectors prior to normalization. In such aspect of the invention, the nucleic acid molecules of the library may be used to synthesize haptenylated nucleic acid molecules using well known techniques. For example, haptenylated nucleic acid molecules may be synthesized in the presence of one or more DNA polymerases, one or more appropriate primers or probes and one or more nucleotides (the nucleotides and/or primers or probes may be haptenylated). In this manner, haptenylated DNA molecules will be produced and may be used to normalized the library in accordance with the invention. Alternatively, one or more promoters may be added to (e.g., ligated, attached using topoisomerase, attached via recombination, etc) the library molecules, thereby allowing synthesis of haptenylated RNA molecules for use to normalize the library in accordance with the invention. For example, adapters containing one or more promoters may be added to one or more ends of double stranded library molecules (e.g., cDNA library prepared from a population of mRNA molecules). Such promoters may then be used to prepare haptenylated RNA molecules complementary to all or a portion of the nucleic acid molecules of the library. In accordance with the invention, the library may then be normalized and, if desired, inserted into one or more vectors.

[0324] While haptenylated RNA is preferably used to normalize libraries, other haptenylated nucleic acid molecules may be used in accordance with the invention. For example, haptenylated DNA may be synthesized from the library and used in accordance with the invention.

[0325] Haptens suitable for use in the methods of the invention include, but are not limited to, avidin, streptavidin, protein A, protein G, a cell-surface Fc receptor, an antibody-specific antigen, an enzyme-specific substrate, polymyxin B, endotoxin-neutralizing protein (ENP), Fe+++, a transferrin receptor, an insulin receptor, a cytokine receptor, CD4, spectrin, fodrin, ICAM-1, ICAM-2, C3bi, fibrinogen, Factor X, ankyrin, an integrin, vitronectin, fibronectin, collagen, laminin, glycophorin, Mac-1, LFA-1, β-actin, gp120, a cytokine, insulin, ferrotransferrin, apotransferrin, lipopolysaccharide, an enzyme, an antibody, biotin and combinations thereof. A particularly preferred hapten is biotin.

[0326] In accordance with the invention, hybridized molecules produced by the above-described methods may be isolated, for example by extraction or by hapten-ligand, interactions. Preferably, extraction methods (e.g. using organic solvents) are used. Isolation by hapten-ligand interactions may be accomplished by incubation of the haptenylated molecules with a solid support comprising at least one ligand that binds the hapten. Preferred ligands for use in such isolation methods correspond to the particular hapten used, and include, but are not limited to, biotin, an antibody, an enzyme, lipopolysaccharide, apotransferrin, ferrotransferrin, insulin, a cytokine, gp120, β-actin, LFA-1, Mac-1, glycophorin, laminin, collagen, fibronectin, vitronectin, an integrin, ankyrin, C3bi, fibrinogen, Factor X, ICAM-1, ICAM-2, spectrin, fodrin, CD4, a cytokine receptor, an insulin receptor, a transferrin receptor, Fe+++, polymyxin B, endotoxin-neutralizing protein (ENP), an enzyme-specific substrate, protein A, protein G, a cell-surface Fc receptor, an antibody-specific antigen, avidin, streptavidin or combinations thereof. The solid support used in these isolation methods may be nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, a latex bead, a magnetic bead, a paramagnetic bead, a superparamagnetic bead or a microtitre plate. Preferred solid supports are magnetic beads, paramagnetic beads and superparamagnetic beads, and particularly preferred are such beads comprising one or more streptavidin or avidin molecules.

[0327] In another aspect of the invention, normalized libraries are subjected to further isolation or selection steps which allow removal of unwanted contamination or background. Such contamination or background may include undesirable nucleic acids. For example, when a library to be normalized is constructed in one or more vectors, a low percentage of vector (without insert) may be present in the library. Upon normalization, such low abundance molecules (e.g. vector background) may become a more significant constituent as a result of the normalization process. That is, the relative level of such low abundance background may be increased as part of the normalization process.

[0328] Removal of such contaminating nucleic acids may be accomplished by incubating a normalized library with one or more haptenylated probes which are specific for the nucleic acid molecules of the library (e.g. target specific probes). In principal, removal of contaminating sequences can be accomplished by selecting those nucleic acids having the sequence of interest or by eliminating those molecules that do not contain sequences of interest. In accordance with the invention, removal of contaminating nucleic acid molecules may be performed on any normalized library (whether or not the library is constructed in a vector). Thus, the probes will be designed such that they will not recognize or hybridize to contaminating nucleic acids. Upon hybridization of the haptenylated probe with nucleic acid molecules of the library, the haptenylated probes will bind to and select desired sequences within the normalized library and leave behind contaminating nucleic acid molecules, resulting in a selected normalized library. The selected normalized library may then be isolated. In a preferred aspect, such isolated selected normalized libraries are single-stranded, and may be made double stranded following selection by incubating the single-stranded library under conditions sufficient to render the nucleic acid molecules double-stranded. The double stranded molecules may then be transformed into one or more host cells. Alternatively, the normalized library may be made double stranded using the haptenylated probe or primer (preferably target specific) and then selected by extraction or ligand-hapten interactions. Such selected double stranded molecules may then be transformed into one or more host cells.

[0329] In another aspect of the invention, contaminating nucleic acids may be reduced or eliminated by incubating the normalized library in the presence of one or more primers specific for library sequences. This aspect of the invention may comprise incubating the single stranded normalized library with one or more nucleotides (preferably nucleotides which confer nuclease resistance to the synthesized nucleic acid molecules), and one or more polypeptides having polymerase activity, under conditions sufficient to render the nucleic acid molecules double-stranded. The resulting double stranded molecules may then be transformed into one or more host cells. Alternatively, resulting double stranded molecules containing nucleotides which confer nuclease resistance may be digested with such a nuclease and transformed into one or more host cells.

[0330] In yet another aspect, the elimination or removal of contaminating nucleic acid may be accomplished prior to normalization of the library, thereby resulting in selected normalized library of the invention. In such a method, the library to be normalized may be subjected to any of the methods described herein to remove unwanted nucleic acid molecules and then the library may then be normalized by the process of the invention to provide for the selected normalized libraries of the invention.

[0331] In accordance with the invention, double stranded nucleic acid molecules are preferably made single stranded before hybridization. Thus, the methods of the invention may further comprise treating the above-described double-stranded nucleic acid molecules of the library under conditions sufficient to render the nucleic acid molecules single-stranded. Such conditions may comprise degradation of one strand of the double-stranded nucleic acid molecules (preferably using gene II protein and Exonuclease III), or denaturing the double-stranded nucleic acid molecules using heat, alkali and the like.

[0332] The invention also relates to normalized nucleic acid libraries, selected normalized nucleic acid libraries and transformed host cells produced by the above-described methods.

[0333] The above-described technique may be used to prepare a normalized library from any organism or tissue source. In some embodiments, normalized libraries may be prepared from tissue of mammalian origin (e.g., human, rat, mouse, dog, etc.). Normalized libraries may be prepared from numerous tissue types from a single organism (e.g., from human heart, lung, liver, kidney, brain, etc.).

[0334] An additional service available in the present invention is the normalization of libraries prepared by a customer. For example, a customer may have previously prepared a library from a particular source. The customer may request that the provider prepare a normalized library from the previously prepared library. The provider may prepare the normalized library using the technique described above or any other suitable technique.

[0335] Research and Development Consulting.

[0336] In some embodiments, the present invention provides the service of analyzing subscriber Research and Development. A provider may provide one or more individuals to a subscriber in order to analyze the methodology used by the subscriber. The individuals may identify portions of the subscriber's Research and Development that might be improved using materials and/or knowledge provided by the provider. For example, a subscriber may, as part of its business, analyze the effects of small molecules on enzymes. The provider may provide improved materials and/or methods to facilitate this type of analysis. For example, the provider may provide improved reaction conditions under which to assay an enzyme of interest. The provider might provide a more suitable assay to assess the effects of the small molecules on enzyme activity than the assay used by the customer.

[0337] It will be understood by one of ordinary skill in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein are readily apparent from the description of the invention contained herein in view of information known to the ordinarily skilled artisan, and may be made without departing from the scope of the invention or any embodiment thereof.

[0338] The entire disclosures of U.S. application Ser. No. 08/486,139, (now abandoned), filed Jun. 7, 1995, U.S. application Ser. No. 08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732), U.S. application Ser. No. 09/233,492, filed Jan. 20, 1999, (now U.S. Pat. No. 6,270,969), U.S. application Ser. No. 09/233,493, filed Jan. 20, 1999, (now U.S. Pat. No. 6,143,557), U.S. application Ser. No. 09/005,476, filed Jan. 12, 1998, (now U.S. Pat. No. 6,171,861), U.S. application Ser. No. 09/432,085 filed Nov. 2, 1999, U.S. application Ser. No. 09/498,074 filed Feb. 4, 2000, U.S. application Ser. No. 60/065,930, filed Oct. 24, 1997, U.S. application Ser. No. 09/177,387, filed Oct. 23, 1998, U.S. application Ser. No. 09/296,280, filed Apr. 22, 1999, (now U.S. Pat. No. 6,277,608), U.S. application Ser. No. 09/296,281, filed Apr. 22, 1999, (now abandoned), U.S. application Ser. No. 09/648,790, filed Aug. 28, 2000, U.S. application Ser. No. 09/855,797, filed May 16, 2001, U.S. application Ser. No. 09/907,719, filed Jul. 19, 2001, U.S. application Ser. No. 09/907,900, filed Jul. 19, 2001, U.S. application Ser. No. 09/985,448, filed Nov. 2, 2001, U.S. application Ser. No. 60/108,324, filed Nov. 13, 1998, U.S. application Ser. No. 09/438,358, filed Nov. 12, 1999, U.S. application Ser. No. 60/161,403, filed Oct. 25, 1999, U.S. application Ser. No. 09/695,065, filed Oct. 25, 2000, U.S. application Ser. No. 09/984,239, filed Oct. 29, 2001, U.S. application Ser. No. 60/122,389, filed March 2, 1999, U.S. application Ser. No. 60/126,049, filed Mar. 23, 1999, U.S. application Ser. No. 60/136,744, filed May 28, 1999, U.S. application Ser. No. 09/517,466, filed March 2, 2000, U.S. application Ser. No. 60/122,392, filed Mar. 2, 1999, U.S. application Ser. No. 09/518,188, filed Mar. 2, 2000, U.S. application Ser. No. 60/169,983, filed December 10, 1999, U.S. application Ser. No. 60/188,000, filed Mar. 9, 2000, U.S. application Ser. No. 09/732,914, filed Dec. 11, 2001, U.S. application Ser. No. 60/284,528, filed Apr. 19, 2001, U.S. application Ser. No. 60/291,973, filed May 21, 2001, U.S. application Ser. No. 60/318,902, filed Sep. 14, 2001, U.S. application Ser. No. 60/333,124, filed Nov. 27, 2001, and U.S. application Ser. No. 10/005,876, filed Dec. 7, 2001, are herein incorporated by reference.

[0339] Having now fully described the present invention in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims.

[0340] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

1TABLE 1GenBank Accession numbers of human sequence recordsidentified as related to nucleic acids encoding protein kinasespotentially connected to the cell cycle.1: NM_0058582: NM_1444903: NM_0162484: M377125: NM_1393236: NM_0034047: NM_0031578: NM_0012559: NM_13901410: NM_13901311: NM_13901212: NT_00890213: NT_02367814: NT_03004015: NT_03398416: NT_03389417: NM_07846718: NM_03198819: NM_00275820: NM_00131521: NT_03394422: XM_00542023: NM_00614224: NT_00649725: NT_00781926: NT_03396427: NM_13892328: NM_00460629: NM_00005130: NM_13829331: NM_13829232: NM_00121133: NM_00118434: NM_00360035: NM_00339036: NM_00139637: NM_13043838: NM_13043739: NM_13043640: NM_10139541: NM_00038942: NM_00179943: NM_00350344: NM_00469045: NM_00719446: NM_00627147: NM_00540048: NM_02401149: NM_03362150: NM_03353751: NM_03353652: NM_03353453: NM_03353254: NM_03353155: NM_03352956: NM_03352857: NM_03352758: AF04910559: NM_01650860: NM_00126161: NM_00125962: NM_05298863: NM_05298764: NM_00126065: NM_00367466: NM_05298467: NM_00007568: NM_05282769: NM_00179870: NM_03349371: NM_03349272: NM_03349173: NM_03349074: NM_03348975: NM_03348876: NM_03348777: NM_03348678: NM_00178779: NM_03337980: NM_00178681: NM_00313782: NM_00657583: AX13604984: NM_03126785: NM_00371886: NM_00590687: NM_00495488: NM_01749089: AJ27754690: NM_00192491: NM_00718692: NM_00485393: NM_00315894: NM_00316095: NM_00249796: NM_00182797: NM_00182698: AF16266799: AF162666100: AF174135101: AF107297102: AB017332103: AF086904104: AF005209105: AF032874106: D84212107: Y13115108: U78073109: Z25437110: Z25436111: Z25435112: Z25434113: Z25433114: Z25432115: Z25431116: Z25430117: Z25429118: Z25428119: Z25427120: Z25426121: Z25425122: Z25424123: Z25423124: Z25422125: Z25421126: X73458127: Z29067128: Z29066129: Y00272130: L19559

[0341]

2

TABLE 2

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to inositol

metabolism and/or signaling.

1: AF469196
2: NM_022468
3: NM_144489
4: NM_144488

5: NM_134427
6: NM_017790
7: NM_021106
8: NM_130795

9: NM_000276
10: NM_001587
11: NM_022718
12: NM_014216

13: AF273055
14: NM_002649
15: NM_054111
16: NT_030828

17: NT_009458
18: NT_008902
19: NT_008769
20: NT_011139

21: NT_024040
22: NT_007972
23: NT_005990
24: NT_005927

25: NT_004525
26: NT_004511
27: NT_006258
28: NT_022760

29: NT_022439
30: NT_033930
31: NM_138687
32: NM_003559

33: NM_005028
34: NM_016532
35: NM_130766
36: NT_011903

37: NM_006085
38: NT_033291
39: NT_011512
40: NT_010692

41: NT_007592
42: XM_165804
43: XM_165697
44: NT_010956

45: NT_009471
46: NT_033944
47: XM_084759
48: XM_056913

49: XM_114817
50: NM_016368
51: XM_095533
52: XM_062470

53: XM_067111
54: XM_067089
55: NM_052885
56: XM_044063

57: XM_028610
58: NT_011526
59: XM_008065
60: XM_006747

61: XM_030060
62: XM_003530
63: NM_006319
64: NT_029991

65: NT_009799
66: XM_018252
67: NT_011288
68: XM_165960

69: XM_114004
70: NT_026437
71: XM_029288
72: NT_005414

73: XM_096169
74: NT_005403
75: XM_115825
76: NT_022197

77: NT_022171
78: XM_002493
79: XM_002279
80: XM_029748

81: BC027960
82: NM_002676
83: NM_017584
84: BC026331

85: NM_004897
86: NM_130785
87: AF009963
88: NM_014845

89: NM_025194
90: NM_006069
91: NM_130385
92: AL365444

93: AY064416
94: NM_078488
95: NM_004665
96: BC018952

97: NM_003866
98: NM_019892
99: NM_014937
100: Y18024

101: AK057550
102: AK056586
103: AF039945
104: BC018192

105: NM_005086
106: BC017189
107: BC017176
108: BC009565

109: BC015496
110: AF393812
111: U84400
112: AF368319

113: AB057723
114: AJ315644
115: NM_007368
116: BC008381

117: BC005274
118: BC004362
119: BC003622
120: BC001864

121: BC001444
122: AJ290975
123: AB057724
124: AF279372

125: AJ242780
126: AY032885
127: AL136579
128: AL050356

129: X83558
130: M88162
131: AF184215
132: NM_004027

133: NM_001566
134: NM_006506
135: AF063823
136: AF063822

137: AB042328
138: AL096840
139: AF207640
140: NM_002222

141: NM_000717
142: NM_005536
143: NM_016291
144: NM_014214

145: NM_006933
146: NM_005541
147: NM_005539
148: NM_005139

149: NM_001567
150: NM_002194
151: NM_003895
152: NM_002224

153: NM_002223
154: NM_002221
155: NM_002220
156: AC023051

157: AK024596
158: AK024045
159: AK022846
160: AK021526

161: AY007091
162: AF251265
163: AH009098
164: AF220249

165: AF220259
166: AF220258
167: AF220257
168: AF220256

169: AF220255
170: AF220254
171: AF220253
172: AF220252

173: AF220251
174: AF220250
175: AF220530
176: AF218361

177: AF187891
178: AF025878
179: AH007532
180: AF014398

181: AP001719
182: AF025886
183: AF025885
184: AF025884

185: AF025883
186: AF085632
187: AF085631
188: AF085630

189: AF085629
190: AF085628
191: AF085627
192: AF025882

193: AF025881
194: AF025880
195: AF025879
196: AF042729

197: AF178754
198: AF016028
199: AB036831
200: AB036830

201: AB036829
202: AK001325
203: AL137749
204: AJ251881

205: D13435
206: AF141325
207: AJ249339
208: AF177145

209: AF200432
210: AF125042
211: D89974
212: AH007823

213: AF157102
214: AF157101
215: AF157100
216: AF157099

217: AF157098
218: AF157097
219: AF157096
220: AF046915

221: AF046914
222: AC007192
223: S82269
224: S74936

225: AF115573
226: AF084944
227: AF084943
228: U53470

229: AB012610
230: U88725
231: AF009040
232: AF009039

233: U51336
234: U50041
235: U50040
236: U01062

237: L38500
238: AF027153
239: X80907
240: U23850

241: Y15056
242: Y14385
243: Y11366
244: Y11365

245: Y11364
246: Y11363
247: Y11367
248: Y11362

249: Y11361
250: Y11360
251: U96922
252: U96919

253: D38169
254: D26070
255: D26351
256: D26350

257: U57650
258: Y11999
259: X89105
260: X98429

261: L38019
262: U26398
263: X66922
264: X57206

265: X77567
266: Z31695
267: X54938
268: L36818

269: M74161
270: L47220
271: M63310
272: L08488

273: AH001430
274: L10955
275: L10954
276: L10953

[0342]

3

TABLE 3

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to

adenylate cyclase metabolism and/or signaling.

1: NM_139247
2: D17516
3: NM_020983
4: NM_015270

5: NT_008769
6: NT_023709
7: NT_028053
8: XM_007897

9: XM_012740
10: XM_028817
11: XM_036725
12: XM_096265

13: XM_113762
14: XM_036671
15: XM_041507
16: NT_006859

17: NT_009984
18: XM_036383
19: NT_010164
20: NT_007819

21: XM_166593
22: XM_039712
23: XM_090617
24: XM_036413

25: BC028085
26: BC027943
27: BC020148
28: NM_001841

29: AK056745
30: NM_033181
31: D86984
32: NM_000681

33: NM_004624
34: AK001637
35: NM_016083
36: NM_001840

37: AY028959
38: AY028957
39: AY028956
40: AY028955

41: AY028954
42: AY028953
43: AY028952
44: AY028951

45: AY028950
46: AY028949
47: AY028948
48: AH010599

49: NM_000872
50: NM_019860
51: NM_019859
52: NM_000025

53: NM_001117
54: NM_004036
55: NM_000866
56: NM_012125

57: NM_000677
58: NM_000054
59: NM_005281
60: NM_005145

61: NM_001116
62: NM_001115
63: NM_001114
64: NM_000741

65: NM_000740
66: NM_000739
67: NM_000738
68: NM_000676

69: NM_000674
70: NM_001118
71: AK022951
72: U09216

73: AJ012074
74: S56143
75: AK001924
76: AK001854

77: AK001438
78: X60435
79: S83513
80: U18810

81: L21195
82: AF088070
83: AF086306
84: AF086230

85: Y12507
86: Y12506
87: Y12505
88: D38299

89: D38301
90: D38300
91: D28472
92: X74210

93: X83956
94: X07036
95: X04408
96: X04409

97: X04828
98: M23533
99: L04962
100: L05597

101: L25124

[0343]

4

TABLE 4

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to

potasium channel metabolism and/or signaling.

1: AF348984
2: AF348983
3: AF348982

4: NM_144633
5: NM_138318
6: NM_138317

7: NM_021161
8: NM_033456
9: NM_033455

10: NM_033348
11: NM_033347
12: NM_005714

13: NM_002249
14: NM_002243
15: NM_001194

16: AF493798
17: AF472412
18: AF000972

19: NM_139318
20: NM_002236
21: NM_033311

22: NM_033310
23: NM_016611
24: NM_002246

25: NM_022358
26: NM_014217
27: AF065163

28: SEG—
29: D50315
30: D50314

HUMUKATPS

31: D50313
32: NM_139137
33: NM_139136

34: NT_009307
35: NT_010376
36: NT_024375

37: NT_030075
38: NT_008104
39: NT_008413

40: NT_004612
41: NT_004416
42: NT_022517

43: NT_021909
44: NT_021877
45: NT_019273

46: NT_033262
47: NT_033200
48: NT_033241

49: AF418206
50: NT_010422
51: NT_011512

52: NT_033899
53: NT_011333
54: NT_010700

55: NT_007592
56: XM_056976
57: XM_001299

58: XM_059493
59: XM_084080
60: XM_115258

61: XM_165593
62: XM_115027
63: XM_113221

64: XM_114797
65: NM_133329
66: NM_133497

67: XM_091498
68: XM_084762
69: XM_090187

70: XM_084388
71: XM_088998
72: NT_011362

73: XM_065997
74: XM_028862
75: XM_006988

76: XM_018513
77: NM_016121
78: NT_011669

79: NT_033316
80: XM_113356
81: NT_030171

82: NT_011233
83: NT_006576
84: XM_116412

85: NT_026437
86: NT_005367
87: NT_005334

88: NT_005612
89: XM_056742
90: NT_015120

91: XM_093482
92: XM_066592
93: XM_042027

94: XM_010829
95: XM_029336
96: AF385400

97: AF385399
98: NM_133490
99: BC028739

100: AF305072
101: AF302044
102: NM_014505

103: NM_002252
104: NM_014407
105: AF482710

106: AH011548
107: AC005833
108: BC025726

109: AF453246
110: AF453244
111: AJ272506

112: M38217
113: AJ272519
114: AJ272518

115: AJ272517
116: AJ272516
117: AJ272515

118: AJ272514
119: AJ272513
120: AJ272512

121: AJ272511
122: AJ272510
123: AJ272509

124: AJ272508
125: AJ272507
126: AF294352

127: AF294351
128: AF294350
129: AK074390

130: NM_031460
131: AF349445
132: NM_001364

133: NM_013348
134: AF055989
135: AF438203

136: AF438202
137: NM_016601
138: NM_033272

139: NM_020122
140: AK055089
141: BC018051

142: AL158822
143: NM_004974
144: AY053503

145: AY040849
146: AF358910
147: AF344826

148: NM_022055
149: NM_032115
150: AF268897

151: AF268896
152: NM_022054
153: AY049734

154: AF074247
155: AJ006128
156: AL157833

157: NM_003740
158: NM_004823
159: NM_002245

160: AF294266
161: BC012779
162: AF397175

163: BC004367
164: BC000178
165: AF257081

166: AF257080
167: AL121829
168: AF315818

169: AF336797
170: AF171068
171: AF319633

172: AJ310479
173: AJ251016
174: AF031815

175: U52155
176: U52154
177: U52153

178: U52152
179: AK027657
180: AK027347

181: NM_031886
182: AF358909
183: AF336342

184: AF153819
185: AF153818
186: AH009400

187: AC005559
188: AL118522
189: AL121827

190: AL353658
191: NM_030779
192: AF339912

193: NM_002251
194: AF129399
195: AF043473

196: AB044585
197: AB044584
198: AF153814

199: AF153813
200: AF153812
201: AF153811

202: AF153810
203: AF153809
204: AH009401

205: AF153820
206: AF153817
207: AF153816

208: AF153815
209: AF082182
210: AL121785

211: AL035685
212: AF287303
213: AF287302

214: NM_020298
215: NM_020297
216: NM_006855

217: NM_016657
218: NM_005691
219: AF029780

220: AF311913
221: AF239613
222: AF305735

223: AF305734
224: AF305733
225: AF305732

226: AF305731
227: AH009923
228: U32376

229: AF248242
230: AF248241
231: AJ297404

232: AJ297405
233: NM_000220
234: NM_019842

235: NM_014379
236: NM_014406
237: NM_012283

238: NM_002248
239: NM_005477
240: NM_004983

241: NM_004982
242: NM_000890
243: NM_004981

244: NM_005136
245: NM_004978
246: NM_004977

247: NM_004976
248: NM_004975
249: NM_004700

250: NM_004519
251: NM_004518
252: NM_004732

253: NM_000238
254: NM_000218
255: NM_000219

256: NM_000217
257: NM_001365
258: NM_002250

259: NM_002247
260: NM_002244
261: NM_002240

262: NM_002239
263: NM_000891
264: NM_002241

265: NM_002238
266: NM_002237
267: NM_003636

268: NM_003471
269: NM_002235
270: NM_002234

271: NM_002233
272: NM_002232
273: AF081466

274: AK024857
275: AK022344
276: AF279890

277: AL136087
278: AF179353
279: AF295530

280: AF295076
281: AF181988
282: AF021139

283: AF032897
284: AF249278
285: AF170917

286: AF170916
287: AF202977
288: AF279809

289: AB021865
290: AF263835
291: AP001730

292: AP001729
293: AP001731
294: AP001720

295: AP000365
296: AF212829
297: U11058

298: AF160967
299: AF166011
300: AF166010

301: AF166009
302: AH009283
303: AF160968

304: AF155652
305: AF166008
306: AF166007

307: AH009258
308: AF166006
309: AF166005

310: AF166004
311: AH009257
312: AF166003

313: AF120491
314: AF247042
315: AB032013

316: AB032012
317: AB032011
318: SEG_AB032011S

319: SEG_AB01514S
320: AB015163
321: AB015162

322: AB015161
323: AB015160
324: AB015159

325: AB015158
326: AB015157
327: AB015156

328: AB015155
329: AB015154
330: AB015153

331: AB015152
332: AB015151
333: AB015150

334: AB015149
335: AB015148
336: AB015147

337: AF011904
338: AJ276317
339: AC010072

340: AF214561
341: AF209747
342: AF207992

343: AL133016
344: AL122115
345: AF199599

346: AF199598
347: AF199597
348: AF155110

349: AF043472
350: AF205857
351: AF205856

352: AC004946
353: AC004888
354: AF167082

355: AF139471
356: Z97056
357: AF207550

358: AB013891
359: AB013889
360: AF078742

361: AF078741
362: U69883
363: AF187964

364: AF187963
365: AJ010969
366: AJ011021

367: AF142568
368: AF117708
369: U65406

370: AF016411
371: AH007779
372: AF131948

373: AF131947
374: AF131946
375: AF131945

376: AF131944
377: AF131943
378: AF131942

379: AF131941
380: AF131940
381: AF131939

382: AF131938
383: AF137071
384: AJ006344

385: AJ006343
386: AF076531
387: AF071002

388: AF135188
389: AF121104
390: AF105373

391: AF105372
392: AF110020
393: AH007377

394: AF105216
395: AF105215
396: AF105214

397: AF105213
398: AF105212
399: AF105211

400: AF105210
401: AF105209
402: AF105208

403: AF105207
404: AF105206
405: AF105205

406: AF105204
407: AF105203
408: AF105202

409: AF035046
410: AF004711
411: AH007067

412: AF071491
413: AF071490
414: AF071489

415: AF071488
416: AF071487
417: AF071486

418: AF071485
419: AF071484
420: AF071483

421: AF071482
422: AF071481
423: AF071480

424: AF071479
425: AF071478
426: AJ012369

427: Y10745
428: AF052728
429: Y13896

430: Y13895
431: AJ001891
432: AJ001366

433: AJ007557
434: S72503
435: AF015607

436: AF015606
437: AF015605
438: AF022797

439: U89364
440: U96110
441: U33429

442: U73193
443: U73192
444: U73191

445: U52432
446: U33428
447: U11717

448: U24660
449: U16953
450: U17968

451: U12507
452: AF033021
453: AF053478

454: AF053477
455: AJ010538
456: L23499

457: AJ005898
458: AF022150
459: AF061118

460: AF033383
461: AF033382
462: AF048713

463: AF048712
464: Y15065
465: AF003743

466: AF044253
467: U76996
468: AF033348

469: AF033347
470: AF026005
471: AF026002

472: AF025999
473: AF029749
474: U61537

475: U61536
476: D87327
477: D87291

478: D50134
479: U86146
480: D50312

481: U39196
482: U39195
483: U90065

484: U24055
485: U50964
486: X83127

487: S78737
488: S56770
489: U42600

490: AH003672
491: U42603
492: U42602

493: U42601
494: U69962
495: U25138

496: L78480
497: X83582
498: X17622

499: X68302
500: Z11585
501: U23767

502: U16861
503: U13913
504: U24056

505: L36069
506: U22413
507: L33815

508: U04270
509: U12545
510: U12544

511: U12543
512: U12542
513: U12541

514: M60451
515: M60450
516: M83254

517: M55514
518: M96747
519: M85217

520: L28168
521: M64676
522: U09384

523: U02632
524: M55515
525: M55513

526: L02840
527: L00621
528: L02752

529: L02751
530: L02750
531: M26685

532: U07364
533: U07918

[0344]

5

TABLE 5

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to sodium

channel metabolism and/or signaling.

1: NM_020039
2: NM_001095
3: NM_001094
4: NM_002976

5: NM_015277
6: NM_004588
7: BC030193
8: NT_009151

9: NT_009731
10: NT_009609
11: NT_006129
12: NT_033049

13: NM_005612
14: NT_033284
15: XM_113296
16: NT_033899

17: NT_010736
18: NT_011085
19: XM_114084
20: XM_113411

21: XM_116055
22: XM_083942
23: XM_028504
24: XM_064330

25: XM_008249
26: XM_032835
27: XM_007990
28: XM_097396

29: NT_007914
30: NT_033178
31: NT_005343
32: XM_010769

33: XM_114281
34: XM_054184
35: XM_033675
36: BQ268051

37: AY043484
38: AF260228
39: AF260227
40: AH011264

41: AF260226
42: NM_006922
43: U81961
44: AY007685

45: BD004564
46: BD004563
47: BD004562
48: E37451

49: AX354521
50: AX354520
51: NM_002837
52: NM_001649

53: BM353290
54: BM352813
55: AJ310898
56: AJ310897

57: AJ310896
58: AJ310895
59: AJ310894
60: AJ310893

61: AJ310892
62: AJ310891
63: AJ310890
64: AJ310889

65: AJ310888
66: AJ310887
67: AJ310886
68: AJ310885

69: AJ310884
70: AJ310883
71: AJ310882
72: BM314926

73: NM_018400
74: BC006526
75: BI964932
76: BI962702

77: AH005909
78: AF049497
79: AF049496
80: AB071179

81: BI789210
82: AF087511
83: AF087510
84: AY038064

85: AH007622
86: AF060913
87: AF060912
88: AF060911

89: AF060910
90: BG108767
91: AJ251507
92: AF356502

93: AF356501
94: AF356500
95: AF356499
96: AF356498

97: AF356497
98: AF356496
99: AF356495
100: AF356494

101: AF356493
102: AH010738
103: AU099675
104: AU099608

105: NM_001091
106: S82622
107: E36123
108: M55662

109: NM_021602
110: NM_000626
111: NM_020322
112: NM_020321

113: NM_004769
114: BG152517
115: AF225987
116: AF225986

117: AF225985
118: AF330135
119: AF330134
120: AF330133

121: AF330132
122: AF330131
123: AF330130
124: AF330129

125: AF330128
126: AF330127
127: AF330126
128: AF330125

129: AF330124
130: AF330123
131: AF330122
132: AF330121

133: AF330120
134: AF330119
135: AF330118
136: AF330117

137: AF330116
138: AH010233
139: AF327246
140: AF327245

141: AF327244
142: AF327243
143: AF327242
144: AF327241

145: AF327240
146: AF327239
147: AF327238
148: AF327237

149: AF327236
150: AF327235
151: AF327234
152: AF327233

153: AF327232
154: AF327231
155: AF327230
156: AF327229

157: AF327228
158: AF327227
159: AF327226
160: AF327225

161: AF327224
162: AH010232
163: BF941784
164: NM_000336

165: NM_000335
166: AF038871
167: AJ002484
168: AJ002483

169: BF195781
170: NM_021007
171: NM_014191
172: NM_014139

173: NM_006514
174: NM_001039
175: NM_002978
176: NM_001038

177: NM_002977
178: NM_000334
179: NM_001037
180: G64248

181: BF061009
182: BF002594
183: AX017233
184: AX017232

185: AX017231
186: AX017230
187: AX017229
188: AX017228

189: AX017227
190: AX017226
191: AX017225
192: AX017224

193: AX017223
194: AX017222
195: AX017221
196: AX017220

197: AX017219
198: BE671436
199: AJ277395
200: AJ277394

201: AJ277393
202: AJ276142
203: AJ276141
204: AJ276140

205: AJ276139
206: BE463571
207: AB037525
208: U48937

209: AW771930
210: AJ252011
211: L48689
212: AF239921

213: AJ243396
214: AW468811
215: AF225988
216: A82786

217: A82597
218: A82595
219: A82593
220: AF150882

221: AF109737
222: AW276630
223: U87555
224: AF188679

225: AC002300
226: AW190344
227: AW170363
228: AF059683

229: AW105326
230: AW025990
231: AW008644
232: AW002349

233: AW001231
234: AF126739
235: AF107028
236: AI932372

237: AI915394
238: AI884536
239: AI862563
240: AI796228

241: AB027567
242: AI683977
243: AI675767
244: AF117907

245: AH007414
246: AF050736
247: AF050735
248: AF050734

249: AF050733
250: AF050732
251: AF050731
252: AF050730

253: AF050729
254: AF050728
255: AF050727
256: AF050726

257: AF050725
258: AF050724
259: AF050723
260: AF050722

261: AF050721
262: AF050720
263: AF050719
264: AF050718

265: AF050717
266: AF050716
267: AF050715
268: AF050714

269: AF050713
270: AF050712
271: AF050711
272: AJ005393

273: AJ005392
274: AJ005391
275: AJ005390
276: AJ005389

277: AJ005388
278: AJ005387
279: AJ005386
280: AJ005385

281: AJ005384
282: AJ005383
283: AI567447
284: AI553866

285: AF049618
286: AI361695
287: S75992
288: AI401486

289: AI280308
290: AI277385
291: AI275868
292: AI377290

293: AI361696
294: AA885031
295: AA885211
296: AI338340

297: AI199647
298: AI241832
299: AI191453
300: AI131238

301: AI146968
302: AH006646
303: U53853
304: U53852

305: U53851
306: U53850
307: U53849
308: U53848

309: U53847
310: U53846
311: U53845
312: U53844

313: U53843
314: U53842
315: U53841
316: U53840

317: U53839
318: U53838
319: U53837
320: U53836

321: U53835
322: U48936
323: U50352
324: U38254

325: U35630
326: AI026646
327: AI027237
328: AI017422

329: AI016157
330: AI005419
331: AA994701
332: AA912739

333: AI091722
334: AF035686
335: AF035685
336: X65362

337: Z92978
338: Z92982
339: Z92981
340: Z92980

341: Z92979
342: AJ002482
343: AF007783
344: X97925

345: AA917500
346: AA913881
347: AA913423
348: AA887514

349: AA984063
350: X65361
351: AB010575
352: U24693

353: AA214661
354: AA211081
355: AF049498
356: AA778416

357: AH005825
358: U12194
359: U12193
360: U12192

361: U12188
362: U12191
363: U12190
364: U12189

365: AA666056
366: AA429417
367: AA428361
368: AA422068

369: AA620400
370: AA595839
371: AA397575
372: AA393950

373: AF007782
374: AF007781
375: AH005307
376: L04236

377: L04235
378: L04234
379: L04233
380: L04232

381: L04231
382: L04230
383: L04229
384: L04228

385: L04227
386: L04226
387: L04225
388: L04224

389: L04223
390: L04222
391: L04221
392: L04220

393: L04219
394: L04218
395: L04217
396: L04216

397: AA449579
398: AA446878
399: AA035472
400: AA035445

401: AA029133
402: AA383040
403: AA360938
404: AA322364

405: AA298508
406: AA297746
407: AA297047
408: AA295926

409: U57352
410: U78181
411: U78180
412: AA206530

413: S71446
414: S69887
415: Z50169
416: U22314

417: X82835
418: X87160
419: X87159
420: N53512

421: AH003201
422: L01968
423: L01964
424: L01983

425: L01982
426: L01981
427: L01980
428: L01979

429: L01978
430: L01977
431: L01976
432: L01975

433: L01974
434: L01973
435: L01972
436: L01971

437: L01970
438: L01969
439: L01967
440: L01966

441: L01965
442: L01963
443: L01962
444: L36593

445: L36592
446: T29303
447: T28389
448: R90820

449: H26938
450: H23297
451: R74525
452: U16023

453: R53503
454: L16242
455: M81758
456: L10338

457: M91556
458: M77235
459: T19733
460: M85046

461: M85045
462: M91804
463: M91803
464: L29007

465: M94055
466: U02693
467: T07957
468: T06279

[0345]

6

TABLE 6

GenBank Accession numbers of human sequence

records identified as related to nucleic acids encoding

polypeptides potentially related to serotonin

metabolism and/or signaling.

1: NM_000870
2: NT_009151
3: NT_009714
4: NT_008769

5: NT_004610
6: NT_029218
7: NT_005791
8: NT_024897

9: NT_010641
10: NT_028405
11: XM_049607
12: NT_025741

13: NT_023399
14: NT_033922
15: XM_165640
16: NT_006859

17: NT_006431
18: NT_007666
19: NT_005403
20: XM_004134

21: XM_003692
22: AF498985
23: AF498984
24: AF498983

25: AF498982
26: AF498981
27: AF498980
28: AF498979

29: AF498978
30: NM_003739
31: NM_000864
32: AJ011371

33: NM_130770
34: AF459285
35: NM_000675
36: AX253256

37: AB041403
38: BC007720
39: BC002354
40: AB061801

41: AB061800
42: AB061799
43: AJ308680
44: AJ308679

45: NM_002383
46: S78723
47: NM_024012
48: NM_000872

49: NM_019860
50: NM_019859
51: AJ131724
52: NM_001088

53: NM_000866
54: NM_000621
55: NM_014626
56: NM_014627

57: NM_006028
58: NM_004179
59: NM_000240
60: NM_001045

61: NM_000871
62: NM_000869
63: NM_000868
64: NM_000867

65: NM_000865
66: NM_000863
67: NM_000524
68: NM_000674

69: AF298814
70: AF149416
71: AL157777
72: AJ005205

73: AB037533
74: AB037513
75: AF208053
76: D49394

77: AB041373
78: AB041370
79: AF233399
80: AL049576

81: AF112461
82: AF112460
83: AJ003080
84: AJ003078

85: AJ243213
86: AB031259
87: AB031258
88: AB031257

89: AB031256
90: AB031255
91: AB031254
92: AB031253

93: AB031252
94: AB031251
95: AB031250
96: AB031249

97: AB031248
98: AB031247
99: AL049595
100: X80763

101: AF169255
102: AH003966
103: S42168
104: S42167

105: AH001421
106: M84601
107: M84592
108: M84591

109: M84590
110: M84589
111: M84588
112: M84599

113: M84598
114: M84595
115: M84597
116: M84596

117: M84594
118: M84593
119: M84600
120: M77828

121: L13665
122: AF126506
123: AI819939
124: X57829

125: AF117826
126: X76753
127: Y13147
128: AF080582

129: Y09586
130: U40391
131: U40347
132: L21195

133: AF072904
134: Y12507
135: Y12506
136: U88828

137: Y12505
138: Y08756
139: AF007141
140: Y13584

141: U86813
142: AA757429
143: Y10437
144: AA722177

145: U79746
146: AA708262
147: AA700086
148: AA700070

149: Z49119
150: Z48150
151: U73443
152: D10995

153: D87030
154: AA365330
155: AA364412
156: U49648

157: U49516
158: X76757
159: X76756
160: X76754

161: X76762
162: X76761
163: X76760
164: X76759

165: X76758
166: X76755
167: X98194
168: X98147

169: X98193
170: S71229
171: C06167
172: Z36748

173: Z11168
174: U33819
175: X81412
176: X81411

177: X77307
178: X52836
179: Z34845
180: X70697

181: X57830
182: Z11166
183: L41147
184: M83181

185: M81778
186: M81590
187: M81589
188: M75128

189: M92826
190: M86841
191: M91467
192: L04962

193: L05597
194: M83180
195: L06179
196: L05568

197: M89955
198: M89478

[0346]

7

TABLE 7

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to

fibroblast growth factors metabolism and/or signaling.

1: BC032697
2: NM_139266
3: NM_007315
4: AF508782

5: AF520763
6: NM_004385
7: NM_006654
8: D14872

9: NT_009151
10: NT_024192
11: NT_024413
12: NT_010194

13: NT_008769
14: NT_030764
15: NT_030040
16: NT_005501

17: NT_006111
18: NT_006109
19: NT_022865
20: NT_016354

21: NT_033229
22: NT_024773
23: NT_010478
24: XM_049890

25: NT_010823
26: NT_033929
27: XM_169242
28: XM_167430

29: NT_033944
30: XM_084481
31: XM_044120
32: XM_064055

33: XM_055784
34: XM_003444
35: XM_017651
36: XM_042695

37: NM_013394
38: NT_011719
39: NT_009799
40: NT_033316

41: NT_024524
42: NT_030171
43: NT_006859
44: XM_096234

45: NT_009952
46: NT_006725
47: NT_008300
48: NT_008251

49: XM_049463
50: NT_007819
51: NT_030737
52: NT_023132

53: NT_023098
54: NT_033210
55: NT_005367
56: XM_090648

57: XM_084273
58: M88272
59: BQ269244
60: AF487554

61: AY094623
62: AF487555
63: NM_007083
64: AF497475

65: NM_133336
66: NM_133335
67: NM_133334
68: NM_133333

69: NM_133332
70: NM_133331
71: NM_133330
72: NM_014919

73: NM_007331
74: AF245114
75: NM_007050
76: NM_133170

77: AF360695
78: AH010989
79: AF410480
80: AX378915

81: AX378914
82: BM874752
83: BM874259
84: NM_080838

85: NM_003882
86: AF359246
87: NM_012201
88: NM_006595

89: BM311972
90: AX318785
91: AX318710
92: AX318684

93: NM_007373
94: NM_006824
95: M34641
96: AX275080

97: AX275079
98: AX275054
99: AX275053
100: AX275042

101: BC017664
102: AF035374
103: AX287610
104: AX287608

105: AX287596
106: BC017448
107: AJ298918
108: AJ298917

109: AJ298916
110: AY049782
111: NM_033649
112: NM_004114

113: NM_033642
114: NM_003862
115: NM_003867
116: AX250592

117: AF359241
118: AB014615
119: AF411527
120: BC014388

121: AX235431
122: NM_005247
123: NM_002006
124: NM_003868

125: NM_006119
126: NM_033165
127: NM_033164
128: NM_033163

129: NM_002009
130: NM_020996
131: NM_004112
132: NM_004465

133: NM_002010
134: AX179562
135: AX179564
136: BC011847

137: NM_004464
138: NM_033143
139: NM_020638
140: NM_000800

141: NM_033137
142: NM_033136
143: NM_020637
144: NM_019113

145: NM_002007
146: BC010956
147: NM_005117
148: NM_019851

149: NM_004115
150: NM_000088
151: BC006245
152: BC002537

153: AX156438
154: AX156436
155: AX156434
156: AL160153

157: AF369213
158: AF369212
159: AF369211
160: AX105677

161: AX105675
162: AX105674
163: AX105673
164: AX105671

165: AX105669
166: AX105667
167: AX105665
168: AX105663

169: AX105661
170: AF110400
171: AU100202
172: AX097639

173: AX092981
174: AF279689
175: S67291
176: NM_023031

177: NM_023030
178: NM_023028
179: NM_022976
180: NM_022975

181: NM_022974
182: NM_022973
183: NM_022972
184: NM_022971

185: NM_022970
186: NM_022969
187: NM_015850
188: NM_023111

189: NM_023110
190: NM_023109
191: NM_023029
192: NM_023108

193: NM_000141
194: NM_023107
195: NM_023106
196: NM_023105

197: NM_000604
198: AF312678
199: AX080371
200: AX080370

201: AX080369
202: AX080368
203: AX080364
204: NM_021923

205: NM_002011
206: NM_022963
207: NM_022965
208: NM_000142

209: AB021925
210: E30326
211: NM_004214
212: AF229254

213: AF229253
214: AF250392
215: AF250391
216: U69263

217: BF739878
218: BF739773
219: AL139378
220: AB037973

221: AB030648
222: NM_021032
223: BF221906
224: NM_004339

225: NM_004219
226: NM_000214
227: NM_007045
228: NM_004113

229: NM_005211
230: NM_004383
231: NM_000428
232: NM_003453

233: NM_003199
234: NM_002660
235: NM_001553
236: AJ277437

237: BF110834
238: BF062689
239: BF059273
240: BF058753

241: BF056554
242: BF002774
243: AK026508
244: BE673878

245: BE673874
246: BE673061
247: BE672701
248: BE672483

249: BE671952
250: BE671715
251: BE552216
252: BE551725

253: BE551556
254: BE550968
255: BE549662
256: AF238374

257: BE504886
258: BE502050
259: BE501873
260: AF171928

261: BE466386
262: BE466124
263: BE208220
264: BE207666

265: BE205845
266: BE350605
267: BE349962
268: BE348962

269: BE328768
270: BE301283
271: BE301278
272: BE221273

273: BE047232
274: AF239155
275: BE019402
276: BE019081

277: S81809
278: AW873016
279: AW779920
280: AW779255

281: AW779029
282: AW778975
283: AH003714
284: S41873

285: AH003713
286: S41870
287: S41845
288: S41355

289: AW770670
290: AC004416
291: AW662345
292: AU077033

293: AU076629
294: AF043644
295: AW629787
296: AW628470

297: AW590506
298: AW583780
299: AF233344
300: AF169399

301: AW571604
302: AW518111
303: AW515079
304: AW514184

305: AW510973
306: AW474533
307: AW474496
308: AF010187

309: AL096753
310: X68559
311: AW418776
312: AF199613

313: AF199612
314: AW341130
315: AW338831
316: AW338787

317: AW338133
318: AF202063
319: AW301094
320: AW299662

321: AF211188
322: AF211169
323: AW275471
324: AW273483

325: AW271784
326: AW271769
327: AW270662
328: AW268519

329: AW264608
330: AW262507
331: AW237589
332: AW237163

333: AW235776
334: AW196650
335: AW196066
336: AJ250952

337: AL031386
338: AW172838
339: AW167176
340: AW157414

341: AW151574
342: AW118881
343: AW086037
344: AW081195

345: AW074378
346: AW074098
347: AW073347
348: AW057787

349: AW052021
350: AW025920
351: AW009550
352: AW003200

353: AW002405
354: AW001782
355: AW000986
356: AI991116

357: AI989589
358: AI989525
359: AI984931
360: AI972087

361: AI971057
362: AI969759
363: AI968746
364: AI962257

365: AI952845
366: AI937526
367: AI936283
368: AI932287

369: AI929112
370: AI927457
371: AI927348
372: AI927305

373: AI926324
374: AI924133
375: AI921760
376: AF036718

377: AF036717
378: AI918567
379: AI918460
380: AI915058

381: AI889594
382: AI887836
383: AI887420
384: AI885536

385: AI884363
386: AI873746
387: AI871363
388: AI871071

389: AI869111
390: AI868556
391: AI858722
392: AI858707

393: AI831133
394: AI828125
395: AI825718
396: U76381

397: AI819406
398: AI815637
399: AI814182
400: Y17131

401: AI811355
402: AI810411
403: AI807481
404: AI807060

405: AI805693
406: AI805484
407: AI804152
408: AI802531

409: AI801468
410: AI796742
411: AI768439
412: AI767738

413: AI762738
414: AI762110
415: AI762100
416: AI743298

417: AH007696
418: AF097354
419: AF097353
420: AF097352

421: AF097351
422: AF097350
423: AF097349
424: AF097348

425: AF097347
426: AF097346
427: AF097345
428: AF097344

429: AF097343
430: AF097342
431: AF097341
432: AF097340

433: AF097339
434: AF097338
435: AF097337
436: AF097336

437: AI721131
438: AI720427
439: AI708818
440: AI703144

441: AI702628
442: AI701349
443: AI699955
444: AI698883

445: AI698843
446: AI695161
447: AI694924
448: AI690405

449: AI689479
450: AI689318
451: AI684499
452: AI683268

453: AI681540
454: AI671094
455: AI670114
456: AB002097

457: AI659722
458: AI655715
459: AI655144
460: AI654503

461: AI653112
462: AI652947
463: AI651153
464: AI650627

465: AI640755
466: AI640605
467: AF019633
468: AF019632

469: AF019634
470: AI638490
471: AI638387
472: AI638356

473: AI638328
474: AI638209
475: AI630825
476: AI628825

477: AI624745
478: AI624729
479: AI621022
480: AI608828

481: AI598047
482: AI587337
483: AI583394
484: AI572541

485: AF108756
486: AI560207
487: AI559529
488: X14071

489: X14073
490: X14072
491: Y18046
492: AI539845

493: AI538706
494: AI521743
495: AI493472
496: AI493152

497: AI500404
498: AI500276
499: AI498743
500: AI480167

501: Y13468
502: AF100144
503: AF100143
504: AI474895

505: AI474284
506: AI472373
507: AI459892
508: AI436212

509: AI433806
510: AI433805
511: AI423809
512: AI423808

513: AI422168
514: AI421090
515: AI374640
516: AI369615

517: AI368565
518: AI367719
519: AI360211
520: AI341373

521: AI341329
522: AI338128
523: AI143675
524: AI140801

525: S82438
526: S76658
527: S47380
528: AI400425

529: AI400423
530: AI264866
531: AI263615
532: AI263602

533: AI263355
534: AI306634
535: AI302760
536: AI266466

537: AI266461
538: AI292351
539: AI290617
540: AI273321

541: AI261528
542: AI245969
543: AI245767
544: AI379638

545: AI379298
546: AI379172
547: AI378807
548: AI377468

549: AI369220
550: AA889062
551: AA843793
552: AI343936

553: AA774439
554: AA772399
555: AA772398
556: AA772257

557: AI341894
558: AI336070
559: AI332806
560: AI284647

561: AI275235
562: AI274671
563: AI247085
564: AI270451

565: AI199217
566: AI218552
567: AI217705
568: AB016517

569: X04431
570: AI083781
571: AA985469
572: AI244735

573: AI219687
574: AI192569
575: AI185500
576: AI192433

577: AI188214
578: AI126344
579: AI127918
580: AI143063

581: AI142488
582: AI168407
583: AI167998
584: AI146896

585: AI146864
586: AA975393
587: AI199931
588: AI189158

589: AI186077
590: U73663
591: U73662
592: U73661

593: U73660
594: AI092048
595: AI092260
596: AF075292

597: AI087269
598: AI087201
599: AI087119
600: AI086966

601: AI086936
602: AI086833
603: AI086748
604: AI086711

605: AI086679
606: AI086487
607: AI084796
608: AI084737

609: AI084723
610: AI083989
611: AI082070
612: AI080060

613: AI079867
614: AI079236
615: AI079226
616: AI076759

617: AI076491
618: AI074202
619: AI074048
620: AI057095

621: AI052395
622: AI052337
623: AI052334
624: AI142967

625: AJ224901
626: AI095303
627: AI094703
628: AI085184

629: AI085149
630: AI081876
631: AI077609
632: AI075639

633: AI074992
634: AI074925
635: AI073629
636: AI042137

637: AI041763
638: AI039864
639: AI038887
640: AI037989

641: AA939239
642: U77720
643: U77914
644: AH006649

645: U47011
646: U47010
647: U47009
648: L49241

649: L49240
650: L49239
651: L49238
652: L49242

653: L49237
654: AF062639
655: L78738
656: L78737

657: L78736
658: L78735
659: L78734
660: L78733

661: L78732
662: L78731
663: L78730
664: L78729

665: L78728
666: L78727
667: L78726
668: L78725

669: L78724
670: L78723
671: L78722
672: L78721

673: L78720
674: L25647
675: AC005592
676: AI085805

677: AI023180
678: AI022940
679: AI073906
680: AI017114

681: AI005377
682: AI005374
683: AI004492
684: AA993569

685: AI086867
686: AI086860
687: AI085968
688: AI080594

689: AI078769
690: AI074256
691: AI066663
692: AB007422

693: AI052335
694: AI050058
695: AI049904
696: AF054828

697: AA939114
698: AA932095
699: AI042628
700: AI041773

701: AA928957
702: AA973525
703: AA922587
704: AA913131

705: AA909405
706: AI002948
707: AA916549
708: AA913622

709: AA912389
710: AA905041
711: AA902794
712: AA987837

713: AA984329
714: AA976463
715: AA975827
716: Y13472

717: AA953586
718: AA873489
719: AA934000
720: AB009249

721: AA910578
722: AA902796
723: AA878913
724: AA878580

725: AC004449
726: AA191059
727: AA190616
728: AA195894

729: AA164882
730: AA489435
731: AA599664
732: AA621648

733: AA621439
734: AA608928
735: AB009391
736: AA776567

737: AA776527
738: Y13901
739: AA757478
740: AA738073

741: AA724695
742: AA731115
743: AA723410
744: AA706746

745: AA131477
746: AA074576
747: AA100216
748: AA083999

749: AA081728
750: AA070651
751: AA070081
752: AA071169

753: AA070677
754: AA069659
755: AA702307
756: AA687581

757: AA658115
758: AA678868
759: AA664355
760: AA284286

761: Y08736
762: AA643845
763: AA635556
764: AA426235

765: AA424505
766: AA424365
767: AA424099
768: AA424022

769: AA417704
770: AA417654
771: AA417586
772: AA419620

773: AA419611
774: AA419508
775: AA419497
776: AA419484

777: AA621461
778: D38752
779: AA613015
780: AA587307

781: AA598537
782: AF007878
783: AA574041
784: AA551848

785: AA514485
786: AA288012
787: AA279375
788: AA516449

789: AA405082
790: AA548551
791: AA236812
792: AA235751

793: AA235346
794: AA256191
795: AA256152
796: AA253505

797: AA253402
798: AA258618
799: A46444
800: AA133849

801: AF015910
802: AF006657
803: U67918
804: Y08087

805: Z69640
806: Z69641
807: AH005423
808: M23534

809: M23536
810: M23535
811: L03840
812: E05102

813: E05101
814: E04557
815: E04552
816: E03194

817: E03043
818: E02544
819: E02243
820: E02144

821: D14838
822: AA446994
823: AA446876
824: AA446431

825: AA446123
826: AA443093
827: AA442053
828: AA442030

829: AA441940
830: AA441920
831: AA411000
832: AA410992

833: AA411626
834: AA406576
835: AA293228
836: AA293012

837: AA088648
838: AA088248
839: AA039680
840: AA033657

841: AA032183
842: AA009507
843: AA002254
844: AA001295

845: AA378797
846: AA377626
847: AA376435
848: AA376353

849: AA376295
850: AA376249
851: AA376219
852: AA376130

853: AA375854
854: AA375922
855: AA375695
856: AA375660

857: AA375650
858: AA375508
859: AA375435
860: AA375356

861: AA375129
862: AA375326
863: AA375309
864: AA375301

865: AA375208
866: AA375181
867: AA375167
868: AA375088

869: AA375052
870: AA374874
871: AA374628
872: AA374626

873: AA374622
874: AA374430
875: AA374371
876: AA374364

877: AA374328
878: AA374263
879: AA374161
880: AA374160

881: AA374044
882: AA374064
883: AA373980
884: AA373990

885: AA373825
886: AA373734
887: AA373568
888: AA373794

889: AA373788
890: AA373723
891: AA373667
892: AA373713

893: AA373674
894: AA373617
895: AA373597
896: AA373565

897: AA373516
898: AA373442
899: AA373379
900: AA373369

901: AA373305
902: AA373315
903: AA373300
904: AA373292

905: AA373257
906: AA373244
907: AA373018
908: AA373233

909: AA373074
910: AA373041
911: AA372212
912: AA366756

913: AA361781
914: AA360690
915: AA360561
916: AA357573

917: AA357468
918: AA356426
919: AA356425
920: AA344199

921: AA341853
922: AA330669
923: AA325962
924: AA323790

925: AA316916
926: AA311070
927: AA309032
928: AA309031

929: AA304140
930: AA298698
931: AA298681
932: AA298593

933: AA298620
934: AA298617
935: AA298614
936: AA298582

937: AA298500
938: AA298567
939: AA298557
940: AA298550

941: AA297966
942: AA297637
943: AA297311
944: AA297287

945: AA297220
946: AA297158
947: Y09852
948: Y08092

949: Y08091
950: Y08090
951: Y08089
952: Y08088

953: Y08086
954: Y08101
955: Y08100
956: Y08099

957: Y08098
958: Y08097
959: Y08096
960: Y08095

961: Y08094
962: Y08093
963: AA235910
964: AA232084

965: AA232083
966: Z50197
967: Z50196
968: Z50201

969: X56191
970: AA039601
971: AA039600
972: AA022484

973: AA022483
974: N77733
975: N58365
976: U46214

977: U46213
978: U46212
979: U46211
980: X84939

981: Z70276
982: Z70275
983: AA169370
984: AA152209

985: AA152243
986: S82451
987: AA037149
988: AA037148

989: W51760
990: W25492
991: W25484
992: W25323

993: W25340
994: S76733
995: AH004637
996: S74129

997: S74128
998: S67294
999: S67292
1000: S36271

1001: S36219
1002: S81661
1003: S41878
1004: AH003712

1005: S41350
1006: AH003711
1007: S40851
1008: S40858

1009: S40853
1010: AA115405
1011: U66200
1012: U66199

1013: U66198
1014: U66197
1015: AH003682
1016: U36228

1017: U36227
1018: U36226
1019: U36225
1020: U36223

1021: W72842
1022: W68006
1023: W61036
1024: W52234

1025: W53020
1026: W52295
1027: W52176
1028: W47310

1029: W47603
1030: W47575
1031: W47408
1032: W47218

1033: W46522
1034: W44678
1035: W44677
1036: W44455

1037: W44341
1038: W45667
1039: W45595
1040: W45594

1041: W45612
1042: W45557
1043: W44900
1044: W39595

1045: AA053699
1046: AA037285
1047: AA037281
1048: AA037338

1049: M37825
1050: U64791
1051: W31071
1052: W23905

1053: N95383
1054: W24057
1055: N91902
1056: U56978

1057: W88635
1058: W88553
1059: W87790
1060: U28811

1061: U49177
1062: U49176
1063: U49175
1064: U49174

1065: U49173
1066: W52380
1067: W52112
1068: X65779

1069: Z14152
1070: Z14151
1071: Z14150
1072: Z14149

1073: X65778
1074: X66945
1075: X64875
1076: X51943

1077: X57121
1078: X57120
1079: X57119
1080: X57122

1081: X62586
1082: X52833
1083: X52832
1084: X57205

1085: X51803
1086: X04433
1087: X04432
1088: X59065

1089: X59612
1090: X59932
1091: W49577
1092: W49555

1093: W49554
1094: A29216
1095: A09132
1096: W47595

1097: W47556
1098: W47051
1099: W45649
1100: W44919

1101: W39566
1102: W37147
1103: W32691
1104: W31180

1105: W25267
1106: R58184
1107: W17139
1108: W07463

1109: W05259
1110: Z37976
1111: M30494
1112: N98876

1113: N92237
1114: N91660
1115: N85292
1116: N85228

1117: N84692
1118: N81103
1119: N75511
1120: N67307

1121: N69800
1122: N68644
1123: N66630
1124: N57287

1125: N55322
1126: M50463
1127: N50410
1128: N22749

1129: H89352
1130: H89359
1131: H88160
1132: H89545

1133: H89538
1134: H87979
1135: H87878
1136: H87341

1137: H84447
1138: H83199
1139: H82967
1140: H82912

1141: H80559
1142: H80508
1143: H74055
1144: H73434

1145: H73493
1146: H62035
1147: T29856
1148: T29711

1149: T29093
1150: T29091
1151: T28903
1152: T28486

1153: M37722
1154: R93497
1155: R93496
1156: R92862

1157: R92676
1158: R92588
1159: R91444
1160: R85021

1161: R84974
1162: R83219
1163: H45566
1164: H45559

1165: H42621
1166: H42118
1167: H26048
1168: H23526

1169: H11702
1170: H03123
1171: R81409
1172: R80670

1173: R80475
1174: R77173
1175: R77151
1176: U22410

1177: R71604
1178: R70205
1179: R68912
1180: U26555

1181: R59269
1182: L31408
1183: R54610
1184: R54846

1185: R48871
1186: R38513
1187: U03877
1188: R33868

1189: R28572
1190: R28404
1191: R25381
1192: U16306

1193: R13671
1194: R10619
1195: R10464
1196: R07270

1197: R07269
1198: T94993
1199: M73240
1200: M73239

1201: T94939
1202: T89898
1203: T89622
1204: T89263

1205: T84335
1206: T83836
1207: T83672
1208: T83170

1209: T82019
1210: T71565
1211: M60828
1212: U17170

1213: J03358
1214: M55614
1215: M87843
1216: M34057

1217: M96956
1218: M30493
1219: J03278
1220: M22734

1221: M17446
1222: M87772
1223: M87771
1224: M87770

1225: M64347
1226: M80635
1227: T12244
1228: T12243

1229: L01488
1230: L01486
1231: M85289
1232: L02931

1233: M23086
1234: M23017
1235: M17599
1236: J04513

1237: L01487
1238: M58051
1239: M97193
1240: M27968

1241: AH002695
1242: M30492
1243: M30491
1244: M30490

1245: L01485
1246: M74028
1247: M60516
1248: AH002592

1249: M60521
1250: M60520
1251: M60515
1252: AH002591

1253: M60519
1254: M60518
1255: AH001553
1256: M63978

1257: M63977
1258: M63976
1259: M63975
1260: M63974

1261: M63973
1262: M63972
1263: M63971
1264: M34667

1265: J02814
1266: M21616
1267: M55379
1268: M80638

1269: M80636
1270: M63889
1271: M63888
1272: M63887

1273: M60485
1274: M34188
1275: M34187
1276: M34186

1277: M34185
1278: L22970
1279: L22969
1280: L22968

1281: L22967
1282: J02683
1283: M78197

[0347]

8

TABLE 8

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to

numbers of human sequences identified as related to arachidonate metabolism

and/or signaling.

1: BC032594
2: NM_138318
3: NM_138317
4: NM_021161

5: NM_033311
6: NM_033310
7: NM_016611
8: BC029032

9: NT_008476
10: NT_004641
11: NT_033241
12: NT_033985

13: NT_033299
14: NT_010823
15: XM_113327
16: XM_115027

17: XM_165564
18: XM_091607
19: XM_034446
20: XM_071012

21: XM_036599
22: NT_033997
23: AJ305028
24: AJ305026

25: AJ305020
26: AJ305031
27: AJ305030
28: AJ305029

29: AJ305027
30: AJ305025
31: AJ305024
32: AJ305023

33: AJ305022
34: AJ305021
35: BC028174
36: AF468054

37: AF468053
38: AF468052
39: AF468051
40: NG_001072

41: NM_000775
42: U37143
43: NM_016601
44: AF039089

45: D12638
46: NM_022054
47: NM_001629
48: NM_004823

49: BI712628
50: BI712395
51: G73175
52: G73174

53: NM_013402
54: NM_023944
55: NM_022977
56: NM_004457

57: NM_004458
58: BF593874
59: BF589297
60: BF445948

61: NM_021628
62: BF435282
63: NM_003647
64: NM_001141

65: NM_000698
66: NM_001140
67: NM_001139
68: NM_000697

69: BF055436
70: BF002497
71: BE676451
72: BE676267

73: BE674834
74: AF221943
75: BE222781
76: BE222767

77: BE222760
78: AF226273
79: AW779220
80: AF247042

81: SEG_HUMCPLA
82: D38177
83: D38176
84: AW594003

85: AW518813
86: AW236332
87: AW169993
88: AB019692

89: AW087663
90: AW082242
91: AW081721
92: AW051026

93: AW044581
94: AW044543
95: AW026639
96: AW007295

97: AI922141
98: AI913434
99: AI911767
100: AI864921

101: AI830710
102: AI824788
103: AI804734
104: AI802680

105: AI799008
106: AI798007
107: AI768011
108: AI762841

109: AI762560
110: AI744699
111: AI698814
112: AI696859

113: AI660644
114: AI598073
115: AI572375
116: AI524200

117: AI523931
118: AI523842
119: AI479105
120: AI439947

121: AI436362
122: AI423500
123: AI372974
124: AI372944

125: AI371675
126: AI365403
127: AI363782
128: AI361850

129: AI360992
130: S68587
131: S68588
132: AI401142

133: AI400783
134: AI393821
135: AI393457
136: AI300995

137: AI288519
138: AI380545
139: AI243470
140: AA897232

141: AA860302
142: AA724768
143: AI282525
144: AI221308

145: AI219534
146: AI093644
147: AI219535
148: AI186139

149: AI148820
150: AI128268
151: AI168502
152: AI147982

153: AI142268
154: AI081242
155: AI075284
156: AI056468

157: U49379
158: AF038461
159: AI125083
160: AI123817

161: AI033442
162: AI025269
163: AA995910
164: AA994068

165: AA938017
166: AA931760
167: AA972081
168: AA922175

169: AA975447
170: AA926891
171: AA909607
172: AA904880

173: AA974928
174: AA961104
175: AA903058
176: AA873295

177: AA904309
178: AA825428
179: AA906097
180: AA905982

181: AA897656
182: AA835927
183: AA834872
184: AA876937

185: AA829467
186: AA810216
187: AA838239
188: AA872924

189: AA164575
190: AA629604
191: AA814032
192: AA835909

193: AA810409
194: AA806779
195: AA812165
196: AA811395

197: AA811107
198: AA765334
199: AA804368
200: AA748796

201: AA748538
202: AA748495
203: AA811906
204: AA808006

205: AA777140
206: AA741244
207: AA760798
208: AA761683

209: AA767202
210: AA765905
211: AA766333
212: AA767516

213: AA736656
214: AA748855
215: AA745655
216: AA743363

217: AA721294
218: AA737609
219: AA707722
220: AA122247

221: AA102430
222: AA702824
223: AA665475
224: AA652440

225: AA649213
226: AA613560
227: AA648464
228: AA632217

229: AA622768
230: AA593628
231: AA587388
232: AA587201

233: AA593920
234: AA569903
235: AA583219
236: AA552491

237: AA552112
238: AA521143
239: AA259174
240: AA228877

241: AA515026
242: AA505143
243: AA504178
244: AA504177

245: AA491374
246: AA279070
247: AA280714
248: AA281429

249: AA281261
250: AA258232
251: AA251106
252: AA262146

253: AA261947
254: AA487554
255: AA487262
256: AA548544

257: AA479055
258: AA410835
259: AA455503
260: AA455502

261: AA411551
262: AA411550
263: AA411441
264: AA411432

265: AA401645
266: AA398435
267: AA001754
268: AA355365

269: AA315865
270: AA021259
271: AA020955
272: AA018827

273: AA019064
274: N78045
275: AA013478
276: W81524

277: W47166
278: AA054258
279: W31083
280: W74172

281: M72393
282: N78291
283: N63856
284: N57659

285: N47673
286: N47638
287: N33729
288: H81930

289: H78331
290: H75692
291: H66675
292: H51574

293: H50910
294: R99246
295: T29353
296: R91299

297: H41485
298: H29144
299: H22440
300: H03094

301: R53728
302: R52945
303: R39192
304: R26797

305: R25994
306: R20635
307: R10655
308: T97526

309: T97446
310: T97387
311: T97276
312: T90253

313: T87977
314: T69964
315: T69914
316: T63581

317: T63549
318: T62206
319: T62015
320: T57850

321: M87004
322: M62982

[0348]

9

TABLE 9

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to

leukotriene metabolism and/or signaling.

1: BC029498
2: NT_008438
3: NT_004434
4: NT_033258

5: XM_088569
6: XM_060500
7: XM_033240
8: NT_011597

9: NT_033922
10: NT_006932
11: NT_025130
12: NT_011281

13: NT_010164
14: XM_065152
15: XM_065151
16: XM_029072

17: NM_080842
18: AX304816
19: AX304815
20: AX304814

21: AX304812
22: AX304811
23: AX304810
24: AX304809

25: AX304808
26: AX304807
27: AX304806
28: AX304804

29: AX250331
30: NM_001629
31: AX211656
32: U62025

33: AF133266
34: AC004597
35: BC004545
36: AF279611

37: AC005336
38: AU100177
39: AU099086
40: NM_001082

41: NM_000896
42: AL137118
43: BF939017
44: AL135787

45: AF308571
46: BF590658
47: BF590373
48: BF438819

49: BF438176
50: BF223033
51: NM_020377
52: NM_019839

53: NM_005036
54: NM_006639
55: NM_004121
56: NM_000897

57: NM_000752
58: NM_000895
59: BF114973
60: BF111542

61: BF109754
62: AB041644
63: BF001557
64: AF254664

65: AB044402
66: AB008193
67: AB029892
68: BE551649

69: AB038269
70: AF277230
71: BE468252
72: BE467347

73: BE465656
74: BE464525
75: BE208128
76: U02388

77: BE206519
78: AF221943
79: BE301515
80: AJ278605

81: BE222208
82: BE222016
83: BE042562
84: BE018008

85: AW780275
86: AW771680
87: AW769807
88: AW768775

89: AW768774
90: AB015307
91: SEG_AB01529S
92: AB015306

93: AB015305
94: AB015304
95: AB015303
96: AB015302

97: AB015301
98: AB015300
99: AB015299
100: AB015298

101: AB015297
102: AB015296
103: AB015295
104: SEG_AB002455S

105: AB002461
106: AB002460
107: AB002459
108: AB002458

109: AB002457
110: AB002456
111: AB002462
112: AB002455

113: AW663477
114: AU076907
115: AW615391
116: AW614119

117: AW612553
118: AW612542
119: AW594576
120: AW572845

121: AW518470
122: AW513073
123: AW474311
124: AW469906

125: AW418845
126: AW418767
127: AW339795
128: AW302266

129: AW301707
130: AW301232
131: AW300035
132: AW274396

133: AW236605
134: AW235789
135: AW235300
136: AW183518

137: AW173557
138: AW089665
139: AW087424
140: AW085086

141: AW075528
142: AW058452
143: AW051945
144: AW024508

145: AI985846
146: AI971682
147: AI962575
148: AI961053

149: AI942264
150: AI927415
151: AI921942
152: AI887357

153: AI867323
154: AI865127
155: D12620
156: D12621

157: AI819899
158: AI819721
159: AI819193
160: AI817081

161: AI810292
162: AI797155
163: AF119711
164: AI769908

165: AI769157
166: AI768316
167: AI767278
168: AI766909

169: AI743746
170: AI741766
171: AI697874
172: AI697850

173: AI696788
174: AI690919
175: AI680647
176: AI675321

177: AI674309
178: AI670926
179: AI658628
180: AI655883

181: AI654958
182: AI653619
183: AI650452
184: AI640249

185: AI638776
186: AI638615
187: AI637513
188: AI636026

189: AI635095
190: AI624995
191: AI621247
192: AI621085

193: AI598016
194: AI589108
195: AI582379
196: AI568633

197: AI567317
198: AI539521
199: AI539253
200: AI538292

201: AI521212
202: AI494342
203: AI498676
204: AI480325

205: AI478687
206: AI471212
207: AI470813
208: AI470397

209: AI476663
210: AI474060
211: AI458191
212: AI453742

213: AI434588
214: AI424409
215: AI419536
216: AI373285

217: AI373189
218: AI366863
219: AI203390
220: AI342740

221: AI299075
222: AI268038
223: AI276610
224: AI244788

225: AI379927
226: H49887
227: AI373191
228: AA868493

229: AA860804
230: AI254358
231: AI197820
232: AI242991

233: AI251847
234: AA995855
235: AI097442
236: AI159898

237: AI092835
238: AI051125
239: AI038752
240: AA938888

241: U77604
242: U50136
243: AH006631
244: U43411

245: U43410
246: AI129804
247: AI027805
248: AI023562

249: AI017689
250: AI017654
251: AI016629
252: AI015315

253: AA992816
254: AA977614
255: AA919105
256: AI095208

257: AI091347
258: AI081983
259: U65080
260: AI025313

261: AA991238
262: AA987920
263: AB002454
264: AC004609

265: AA857997
266: AA903138
267: AA896996
268: AA830693

269: AC004523
270: AA847890
271: AA227874
272: AA227873

273: AA857983
274: AA486929
275: AA628131
276: AA743405

277: AA100843
278: AA677046
279: AA703053
280: AA694114

281: AA649092
282: AA143730
283: AA658381
284: AA649335

285: AA626145
286: AA594870
287: AA582641
288: AA559954

289: AA534720
290: AA533595
291: AA565266
292: AA286910

293: AA513348
294: AA281397
295: AA465366
296: AA204704

297: D89079
298: D89078
299: AA452952
300: D49387

301: D26480
302: AA447884
303: AA443448
304: AA443313

305: AA411483
306: AA293255
307: AA291372
308: AA122237

309: AA115940
310: AA381256
311: AA381240
312: AA376869

313: AA375164
314: AA361649
315: AA347345
316: AA346986

317: AA333760
318: AA316671
319: AA314593
320: AA303424

321: AA298616
322: AA297531
323: AA297320
324: AA297314

325: AA296166
326: AD000091
327: N76885
328: N55276

329: AA101453
330: AA100471
331: AA135238
332: AA135125

333: AA011245
334: AA010417
335: W80460
336: W67534

337: W67533
338: W45520
339: W45533
340: X52195

341: R57602
342: N79883
343: N89761
344: N86553

345: N84188
346: N62977
347: AH003354
348: U27293

349: U27292
350: U27291
351: U27290
352: U27289

353: U27288
354: U27287
355: U27286
356: U27285

357: U27284
358: U27283
359: U27282
360: U27281

361: U27280
362: U27279
363: U27278
364: U27277

365: U27276
366: U27275
367: N47508
368: N47507

369: N46659
370: N46112
371: N46111
372: N40365

373: N27550
374: N25087
375: N24395
376: H99146

377: H98865
378: H98864
379: H65433
380: H95493

381: H94973
382: H65432
383: H70526
384: H59380

385: T29585
386: R86096
387: R83819
388: R83378

389: H45442
390: H45141
391: H27032
392: H11149

393: R73358
394: R43438
395: R43393
396: R41544

397: R39103
398: R37480
399: R33232
400: R22687

401: R17948
402: R15120
403: R14197
404: R11911

405: R11267
406: R11209
407: R08919
408: R08229

409: R02521
410: R00042
411: T98002
412: T85456

413: T85359
414: T84363
415: T77751
416: T77750

417: T58950
418: T58888
419: T55357
420: U11552

421: J02959
422: J03459
423: U09353

[0349]

10

TABLE 10

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to

interleukin metabolism and/or signaling.

1: BC032474
2: NM_012448
3: NM_003152
4: NM_003151

5: NM_005546
6: NM_001570
7: NM_145071
8: NM_013324

9: NM_003153
10: NM_033339
11: NM_033338
12: NM_003745

13: NM_004857
14: AF517934
15: BC030975
16: AY090769

17: NM_144701
18: AF293463
19: AF293462
20: NM_000155

21: NM_019009
22: NM_014339
23: AY099265
24: AF461422

25: NM_012455
26: AF512686
27: BC029569
28: BC029273

29: BC029493
30: BC029121
31: NT_009151
32: NT_009781

33: NT_009506
34: NT_009485
35: NT_009458
36: NT_010356

37: NT_029419
38: NT_011176
39: NT_008186
40: NT_011104

41: NT_024115
42: NT_008476
43: NT_004861
44: NT_004858

45: NT_030040
46: NT_005986
47: NT_005927
48: NT_004636

49: NT_005883
50: NT_006258
51: NT_004391
52: NT_030577

53: NT_029258
54: NT_028054
55: NT_021877
56: NT_016354

57: NT_015169
58: NT_033930
59: NT_033983
60: NT_033982

61: NM_138578
62: NM_001191
63: AY071841
64: AY071840

65: NM_032989
66: NM_004322
67: NM_006428
68: NT_010591

69: NT_010552
70: NT_010404
71: NT_011512
72: XM_114185

73: XM_090078
74: XM_006447
75: NT_011387
76: NT_033899

77: NT_010718
78: NT_010663
79: NT_007592
80: NT_011005

81: NT_033321
82: NT_030889
83: NT_028406
84: NT_028405

85: NT_025965
86: NT_025307
87: XM_034304
88: XM_055737

89: XM_059563
90: XM_010533
91: XM_040009
92: XM_113270

93: XM_116140
94: XM_165550
95: NM_032556
96: XM_064619

97: XM_085726
98: XM_084856
99: XM_061442
100: XM_067380

101: XM_086576
102: XM_029434
103: XM_089078
104: NT_011519

105: XM_066253
106: XM_062004
107: XM_062003
108: XM_063176

109: XM_035511
110: NT_011520
111: XM_049427
112: XM_027568

113: XM_028349
114: XM_032349
115: NM_032732
116: XM_013114

117: XM_015989
118: NM_016584
119: NM_012219
120: NM_007199

121: NM_004620
122: NM_004515
123: NT_025741
124: NT_011651

125: NT_009799
126: NT_007072
127: XM_098435
128: XM_085927

129: NT_006859
130: NT_025133
131: XM_115636
132: NT_006788

133: NT_011288
134: NT_011255
135: XM_035638
136: NT_011225

137: NT_010164
138: NT_023195
139: XM_096226
140: NT_016864

141: NT_033965
142: NT_005403
143: NT_005337
144: XM_115806

145: NT_005612
146: NT_005229
147: NT_005567
148: XM_087367

149: NT_005034
150: NT_022171
151: XM_002686
152: NT_019306

153: XM_114217
154: XM_114220
155: XM_031204
156: XM_031221

157: XM_034808
158: XM_008906
159: XM_004011
160: XM_004438

161: XM_002685
162: AF465829
163: BC027733
164: BC028082

165: BC028221
166: BC027599
167: NM_016123
168: NM_138284

169: AF213987
170: AF445802
171: AJ271338
172: AJ242738

173: AJ242737
174: AF276916
175: AF494012
176: NM_004512

177: NM_014439
178: NM_002994
179: NM_016026
180: NM_014143

181: NM_015650
182: NM_014438
183: NM_004103
184: NM_001561

185: NM_004513
186: NM_000628
187: NM_000577
188: NM_133336

189: NM_134470
190: NM_033307
191: NM_033306
192: NM_002182

193: NM_000635
194: NM_134433
195: NM_003268
196: NM_003264

197: NM_003263
198: AL136852
199: AF242456
200: NM_052872

201: AY078238
202: AF362378
203: AF481335
204: BC024747

205: BC025691
206: AY079002
207: AC007165
208: AF053412

209: L37036
210: NM_006504
211: NM_130435
212: AF469756

213: AF469755
214: AF469754
215: NM_001225
216: AF190052

217: AF172150
218: AF172149
219: NM_001560
220: AF093065

221: U58197
222: U58196
223: BC022315
224: AY071830

225: AL391280
226: BC020739
227: BC020717
228: NM_018725

229: NM_001247
230: NM_080591
231: NM_000962
232: NM_000963

233: AF247608
234: AF247607
235: AF247606
236: AF247605

237: AF247604
238: AF247603
239: AY029413
240: AJ297262

241: AY064474
242: NM_022304
243: AL121878
244: NM_004448

245: NM_003680
246: NM_002051
247: NM_001465
248: NM_001806

249: AF077611
250: NM_030804
251: NM_021258
252: NM_018402

253: AF206696
254: AF230377
255: AF039224
256: NM_004926

257: AL158080
258: AY062931
259: AB017505
260: SEG_HUMIL3RA

261: D49412
262: D49410
263: D49408
264: D49409

265: D49407
266: D49406
267: D49404
268: D49403

269: D49402
270: D49401
271: D49411
272: D49405

273: AF416600
274: NM_005755
275: AF054013
276: BC009681

277: BC015768
278: BC014972
279: NM_052962
280: NM_052887

281: BC016141
282: BC009572
283: AF420465
284: AF420464

285: AF420463
286: NM_004347
287: BC015863
288: AF417842

289: AF401315
290: AF384857
291: U57613
292: AF421855

293: AJ289235
294: BC015511
295: X78437
296: NM_000575

297: AF302043
298: AF302042
299: AJ277248
300: AY008847

301: AY008332
302: AY008331
303: AF276915
304: AH008153

305: AF146427
306: AF146426
307: AF172151
308: L41142

309: AF418271
310: NM_033358
311: NM_033357
312: NM_033356

313: NM_033355
314: NM_001228
315: NM_033340
316: NM_001227

317: BC014096
318: AF349574
319: BC013615
320: NM_033295

321: NM_033294
322: NM_033293
323: NM_033292
324: NM_001223

325: U63015
326: AY044641
327: BC013142
328: BC012506

329: AY040367
330: BC012580
331: BC012346
332: AF005485

333: AY040568
334: AY040567
335: AY040566
336: AF404773

337: AF402002
338: BC012071
339: BC011624
340: AF346607

341: NM_032977
342: NM_032976
343: NM_032974
344: NM_001230

345: NM_032992
346: NM_001226
347: NM_032996
348: NM_001229

349: NM_004346
350: NM_032991
351: BC009960
352: BC009745

353: BC008678
354: AY026753
355: BC007461
356: BC007007

357: BC001770
358: BC005823
359: BC004973
360: BC004348

361: BC003110
362: BC001903
363: BC000382
364: AF395008

365: NM_004759
366: NM_032960
367: NM_006850
368: AF334756

369: AF334755
370: NM_006134
371: AF390905
372: AF386077

373: AF385628
374: AF387519
375: AF366364
376: AF366363

377: AF366362
378: AF377331
379: AF372214
380: AF365976

381: AF380360
382: AL135902
383: AF251120
384: AF251119

385: AF251118
386: U91746
387: AJ293654
388: AJ293653

389: AJ293652
390: AJ293651
391: AJ293650
392: AJ293649

393: AJ293648
394: AJ293647
395: AY029171
396: AF361105

397: AF359939
398: AF353265
399: NM_004248
400: AL035252

401: NM_030751
402: NM_002183
403: NM_002186
404: AF295024

405: S61784
406: NM_000104
407: NM_018724
408: Z30175

409: NM_014432
410: S81601
411: S71404
412: AJ271747

413: AJ271746
414: AJ271745
415: AJ271744
416: AJ271741

417: AF283296
418: NM_016232
419: NM_020525
420: NM_012218

421: NM_004516
422: NM_003856
423: AF043337
424: AF228636

425: AF224266
426: NM_022789
427: AF203083
428: AF114158

429: AF305200
430: U52112
431: AF218727
432: AF218728

433: AJ277247
434: AF110385
435: AF301620
436: U64198

437: AF079806
438: NM_017416
439: NM_021803
440: NM_021798

441: AF254069
442: AF254067
443: NM_002309
444: NM_021571

445: NM_005699
446: NM_000585
447: NM_000586
448: NM_000576

449: NM_000572
450: NM_000564
451: NM_000641
452: NM_000640

453: NM_000600
454: NM_000590
455: NM_000584
456: NM_020994

457: NM_006705
458: NM_019618
459: NM_018949
460: NM_014271

461: NM_014443
462: NM_014440
463: NM_005565
464: NM_002298

465: NM_013371
466: NM_013278
467: NM_012275
468: NM_012099

469: NM_006664
470: NM_006165
471: NM_005535
472: NM_005384

473: NM_005263
474: NM_004590
475: NM_004514
476: NM_004633

477: NM_001569
478: NM_000395
479: NM_000206
480: NM_000215

481: NM_000418
482: NM_000417
483: NM_001192
484: NM_002852

485: NM_003954
486: NM_003749
487: NM_001557
488: NM_000634

489: NM_002185
490: NM_000880
491: NM_002184
492: NM_000565

493: NM_000879
494: NM_000589
495: NM_000588
496: NM_000878

497: NM_003854
498: NM_000877
499: NM_003853
500: NM_003855

501: NM_001562
502: NM_002190
503: NM_002189
504: NM_002188

505: NM_001559
506: NM_002187
507: NM_000882
508: NM_001558

509: NM_001504
510: NM_001901
511: U55847
512: AF208005

513: AF269133
514: AF212016
515: AF284436
516: AF284435

517: AF284434
518: AF286095
519: AF279437
520: AF176907

521: L07295
522: AJ295724
523: AF244575
524: AF242300

525: AF193840
526: AF193839
527: AF193838
528: AF276953

529: AF121105
530: AF202445
531: AJ271736
532: AF035279

533: AJ242972
534: AF212311
535: AF235038
536: AF216693

537: AF045606
538: AF039906
539: AF167342
540: AF167341

541: AF167340
542: AF167339
543: AF167338
544: AF167337

545: AF167336
546: AF167335
547: AF167334
548: AF167333

549: AH009309
550: AF167343
551: AF200496
552: AF200494

553: AF200492
554: AF030876
555: AB015961
556: AB015021

557: D82874
558: D31968
559: D16358
560: D14283

561: AJ251550
562: AJ251551
563: AJ251549
564: AF215907

565: AF181286
566: AF181285
567: AF181284
568: AJ272096

569: U62858
570: U48258
571: U48257
572: U48256

573: AF098934
574: AF098933
575: AL034343
576: D11086

577: AF152099
578: AF152098
579: AF177937
580: AF201833

581: AF201832
582: AF201831
583: AF201830
584: AB022176

585: U67206
586: AF031075
587: AL022314
588: AB010445

589: AL031575
590: Z72522
591: Z69719
592: AF152113

593: AC004525
594: AJ012835
595: AJ012834
596: AJ012833

597: AF186094
598: AF038163
599: AF029213
600: AF180563

601: AF180562
602: AF001862
603: AJ243874
604: U81379

605: AF113136
606: AF168416
607: J00264
608: AF017633

609: U81380
610: AF118452
611: AF005095
612: U58146

613: AF039904
614: AF039905
615: AF039907
616: S77834

617: AF077011
618: D64068
619: AB019504
620: X06750

621: AJ005835
622: X67285
623: AF110801
624: AF110800

625: AF110799
626: AF110798
627: AF110460
628: AF101062

629: AH007439
630: AF085452
631: AF085451
632: U43895

633: AF054830
634: S81555
635: L27475
636: AH007359

637: S77835
638: S71420
639: 551359
640: S71419

641: S56892
642: AF069543
643: AF083251
644: AF043938

645: AF017653
646: U94587
647: U93690
648: U74649

649: U63127
650: AF104230
651: AH007043
652: AF043129

653: AF043128
654: AF043127
655: AF043126
656: AF043125

657: AF043124
658: AF043123
659: X53093
660: AF077346

661: AH006906
662: M29053
663: M29052
664: M29051

665: M29050
666: M29049
667: M29048
668: S72848

669: AF035593
670: AF035592
671: U67320
672: U67319

673: U60521
674: U60519
675: U60520
676: U47686

677: U43672
678: U40281
679: U32659
680: U31628

681: U37449
682: U37448
683: U32674
684: U32672

685: U20537
686: U20536
687: U23852
688: U20240

689: U13700
690: U13699
691: U13698
692: U13697

693: L76191
694: M54894
695: AF043143
696: AF016261

697: L10616
698: L19546
699: AF051152
700: AF051151

701: U88881
702: U88880
703: U88879
704: U88878

705: U88540
706: AC005578
707: L39064
708: AF078533

709: AJ002523
710: AF029894
711: AC004763
712: M99412

713: AF057168
714: AB006537
715: AC004511
716: AF048692

717: AF050083
718: M98335
719: AF043336
720: AF043335

721: AF043334
722: AF043333
723: U58917
724: AC004039

725: AC004042
726: AF031167
727: D13720
728: AF039228

729: AF039227
730: AF039226
731: AF039225
732: D00044

733: X01586
734: AF026273
735: AF031845
736: AC003112

737: X97748
738: AF023338
739: AF021799
740: AF008556

741: X64532
742: X65858
743: Z70243
744: AH005384

745: U11869
746: U11868
747: U11867
748: U11866

749: U18373
750: U13738
751: K03122
752: L19593

753: L19591
754: Y08768
755: D28118
756: Y09908

757: U97679
758: U97678
759: U97677
760: U97676

761: U82972
762: U49065
763: D78260
764: U90652

765: U89323
766: X80878
767: X69079
768: X03131

769: S82692
770: U86214
771: L39063
772: L39062

773: Z84723
774: M63099
775: X91233
776: U78798

777: U32324
778: U32323
779: S81089
780: S79880

781: S67780
782: S36271
783: S36219
784: S75511

785: S75512
786: S75513
787: S75514
788: S75515

789: S75516
790: S75517
791: S64248
792: X99404

793: U70981
794: X94223
795: X94222
796: Z58820

797: U43185
798: L78780
799: L78779
800: L78778

801: L78777
802: L78776
803: L78775
804: L78774

805: L78773
806: L78770
807: L78760
808: L78754

809: L78753
810: L78751
811: L78752
812: L78750

813: L78746
814: L78745
815: L78744
816: L78743

817: L78742
818: U64094
819: X95302
820: U58198

821: U31120
822: Z14320
823: Z14319
824: Z14318

825: Z14317
826: Z14954
827: X04664
828: X05232

829: X62156
830: X63053
831: X63613
832: Z48810

833: X52430
834: Y00787
835: X13967
836: X65859

837: Z11686
838: X81851
839: X60787
840: X04602

841: X12830
842: X61176
843: X61178
844: X61177

845: X04688
846: X52425
847: X03138
848: X03137

849: X03136
850: X03135
851: X03134
852: X03133

853: X03132
854: X01057
855: X84348
856: Z38000

857: Z46595
858: Z38102
859: Z46596
860: Z14955

861: X16896
862: X59770
863: X02851
864: X65019

865: X02532
866: X02531
867: X03833
868: X00695

869: V00564
870: X77090
871: X58298
872: X53296

873: X52015
874: X64802
875: Z47277
876: Z47276

877: Z47275
878: Z47274
879: Z47273
880: Z47272

881: Z47271
882: Z47270
883: Z47269
884: Z47268

885: Z47267
886: Z47266
887: Z47265
888: Z47264

889: Z47263
890: Z47262
891: Z47261
892: Z47260

893: Z47259
894: Z47258
895: Z47257
896: Z47256

897: Z47255
898: Z47254
899: Z47253
900: Z47252

901: Z47251
902: Z47250
903: Z47249
904: Z47248

905: Z47247
906: Z47246
907: Z47245
908: Z47244

909: X58377
910: K02056
911: J02971
912: X94993

913: U41806
914: L08187
915: L77073
916: L77072

917: L77071
918: L77070
919: L77069
920: L77068

921: L77067
922: L77060
923: L77044
924: L77040

925: L77039
926: L77036
927: L77035
928: L77034

929: L77033
930: L77032
931: L77031
932: X73536

933: M87879
934: U25804
935: U10307
936: M73969

937: L49046
938: U16720
939: L48479
940: L48478

941: L48477
942: L48476
943: L48475
944: L48474

945: L48473
946: L48472
947: U14750
948: U28015

949: U28014
950: L46904
951: L46900
952: L46899

953: J03478
954: M15840
955: U25676
956: L43412

957: L43411
958: L43399
959: L43398
960: L43393

961: L43392
962: L43391
963: L43387
964: L43386

965: U26540
966: AH003109
967: M11065
968: M11066

969: M11064
970: M11063
971: M11062
972: M11061

973: M11060
974: M10322
975: M87507
976: L42104

977: L42103
978: L42102
979: L42098
980: L42097

981: L42096
982: L42095
983: L42094
984: L42091

985: L42090
986: L42089
987: L42088
988: L42087

989: L42086
990: L42085
991: L42080
992: L42079

993: L42078
994: U13737
995: U11878
996: U11877

997: U11876
998: U11875
999: U11874
1000: U11873

1001: U11872
1002: U11871
1003: U11870
1004: J02923

1005: M57627
1006: M91557
1007: L19592
1008: M94654

1009: M15864
1010: M86593
1011: M97502
1012: M68932

1013: M28130
1014: AH002843
1015: L12183
1016: L12182

1017: L12181
1018: L12180
1019: L12179
1020: L12177

1021: L12176
1022: L12178
1023: M29696
1024: J04156

1025: M29150
1026: M22111
1027: M96652
1028: M96651

1029: M23442
1030: M13982
1031: M60870
1032: M74782

1033: M20137
1034: M14743
1035: M16285
1036: M26062

1037: M32979
1038: M14098
1039: M13879
1040: M22005

1041: AH002842
1042: M33198
1043: M33199
1044: M97748

1045: M55646
1046: M27492
1047: M54933
1048: M15330

1049: M28983
1050: M15329
1051: M81890
1052: M57765

1053: U13022
1054: U13021
1055: M84747
1056: L05921

1057: U16031
1058: U06844
1059: M18403
1060: J03049

1061: M14584
1062: M75914
1063: M94582
1064: L09701

1065: M13784
1066: L13029
1067: L06801
1068: K02770

1069: L07488
1070: M17115
1071: M65272
1072: M65271

1073: U14407
1074: U10324
1075: U10323
1076: U03688

1077: U00672
1078: U08191

[0350]

11

TABLE 11

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to G-

protein-coupled receptors metabolism and/or signaling.

1: AX429467
2: AX429465
3: AX427634
4: NM_021634

5: AX417288
6: AX417287
7: AX417286
8: AX417285

9: AX417284
10: AX417283
11: AX417281
12: AX417279

13: NM_144766
14: NM_002927
15: NM_013936
16: AX411685

17: AX411548
18: AX411478
19: AX411477
20: AX411476

21: AX411475
22: AX411474
23: AX411473
24: AX411472

25: AX411471
26: AX411470
27: AX411469
28: AX411468

29: AX411467
30: AX411464
31: AX407143
32: AX407142

33: AX407139
34: AX404911
35: NM_144773
36: BC030948

37: NM_002921
38: AF369708
39: AF232905
40: L12116

41: NM_032554
42: NM_004054
43: NM_005300
44: NM_054021

45: AX399470
46: AX399466
47: NM_139201
48: NM_057170

49: NM_057169
50: NM_014776
51: NM_139209
52: NM_017572

53: NM_013345
54: NM_006564
55: NM_004778
56: D17516

57: D13168
58: D13167
59: D13166
60: D13165

61: D13164
62: D13163
63: D13162
64: D11151

65: D11150
66: D11149
67: D11148
68: D11147

69: D11146
70: D11145
71: D11144
72: AF385432

73: AF385431
74: AB083632
75: AB083631
76: AB083630

77: AB083629
78: AB083628
79: AB083627
80: AB083626

81: AB083625
82: AB083624
83: AB083623
84: AB083622

85: AB083621
86: AB083620
87: AB083619
88: AB083618

89: AB083617
90: AB083616
91: AB083615
92: AB083614

93: AB083613
94: AB083612
95: AB083611
96: AB083610

97: AB083609
98: AB083608
99: AB083607
100: AB083606

101: AB083605
102: AB083604
103: AB083603
104: AB083602

105: AB083601
106: AB083600
107: AB083599
108: AB083598

109: AB083597
110: AB083596
111: AB083595
112: AB083594

113: AB083593
114: AB083592
115: AB083591
116: AB083590

117: AB083589
118: AB083588
119: AB083587
120: AB083586

121: AB083585
122: AB083584
123: AB083583
124: AX395171

125: AX395169
126: NM_018485
127: BC030147
128: BC029363

129: NT_009368
130: NT_009307
131: NT_009770
132: NT_009731

133: NT_009714
134: NT_030828
135: NT_009528
136: NT_009485

137: NT_009464
138: NT_008902
139: NT_011176
140: NT_011148

141: NT_011139
142: NT_011109
143: NT_011091
144: NT_024064

145: NT_030032
146: NT_023868
147: NT_008438
148: NT_004858

149: NT_019483
150: NT_004836
151: NT_004668
152: NT_004612

153: NT_005849
154: NT_005832
155: NT_005825
156: NT_006302

157: NT_004434
158: NT_006216
159: NT_004350
160: NT_005527

161: NT_004308
162: NT_006081
163: NT_006051
164: NT_025667

165: NT_028053
166: NT_026943
167: NT_022411
168: NT_033903

169: NT_033902
170: NT_033900
171: NT_022454
172: NT_022740

173: AY089976
174: 20143796
175: 20142348
176: NM_078473

177: NM_031940
178: NM_032027
179: NM_007264
180: AC008115

181: NM_003717
182: NT_024812
183: XM_115412
184: NT_024776

185: XM_064062
186: XM_165649
187: NT_010393
188: XM_061650

189: XM_089844
190: XM_045812
191: XM_085672
192: XM_089954

193: XM_089955
194: NT_011333
195: NT_033302
196: XM_115586

197: NT_010672
198: NT_007592
199: XM_167160
200: XM_167080

201: XM_167214
202: XM_167129
203: NT_033363
204: NT_009702

205: XM_115948
206: XM_114696
207: XM_090428
208: NT_033340

209: XM_166070
210: NT_033321
211: NT_009563
212: NT_028405

213: NT_007422
214: XM_090326
215: XM_015921
216: NT_011793

217: NT_011786
218: NT_033944
219: XM_061555
220: XM_005969

221: XM_085864
222: XM_085103
223: XM_070357
224: XM_097508

225: XM_067593
226: XM_003091
227: XM_001499
228: XM_068013

229: XM_093332
230: XM_115096
231: XM_115095
232: XM_115094

233: XM_115082
234: XM_115600
235: XM_116729
236: XM_166794

237: XM_166195
238: XM_113529
239: XM_116678
240: XM_116151

241: XM_116127
242: XM_113420
243: XM_116279
244: XM_114092

245: XM_057872
246: XM_115966
247: NM_138964
248: NM_130806

249: NM_031936
250: XM_045532
251: XM_006549
252: XM_089843

253: XM_060898
254: XM_010608
255: XM_086232
256: NM_080818

257: XM_066873
258: XM_066104
259: XM_064958
260: XM_064909

261: XM_064908
262: XM_047911
263: XM_062248
264: NT_030871

265: XM_064220
266: XM_068231
267: XM_060177
268: XM_057984

269: NT_011520
270: NM_020960
271: XM_001907
272: XM_009140

273: XM_001543
274: NM_020400
275: NM_013308
276: NM_006056

277: NM_004767
278: NT_011719
279: NT_011669
280: NT_025741

281: NT_009799
282: NT_033922
283: NT_019424
284: NT_024524

285: NT_006859
286: NT_009984
287: NT_011296
288: NT_011295

289: NT_011294
290: NT_009952
291: NT_011277
292: XM_044591

293: NT_011268
294: NT_011258
295: NM_000710
296: NT_026437

297: NT_007968
298: NT_007933
299: NT_010164
300: NT_028179

301: XM_057299
302: NT_023085
303: NT_029366
304: NT_005472

305: NT_005403
306: NT_005370
307: NT_005367
308: NT_005612

309: XM_067401
310: NT_005204
311: NT_005151
312: XM_115784

313: XM_051522
314: NT_005079
315: NT_005034
316: XM_115750

317: NT_022140
318: XM_115681
319: XM_116850
320: XM_092364

321: XM_007392
322: XM_018505
323: XM_096288
324: XM_092406

325: XM_086954
326: XM_066655
327: XM_062863
328: XM_066605

329: XM_063192
330: XM_033082
331: XM_068829
332: NM_053278

333: XM_057250
334: XM_003736
335: XM_046588
336: XM_033529

337: XM_010228
338: XM_002624
339: NM_080819
340: NM_080817

341: NM_030784
342: AF502962
343: NM_005302
344: BC028163

345: BC027597
346: AF498922
347: AF498919
348: AF498918

349: AF498917
350: AF498916
351: AF498915
352: NM_002054

353: AF502281
354: NG_001272
355: NG_001217
356: NG_001132

357: NG_001131
358: AF498961
359: AF498921
360: AF498920

361: AF458154
362: AF458153
363: AF458152
364: AF458151

365: AF458150
366: AF458149
367: AH011576
368: NM_005458

369: BC026357
370: NM_018969
371: NM_007227
372: NM_005682

373: NM_030774
374: NM_018697
375: NM_001337
376: NM_032119

377: AF293323
378: AF293322
379: AH011557
380: AX393069

381: AX392789
382: AX385030
383: AX391087
384: AX391083

385: AX385042
386: AX385040
387: AX385037
388: AX385035

389: AX385032
390: AX385027
391: AX384675
392: AX384666

393: AX384665
394: AX384664
395: AX384663
396: AX384661

397: AX384211
398: AX384210
399: AX384209
400: AX384207

401: AX379474
402: AX379473
403: AX379472
404: AX379470

405: AX379468
406: AX378810
407: AX378806
408: AX378804

409: AX378802
410: AC078860
411: BC025695
412: AF474992

413: AF474991
414: AF474990
415: AF474989
416: AF474988

417: AF474987
418: AX376587
419: AX376585
420: AX376583

421: AX376581
422: AX376579
423: AX376577
424: AX376575

425: BI480949
426: AX365511
427: AX369353
428: AX369349

429: AX369310
430: NM_006794
431: AF439409
432: AX365515

433: AX365514
434: AX360197
435: AX360195
436: AX358252

437: AX357037
438: BM503956
439: NM_057159
440: NM_001401

441: AH003177
442: L31584
443: L31583
444: L31582

445: NM_054032
446: NM_054031
447: NM_054030
448: BD010057

449: BD010056
450: BD010055
451: BD010054
452: BD010053

453: BD010052
454: BD010051
455: BD010050
456: BD010049

457: BD010046
458: BD010035
459: BD010034
460: BD010028

461: BD010022
462: E51301
463: E51300
464: E51299

465: E51298
466: E51297
467: E51296
468: E50838

469: E50837
470: E50836
471: E50835
472: E50834

473: E50833
474: BD003056
475: E55122
476: E55121

477: E55120
478: E55119
479: E55118
480: E55117

481: E58499
482: E58495
483: E58494
484: E58488

485: E58485
486: E58484
487: E58479
488: E44151

489: E44032
490: AX356204
491: AX355996
492: AX355871

493: AX355868
494: AX355867
495: AX355841
496: AX355837

497: AX354961
498: AX354959
499: AX353651
500: AX353650

501: AX353649
502: AX353643
503: AX351008
504: AX350707

505: AX350705
506: AX350702
507: AX350701
508: AX350698

509: AX350697
510: AX350694
511: AX350693
512: AX350689

513: AX350686
514: AX350685
515: AX350683
516: AX350679

517: AX350675
518: AX350673
519: AX350672
520: AX350669

521: AX350668
522: AX350664
523: AX350663
524: AX350661

525: AX350659
526: AX350653
527: AX350651
528: AX350647

529: AX350645
530: AX350643
531: AX350641
532: AX350639

533: AX350637
534: AX350635
535: AX350633
536: AX350631

537: AX350629
538: AX350627
539: AX350625
540: AX350623

541: AX350374
542: AX350372
543: AX343924
544: AX343922

545: AX343921
546: AX343917
547: AF453828
548: NM_023915

549: NM_018490
550: NM_003667
551: NM_016235
552: NM_006055

553: BC021553
554: BC020752
555: BC020614
556: BC020678

557: AJ298292
558: AX342691
559: AX342465
560: NM_030760

561: AX339742
562: AX339740
563: AX338965
564: AX338964

565: AX338963
566: AX338960
567: AX338958
568: AX338219

569: AX338078
570: AX338076
571: AX329226
572: AX327312

573: AX327310
574: AF258342
575: AF435925
576: NM_019888

577: NM_000795
578: NM_016574
579: AY062031
580: AY062030

581: AX318782
582: AX317852
583: AX317850
584: AX317848

585: AX317846
586: AX317844
587: AX317842
588: AX317840

589: AX317838
590: AX317836
591: AX317834
592: AX317832

593: AX317830
594: AX317828
595: AX317826
596: AX316190

597: AX316189
598: NM_078474
599: NM_025141
600: NM_014286

601: AX305114
602: AX305113
603: AX305111
604: L78805

605: NM_032966
606: NM_001716
607: NM_004951
608: NM_022304

609: NM_007232
610: NM_005307
611: NM_004230
612: NM_001841

613: NM_025195
614: AF257182
615: NM_007369
616: NM_007223

617: NM_006018
618: AL590083
619: AF411117
620: AF411116

621: AF411115
622: AF411114
623: AF411113
624: AF411112

625: AF411111
626: AF411110
627: AF411109
628: AF411108

629: AF411107
630: AK056697
631: AK056040
632: AX276991

633: AX276989
634: AX275089
635: AX275088
636: AX275087

637: AX275085
638: AX275083
639: AX268495
640: AX268494

641: AX268493
642: AX268492
643: AX268491
644: AX268489

645: AX262404
646: AX262402
647: AX259499
648: AX259498

649: AX259496
650: AX259494
651: AF406692
652: NM_023922

653: NM_023921
654: NM_023920
655: NM_023919
656: NM_023918

657: NM_023917
658: AL445495
659: BM141985
660: NM_000675

661: AX299707
662: AX299705
663: AX299475
664: AX299473

665: AX298070
666: BM129715
667: BM129426
668: BM128329

669: AF282269
670: AX286290
671: AX286289
672: AX286288

673: AX286287
674: AX286286
675: AX286285
676: AX286284

677: AX286283
678: AX286282
679: AX286281
680: AX286280

681: AX286279
682: AX286278
683: AX286277
684: AX286276

685: AX286275
686: AX286274
687: AX286272
688: AX283620

689: BM091360
690: BM091055
691: AF310685
692: AY033942

693: BC016860
694: BM053023
695: BM052746
696: AX282666

697: AX282663
698: AX282661
699: AX282660
700: AX282659

701: AX282658
702: AX282656
703: AX282654
704: AX282380

705: AX282378
706: AX282376
707: AX282374
708: AX282372

709: AX282370
710: AX282369
711: AX282367
712: AX282365

713: AX282363
714: AX282361
715: AX282359
716: AX282357

717: AX282355
718: AX282353
719: AX282351
720: AX281258

721: AX281256
722: AX277635
723: NM_053036
724: NM_032551

725: NM_000798
726: NM_000794
727: NM_014879
728: NM_000797

729: BI962766
730: BC009540
731: AF055084
732: AX254762

733: AX254760
734: AX254742
735: AX254632
736: AX254348

737: AX253448
738: AX253256
739: NM_033050
740: NM_023914

741: NM_020370
742: NM_005756
743: AX253152
744: AX253150

745: AX253148
746: AX253146
747: AX252471
748: AX252469

749: AX252467
750: AX252386
751: AX252384
752: AX252382

753: AX250688
754: AX250685
755: AX250683
756: AX250547

757: AX250545
758: AX250543
759: AX250541
760: AX250539

761: AX250331
762: AF303576
763: AY008280
764: BI792406

765: BI789257
766: AX240018
767: AX240016
768: AX240014

769: AX240012
770: AX240010
771: AX240008
772: AX240004

773: AX240002
774: AX240000
775: AX239998
776: AX239996

777: AX239993
778: AX239991
779: AX239989
780: AX239987

781: AX239985
782: AX239983
783: AX239981
784: AL035542

785: NM_000024
786: NM_000683
787: NM_000682
788: NM_000681

789: BI715205
790: BI712099
791: AF399937
792: AY029541

793: AY042216
794: AY042215
795: AY042214
796: AY042213

797: AX235352
798: AX235351
799: AX235350
800: AX235348

801: AX235262
802: AX235260
803: Y11395
804: AX214118

805: AX214117
806: AX214110
807: AX214107
808: AX214105

809: AX214103
810: AX214101
811: AX214099
812: AX214097

813: AX214095
814: AX214093
815: AX214091
816: AX214089

817: AX214087
818: AX211539
819: NM_000678
820: NM_000679

821: NM_033304
822: NM_033303
823: NM_033302
824: NM_000680

825: AX208080
826: AX208078
827: AX208076
828: AF317654

829: AF330055
830: AF330053
831: AF190501
832: AF190500

833: AJ309020
834: BC011634
835: AF343725
836: AF380193

837: AF380192
838: AF380189
839: AF380185
840: BC011349

841: NM_005292
842: AF395806
843: NM_032503
844: BC008770

845: AF345566
846: AF345565
847: BC008094
848: BC004555

849: BC004925
850: BC003187
851: BC000181
852: BC001736

853: BC001379
854: BC009277
855: AL121581
856: AX167470

857: AX167242
858: AF279611
859: AX163735
860: AX151331

861: AX151329
862: AX151327
863: AX151325
864: AX151323

865: AX151321
866: AX151319
867: AX151264
868: AX151263

869: AX151262
870: AX151260
871: AX151258
872: AX151256

873: AX151254
874: AX151252
875: AX151250
876: AX151248

877: AX151246
878: AX151244
879: AX151242
880: AX151240

881: AX151238
882: AX151236
883: AX151232
884: AX151230

885: AX151228
886: AX151226
887: AX151224
888: AX151222

889: AX151220
890: AX151218
891: AX151216
892: U73141

893: AF236083
894: AX139466
895: AX139465
896: AX139463

897: AX139441
898: AX139440
899: AX139438
900: AX139122

901: AX139121
902: AX139120
903: AX139119
904: AX139118

905: AX139117
906: AX139116
907: AX139115
908: AX139113

909: AX139112
910: AX139111
911: AX139110
912: AX139109

913: AX139107
914: AX139103
915: AX138881
916: AX138880

917: AX138878
918: AX138829
919: AX138796
920: AX138589

921: AX138588
922: AX138586
923: AB051065
924: AF347063

925: AX135421
926: AX134204
927: AH003248
928:U40771

929: AB060151
930: NM_031409
931: NM_004367
932: AK027784

933: AK027780
934: AF209923
935: AF207989
936: NM_018980

937: NM_016945
938: AF363791
939: AX109244
940: AX109242

941: AX109240
942: AX109238
943: AX109236
944: AX109234

945: AX107042
946: AX107041
947: AX107037
948: AF329449

949: AY029324
950: AF346711
951: AF346710
952: AF346709

953: AH010608
954: NM_030968
955: AU100154
956: AU099841

957: AU099821
958: AU099377
959: AU098961
960: AF295368

961: AF237763
962: AF237762
963: NM_004248
964: AX099247

965: AF348078
966: NM_019599
967: AX088165
968: AX087894

969: AX087885
970: NM_016944
971: NM_016943
972: AB038237

973: AF178982
974: AF321815
975: AL121755
976: BG370235

977: U48958
978: AX081250
979: AX081248
980: AX081246

981: AX080495
982: AX077889
983: AF317655
984: AF317653

985: AF317652
986: AX077691
987: NM_022036
988: NM_018653

989: NM_018654
990: AF312230
991: NM_001400
992: AF316895

993: AX076182
994: NM_000916
995: AF316894
996: NM_018971

997: NM_005242
998: NM_016334
999: NM_016602
1000: NM_000115

1001: NM_002980
1002: NM_003991
1003: BG150191
1004: AX068839

1005: BG057775
1006: BG057661
1007: BF941117
1008: BF940605

1009: BF939693
1010: AF313449
1011: BF733007
1012: BF732711

1013: BF732412
1014: NM_003979
1015: AJ272138
1016: NM_012152

1017: AF285095
1018: AF285094
1019: AF285093
1020: AL137000

1021: AF268899
1022: AF268898
1023: Y19228
1024: Y19231

1025: Y19230
1026: Y19229
1027: AJ272207
1028: AF311306

1029: NM_004885
1030: BF594242
1031: BF592107
1032: BF591300

1033: BF588506
1034: AF292402
1035: AL096774
1036: AF317676

1037: BF477409
1038: BF476145
1039: NM_022049
1040: AF281308

1041: BF447902
1042: BF447858
1043: BF447783
1044: BF446953

1045: BF446952
1046: AF205437
1047: BF439382
1048: BF439363

1049: BF435092
1050: BF434415
1051: BF434140
1052: BF432690

1053: BF432379
1054: BF431669
1055: BF431528
1056: AX041939

1057: AX041937
1058: AX041935
1059: AX041933
1060: AX041931

1061: AX041929
1062: AX041927
1063: AX041925
1064: AX041923

1065: AJ249248
1066: AB042411
1067: AB042410
1068: NM_004720

1069: NM_005226
1070: AF307973
1071: NM_005508
1072: NM_005283

1073: BF195014
1074: AF197929
1075: AF280400
1076: AF280399

1077: NM_018970
1078: NM_018949
1079: NM_016568
1080: NM_016540

1081: NM_014030
1082: NM_014626
1083: NM_014627
1084: NM_014373

1085: NM_013937
1086: NM_013941
1087: NM_001992
1088: NM_001526

1089: NM_006583
1090: NM_006143
1091: NM_005683
1092: NM_005684

1093: NM_000054
1094: NM_005308
1095: NM_005286
1096: NM_005285

1097: NM_005284
1098: NM_005282
1099: NM_005306
1100: NM_005305

1101: NM_005304
1102: NM_005303
1103: NM_005281
1104: NM_005301

1105: NM_005299
1106: NM_005298
1107: NM_005297
1108: NM_005296

1109: NM_005295
1110: NM_005294
1111: NM_005293
1112: NM_005279

1113: NM_005291
1114: NM_005290
1115: NM_005288
1116: NM_005161

1117: NM_005048
1118: NM_004224
1119: NM_004246
1120: NM_004072

1121: NM_001525
1122: NM_003272
1123: NM_003608
1124: NM_003485

1125: NM_000910
1126: NM_000752
1127: NM_000868
1128: NM_002082

1129: NM_001504
1130: NM_001508
1131: NM_001507
1132: NM_001506

1133: NM_001505
1134: NM_000164
1135: NM_003775
1136: NM_001838

1137: NM_000674
1138: AB019000
1139: AH007076
1140: AF019765

1141: AF019764
1142: AF272363
1143: AF272362
1144: BF109118

1145: BF062418
1146: BF061464
1147: BF061085
1148: BF060724

1149: BF058335
1150: BF055267
1151: BF054837
1152: BF054680

1153: AF239668
1154: AF029759
1155: AF089087
1156: AF254664

1157: AK024416
1158: BE858655
1159: BE858216
1160: AB041228

1161: AF250237
1162: AX018430
1163: AX018429
1164: AX018428

1165: AX018426
1166: AX014744
1167: AX014742
1168: BE677821

1169: BE671344
1170: BE671261
1171: BE671257
1172: BE670057

1173: BE646269
1174: AF257210
1175: AF233092
1176: BE503731

1177: BE503724
1178: BE502880
1179: BE502852
1180: BE502582

1181: BE501091
1182: AF282693
1183: AF236117
1184: BE467925

1185: BE466690
1186: BE465916
1187: BE464797
1188: BE464297

1189: AL121935
1190: BE208338
1191: BE350014
1192: BE328133

1193: BE328109
1194: BE328060
1195: BE219456
1196: BE218901

1197: BE218235
1198: BE218140
1199: BE218139
1200: AB040801

1201: AB040800
1202: AB040799
1203: BE049570
1204: BE046086

1205: BE042841
1206: BE041936
1207: AF208237
1208: AF073924

1209: D88437
1210: AW873727
1211: AW827198
1212: AW779207

1213: AW771926
1214: AW771412
1215: AW770712
1216: AW770705

1217: AW768971
1218: AF202640
1219: AF236081
1220: AF030335

1221: AF215981
1222: AF056085
1223: AW665207
1224: AW664477

1225: AU076620
1226: AW631295
1227: AW627455
1228: AW614983

1229: AW613556
1230: AW612883
1231: AW612249
1232: AW594595

1233: AW594481
1234: AW590950
1235: AW590629
1236: AF227139

1237: AF227138
1238: AF227137
1239: AF227136
1240: AF227135

1241: AF227134
1242: AF227133
1243: AF227132
1244: AF227131

1245: AF227130
1246: AF227129
1247: AW583167
1248: AW573093

1249: AF112462
1250: AF112461
1251: AF112460
1252: AW515813

1253: AW468602
1254: AW468498
1255: AW467603
1256: AW418550

1257: X89271
1258: AJ243213
1259: AC002381
1260: AW339203

1261: AW338938
1262: AW338568
1263: AW316632
1264: AW299960

1265: AW299685
1266: Z86090
1267: AW272269
1268: AW271290

1269: U78723
1270: AC004925
1271: AW239400
1272: AW239010

1273: AW197479
1274: AW193726
1275: AW191974
1276: AL022171

1277: AL009181
1278: Z85996
1279: Z69387
1280: Z68281

1281: Z68273
1282: Z68192
1283: AW188960
1284: AW188400

1285: AW173257
1286: AW173009
1287: AW170317
1288: AW150789

1289: AW149665
1290: AW148557
1291: AF181862
1292: X68149

1293: AW129012
1294: AW128849
1295: AW118213
1296: AW102735

1297: AW087372
1298: AW083550
1299: AW083541
1300: AW075850

1301: AW075598
1302: AW075549
1303: AW072548
1304: AW071110

1305: AF140631
1306: AF040752
1307: AF040751
1308: AF040753

1309: AF186380
1310: AF147204
1311: AW058177
1312: AF127138

1313: AF104939
1314: AF104266
1315: AW051846
1316: AW050562

1317: AF104938
1318: AW024131
1319: AH008056
1320: AF129514

1321: AW004908
1322: AW004735
1323: AF101472
1324: AF072693

1325: AW000832
1326: AI990500
1327: AI979039
1328: AI969765

1329: AI969011
1330: AI968199
1331: AI968062
1332: AF039686

1333: AI963290
1334: AI962628
1335: AI962439
1336: AI952936

1337: AI951598
1338: AJ238044
1339: AF083955
1340: E16188

1341: E16187
1342: E16186
1343: E14219
1344: E14218

1345: E14217
1346: AI937602
1347: AI936826
1348: AI936528

1349: AI934968
1350: AI929343
1351: AI921242
1352: AI920946

1353: AI910975
1354: AI890025
1355: AI889324
1356: AI884686

1357: AI884548
1358: AH005868
1359: AF044601
1360: AF044600

1361: AI870119
1362: AI869176
1363: AI867390
1364: AI866909

1365: AI864743
1366: AI861901
1367: AF153500
1368: AI859538

1369: AI858943
1370: AI857339
1371: AI831861
1372: AI830135

1373: AI817194
1374: X13556
1375: AI807566
1376: AI801319

1377: AI798928
1378: AI796432
1379: AF119711
1380: AI767062

1381: AI765236
1382: AI762692
1383: AI745026
1384: AI743546

1385: AI742092
1386: AI740732
1387: AI738477
1388: AF145207

1389: AI719098
1390: AI703458
1391: AI703188
1392: AI700112

1393: AI699236
1394: AI698562
1395: AI697249
1396: AI697103

1397: AI696158
1398: AI695339
1399: AI694940
1400: AI693678

1401: AI692576
1402: AF144308
1403: AI683322
1404: AI682902

1405: AI682706
1406: AI681718
1407: AI678669
1408: AI675038

1409: AI672910
1410: AI672677
1411: AI672434
1412: AI670734

1413: AF106858
1414: AI660355
1415: AI659965
1416: AI659657

1417: AI656746
1418: AI655538
1419: AI653213
1420: AI640447

1421: AI640213
1422: AI636061
1423: AI611298
1424: AI610565

1425: AF069755
1426: AI583169
1427: AI583146
1428: AI582682

1429: AI581657
1430: AF058762
1431: AF096786
1432: AF096785

1433: AF096784
1434: AI568975
1435: AF119815
1436: AI566829

1437: AC007136
1438: AF118266
1439: AF118265
1440: AI524429

1441: AI524007
1442: AF118670
1443: AI493618
1444: AT498729

1445: X97881
1446: X97880
1447: X97879
1448: AF105367

1449: AI470243
1450: AI470241
1451: AI470231
1452: AI468820

1453: AI476811
1454: AI473656
1455: AI457930
1456: AI439188

1457: AI434652
1458: AI422268
1459: AI370816
1460: AI368913

1461: AI359560
1462: AI358974
1463: AI358446
1464: AI355648

1465: AI308145
1466: AI338666
1467: AI338653
1468: AI123732

1469: U68031
1470: AI417609
1471: AI417456
1472: AI417427

1473: AI253178
1474: AI249788
1475: AI348152
1476: AI344724

1477: AI344626
1478: AI300807
1479: AI300764
1480: AI289854

1481: AI292165
1482: AI290226
1483: AI268995
1484: AI379767

1485: AI379745
1486: AI376916
1487: AI284206
1488: AI263529

1489: AI240328
1490: AI375269
1491: AF080586
1492: AA694447

1493: AF074483
1494: AA890050
1495: AA883367
1496: AF106941

1497: AI346265
1498: AA844623
1499: AA781110
1500: AA772427

1501: AF034780
1502: AI342261
1503: AI337353
1504: AI334621

1505: AI334042
1506: AF099148
1507: AF095448
1508: AC006132

1509: AI249966
1510: AI243295
1511: AH007062
1512: U90660

1513: U90659
1514: U90658
1515: AI243951
1516: AI239970

1517: AI218191
1518: AI215993
1519: AI208357
1520: Y12476

1521: AJ000479
1522: Y12477
1523: AF061444
1524: AI002547

1525: AI193140
1526: AI192675
1527: AI138606
1528: AI126520

1529: AI161367
1530: AI160744
1531: AI159856
1532: AI143180

1533: AI148328
1534: AI167285
1535: AF091890
1536: AI050884

1537: AI041787
1538: AF032132
1539: AF027957
1540: AF027956

1541: AF022137
1542: AF002986
1543: AF015257
1544: U83326

1545: AF012270
1546: U65402
1547: U94320
1548: U66581

1549: U66580
1550: U66579
1551: U66578
1552: U79527

1553: U79526
1554: U77827
1555: U68032
1556: U68030

1557: AH006663
1558: U50146
1559: U66275
1560: U62027

1561: U48405
1562: AH006647
1563: U47129
1564: U47128

1565: U47127
1566: U47126
1567: U34806
1568: U25341

1569: U28488
1570: U40223
1571: U32672
1572: AH006630

1573: U33168
1574: U33167
1575: U33166
1576: U33165

1577: U33164
1578: U33163
1579: U33162
1580: U33161

1581: U33160
1582: U33159
1583: U33158
1584: U33157

1585: U33156
1586: U33155
1587: U33154
1588: U33153

1589: U33056
1590: U33055
1591: U33054
1592: U22492

1593: U22491
1594: U31332
1595: U31099
1596: U31098

1597: U25128
1598: L40764
1599: AF045767
1600: AF045765

1601: AF045764
1602: AF027826
1603: AF041245
1604: AF041243

1605: AF073799
1606: D10202
1607: Y12546
1608: AI050992

1609: AI051919
1610: AI051863
1611: AI022030
1612: Z94155

1613: Z94154
1614: AF086432
1615: AI017452
1616: AA994898

1617: AA992531
1618: AA936395
1619: AI097347
1620: AI077789

1621: AF080214
1622: AF062006
1623: AF011466
1624: AI032237

1625: AI032226
1626: AA989434
1627: AF034633
1628: AF034632

1629: AI050023
1630: AA970139
1631: AA935899
1632: AA935648

1633: AA934643
1634: E12487
1635: E12484
1636: AA953688

1637: AA931357
1638: AA923762
1639: AA933596
1640: Y14838

1641: AA927880
1642: AA834277
1643: AA825595
1644: AF067733

1645: AA905915
1646: AA863264
1647: AA862435
1648: U71092

1649: AA857647
1650: Y16280
1651: AA834537
1652: AA826204

1653: AA808103
1654: AA829514
1655: AA883661
1656: AA836111

1657: AA836067
1658: AA832466
1659: AA824607
1660: AA205847

1661: AA197280
1662: AA181641
1663: AA634862
1664: AA634211

1665: AA451915
1666: AA827835
1667: AA804628
1668: AA811093

1669: AA760743
1670: AA748438
1671: AA804282
1672: AA779703

1673: AA780337
1674: AA731086
1675: AA744637
1676: AA760855

1677: AA769730
1678: AA768086
1679: U78192
1680: Z73157

1681: AA773241
1682: AA747545
1683: AA743645
1684: AA743379

1685: Y10530
1686: Y10529
1687: AA713608
1688: AA732228

1689: AF014826
1690: AA707668
1691: AA705077
1692: AA112062

1693: AA083607
1694: AH005747
1695: U15790
1696: U15789

1697: U15788
1698: U15787
1699: U15786
1700: U15785

1701: U14911
1702: AA661523
1703: AF007171
1704: U63917

1705: Y13583
1706: AF024690
1707: AF024689
1708: AF024688

1709: AF024687
1710: AA421523
1711: AA421558
1712: AA417176

1713: AA610463
1714: AA650037
1715: AF025375
1716: AA621854

1717: AA634201
1718: AA630455
1719: AA426566
1720: AA426644

1721: AA424850
1722: AA419064
1723: AA583854
1724: AF017263

1725: AF017264
1726: AF017262
1727: AA576017
1728: AA554406

1729: AC002511
1730: AA573161
1731: AA534523
1732: AA259199

1733: AA225739
1734: AA507254
1735: AA502605
1736: AA501992

1737: AA490436
1738: AA490329
1739: AA558023
1740: AA479467

1741: AA479357
1742: AA477030
1743: AA476919
1744: AA284569

1745: AA284857
1746: L42324
1747: AA148292
1748: AA148291

1749: AA523398
1750: AA059452
1751: AA059451
1752: AF007545

1753: Z79783
1754: AF004021
1755: U45984
1756: AF000546

1757: U90322
1758: U90323
1759: U45983
1760: X65857

1761: X65858
1762: AC002306
1763: U73531
1764: U73530

1765: U73529
1766: D89079
1767: D89078
1768: AH005415

1769: U48231
1770: U18550
1771: D38449
1772: Y09479

1773: AA436258
1774: AA194811
1775: AA194998
1776: X95876

1777: AF000545
1778: AA411265
1779: AA137186
1780: AA137185

1781: AA129610
1782: AA129609
1783: AA121357
1784: AA121265

1785: AA099858
1786: AA099323
1787: AA058812
1788: AA045235

1789: AA037526
1790: AA037376
1791: AA036907
1792: AA036853

1793: X98510
1794: AA314786
1795: AA298791
1796: AA297171

1797: U91939
1798: U64871
1799: U34038
1800: X70070

1801: AA193392
1802: N58609
1803: N54441
1804: U49516

1805: X98118
1806: X83864
1807: X70812
1808: AA127402

1809: AA127401
1810: X69680
1811: S45489
1812: Z79784

1813: Z79782
1814: U73304
1815: X98356
1816: W79920

1817: W77864
1818: W72081
1819: W73685
1820: U67784

1821: U33448
1822: U33447
1823: U49727
1824: X99393

1825: AA041219
1826: W40430
1827: W21494
1828: N93476

1829: W23870
1830: N95025
1831: AA007184
1832: AA007183

1833: L03718
1834: X96597
1835: N62053
1836: H97311

1837: X81121
1838: X81120
1839: X69920
1840: X69168

1841: X83956
1842: X72089
1843: X65181
1844: X65180

1845: X65179
1846: X65177
1847: X65178
1848: X68596

1849: X71635
1850: X65176
1851: X65175
1852: X65174

1853: X65173
1854: X65172
1855: X68829
1856: X52068

1857: X65859
1858: X64993
1859: X64992
1860: X64991

1861: X64990
1862: X64989
1863: X64988
1864: X64987

1865: X64986
1866: X64985
1867: X64984
1868: X64983

1869: X64982
1870: X64981
1871: X64980
1872: X64979

1873: X64974
1874: X64978
1875: X64977
1876: X64976

1877: X64975
1878: X64995
1879: X64994
1880: X75897

1881: X54937
1882: U55312
1883: W24753
1884: W17011

1885: U21051
1886: W01442
1887: U47124
1888: N93987

1889: N90783
1890: U45982
1891: N86436
1892: U32500

1893: U20350
1894: U18549
1895: U18548
1896: AH003369

1897: U23430
1898: U23429
1899: U23428
1900: M73481

1901: N49854
1902: U20760
1903: U20759
1904: N23898

1905: U39231
1906: H88656
1907: H88701
1908: U35399

1909: U35398
1910: L35318
1911: T29782
1912: T29676

1913: T28268
1914: R91585
1915: H37859
1916: L31581

1917: L32831
1918: L32830
1919: H45306
1920: H29103

1921: H29001
1922: H27787
1923: H14301
1924: H21565

1925: H20663
1926: H16711
1927: H16710
1928: H12955

1929: H06644
1930: R80054
1931: R78657
1932: R78620

1933: R76070
1934: R73329
1935: R72859
1936: R55156

1937: R55018
1938: R48699
1939: R48597
1940: H27256

1941: R23115
1942: R23114
1943: R20666
1944: R20475

1945: R15256
1946: R13546
1947: U13668
1948: U13667

1949: U13666
1950: T99860
1951: T98622
1952: U11878

1953: U11877
1954: U11876
1955: U11875
1956: U11874

1957: U11873
1958: U11872
1959: T87010
1960: L36150

1961: L36148
1962: T72605
1963: T64864
1964: L36149

1965: T62636
1966: T62491
1967: U17473
1968: T51359

1969: T51244
1970: U19487
1971: M74290
1972: L16862

1973: M73482
1974: L09237
1975: L15388
1976: L08176

1977: U14910
1978: M95489
1979: M67439
1980: L14856

1981: L10918
1982: L08177
1983: U03642
1984: L10820

1985: U00686
1986: L06797

[0351]

12

TABLE 12

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding polypeptides potentially related to orphan

G-protein-coupled receptors metabolism and/or signaling.

1: NM_005300
2: NM_004778
3: NM_018485
4: NT_009714

5: NT_009528
6: NT_008902
7: NT_005849
8: NT_028053

9: AY089976
10: NM_003717
11: NT_010672
12: NT_033363

13: XM_114696
14: XM_061555
15: NM_138964
16: NT_011520

17: NM_004767
18: NT_033922
19: NT_005612
20: NT_005151

21: XM_086954
22: NM_007227
23: NM_001337
24: AC078860

25: NM_006794
26: BM503956
27: NM_003667
28: NM_016235

29: NM_053036
30: NM_032551
31: NM_033050
32: NM_023914

33: AY029541
34: AF343725
35: U73141
36: AF209923

37: AF207989
38: AU099377
39: AF295368
40: AF237763

41: AF237762
42: AF348078
43: AF321815
44: NM_022036

45: NM_018653
46: NM_018654
47: NM_016602
48: NM_003979

49: Y19228
50: Y19231
51: Y19230
52: Y19229

53: NM_004885
54: BF592107
55: NM_018949
56: NM_005281

57: NM_005291
58: NM_001508
59: NM_001507
60: AF250237

61: AF257210
62: AF208237
63: AF202640
64: AF236081

65: AF215981
66: X89271
67: AF140631
68: AF101472

69: AF072693
70: AI969765
71: AI968199
72: AI962439

73: AI951598
74: AH005868
75: AF044601
76: AF044600

77: AI831861
78: AI703458
79: AI699236
80: AI697103

81: AI694940
82: AI692576
83: AI681718
84: AI640447

85: AF069755
86: AF118266
87: AF118265
88: AF118670

89: AI215993
90: AF091890
91: AF027957
92: AF027956

93: U79527
94: U79526
95: U77827
96: U32672

97: AF045764
98: Y12546
99: Z94155
100: Z94154

101: AF062006
102: AF034633
103: AF034632
104: Y14838

105: Y16280
106: U67784
107: X96597
108: X83956

109: U20350
110: U17473
111: L06797

[0352]

13

TABLE 13

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding protein kinases potentially involved in

transcription metabolism and/or signaling.

1: NM_020168
2: NM_004857
3: NM_139070
4: NM_139069

5: NM_139068
6: NM_002752
7: D10022
8: NM_138957

9: NM_002745
10: NM_002754
11: NM_138993
12: NM_002751

13: NM_139049
14: NM_139047
15: NM_139046
16: NM_005456

17: NM_139014
18: NM_139013
19: NM_139012
20: NM_138982

21: NM_138981
22: NM_138980
23: NM_002753
24: NM_139034

25: NM_139033
26: NM_139032
27: NM_002749
28: NM_002750

29: NT_009307
30: NT_009237
31: NT_024229
32: NT_009770

33: NT_024654
34: NT_010274
35: NT_010194
36: NT_030059

37: NT_011139
38: NT_011109
39: NT_007993
40: NT_010019

41: NT_008413
42: NT_004858
43: NT_030040
44: NT_004734

45: NT_004658
46: NT_006397
47: NT_004525
48: NT_006371

49: NT_021877
50: NT_019273
51: NT_033927
52: NT_033241

53: NT_028327
54: NT_033984
55: NT_033982
56: NT_033892

57: NM_002401
58: NM_032989
59: NM_004322
60: NM_031988

61: NM_002758
62: NM_001315
63: NT_033291
64: NT_010552

65: NT_010478
66: NT_010441
67: NT_011512
68: NT_011387

69: NT_010808
70: NT_010783
71: NT_010755
72: NT_010748

73: NT_010736
74: NT_010718
75: NT_031911
76: NT_007592

77: NT_009563
78: NT_009526
79: NT_025965
80: NT_007422

81: NT_025273
82: NT_007299
83: MT 033944
84: NT_011362

85: NT_011520
86: NT_033167
87: NT_030710
88: NT_025741

89: NT_009799
90: NT_023399
91: NT_007072
92: NT_006859

93: NT_011295
94: NT_011271
95: NT_011255
96: NT_009910

97: NT_006654
98: NT_006497
99: NT_026437
100: NT_007968

101: NT_007933
102: NT_008046
103: NT_025892
104: NT_010164

105: NT_007758
106: NT_008580
107: NT_007688
108: NT_033965

109: NT_033964
110: NT_030001
111: NT_029366
112: NT_017168

113: NT_005367
114: NT_005334
115: NT_005332
116: NT_005190

117: NT_005151
118: NT_022171
119: NT_022135
120: NM_138923

121: NM_004606
122: NM_080601
123: NM_002834
124: NM_022740

125: NM_005806
126: NM_001799
127: NM_022304
128: NM_002005

129: NM_037370
130: NM_012142
131: NM_012333
132: AY028384

133: NM_001261
134: NM_052988
135: NM_052987
136: NM_001260

137: NM_003674
138: NM_052827
139: NM_001798
140: NM_021104

141: NM_000024
142: NM_000681
143: NM_002006
144: NM_012138

145: NM_002755
146: NM_004635
147: AD000092
148: NM_031965

149: AF289865
150: NM_022550
151: NM_022406
152: NM_003401

153: NM_005734
154: AJ277546
155: NM_001924
156: NM_013311

157: NM_005163
158: NM_000165
159: NM_002227
160: AF184924

161: AP001751
162: U83994
163: U87803
164: AH007140

165: U87276
166: U87275
167: U87274
168: U87273

169: U87272
170: U87271
171: AF074715
172: AF015256

173: AF009225
174: U64573
175: U35005
176: U35004

177: U35003
178: U35002
179: U34822
180: U34821

181: U34820
182: U34819
183: Z92868
184: AF049893

185: Y10256
186: Y07641
187: AH004914
188: U03874

[0353]

14

TABLE 14

GenBank Accession numbers of human sequence records identified

as related to nucleic acids encoding protein kinases potentially involved in G-

protein coupled receptor metabolism and/or signaling.

1: NM_007202
2: NM_144489
3: NM_144488
4: NM_134427

5: NM_017790
6: NM_021106
7: NM_130795
8: NM_138957

9: NM_002745
10: NM_139034
11: NM_139033
12: NM_139032

13: NM_002749
14: NT_009307
15: NT_009770
16: NT_030828

17: NT_010194
18: NT_008902
19: NT_011151
20: NT_011139

21: NT_011109
22: NT_008413
23: NT_004858
24: NT_006014

25: NT_004771
26: NT_004434
27: NT_004350
28: NT_006051

29: NT_025667
30: NT_029860
31: NT_028053
32: NT_026943

33: NT_033903
34: NT_010552
35: NT_010823
36: NT_010808

37: NT_010783
38: NT_007592
39: NT_009563
40: NT_007422

41: NT_007299
42: NT_011793
43: NT_033944
44: NT_011362

45: NT_011520
46: NT_011719
47: NT_011669
48: NT_025741

49: NT_009799
50: NT_033922
51: NT_006859
52: NT_011295

53: NT_006519
54: NT_026437
55: NT_007968
56: NT_007933

57: NT_007914
58: NT_010164
59: NT_008580
60: NT_029366

61: NT_017168
62: NT_005367
63: NT_005151
64: NT_005079

65: NM_022304
66: NM_006098
67: AF282269
68: NM_002880

69: NM_000024
70: NM_000681
71: NM_032938
72: NM_004489

73: NM_032442
74: NM_004127
75: NM_004041
76: NM_020251

77: NM_005160
78: AL031282
79: U20285
80: AC007136

81: U28963

[0354]

15

TABLE 15

GenBank Accession numbers of human sequence records

identified as related to nucleic acids encoding protein kinases potentially

involved in a apotosis.

1: NM_005923
2: NM_020168
3: NM_144489
4: NM_144488

5: NM_134427
6: NM_017790
7: NM_021106
8: NM_130795

9: NM_139070
10: NM_139069
11: NM_139068
12: NM_002752

13: NM_006712
14: NM_033015
15: NM_025096
16: NM_139049

17: NM_139047
18: NM_139046
19: NM_005456
20: NM_139014

21: NM_139013
22: NM_139012
23:NM_138982
24: NM_138981

25: NM_138980
26: NM_002753
27: NM_002750
28: NT_024192

29: NT_009770
30: NT_010194
31: NT_030059
32: NT_011109

33: NT_021877
34: NM_078467
35: NM_032989
36: NM_004322

37: NM_031988
38: NM_002758
39: NM_001315
40: NT_010552

41: NT_010478
42: NT_010823
43: NT_010755
44: NT_010748

45: NT_007592
46: NT_033944
47: NT_011520
48: NT_011694

49: NT_006497
50: NT_026437
51: NT_010164
52: NT_007819

53: NT_007758
54: NT_033181
55: NT_005190
56: XM_050441

57: NM_003821
58: NM_004103
59: NM_131917
60: NM_007051

61: NM_003682
62: NM_130476
63: NM_130475
64: NM_130474

65: NM_130473
66: NM_130472
67: NM_130471
68: NM_130470

69: AB040057
70: NM_014326
71: NM_000389
72: NM_005400

73: NM_004226
74: NM_024011
75: NM_033621
76: NM_033537

77: NM_033536
78: NM_033534
79: NM_033532
80: NM_033531

81: NM_033529
82: NM_033528
83: NM_033527
84: AF305840

85: NM_033493
86: NM_033492
87: NM_033491
88: NM_033490

89: NM_033489
90: NM_033488
91: NM_033487
92: NM_033486

93: NM_001787
94: NM_006947
95: NM_002880
96: NM_012138

97: NM_031267
98: NM_003718
99: NM_014245
100: NM_005163

101: NM_004760
102: NM_001348
103: AF052941
104: AB018001

105: AB011421
106: AB011420
107: AF027706
108: AF021792

[0355]

16

TABLE 16

Modifications of the First Three Nucleotides of

the att Site Seven Base Pair Overlap Region that

Alter Recombination Specificity.

AAA
CAA
GAA
TAA

AAC
CAC
GAC
TAC

AAG
CAG
GAG
TAG

AAT
CAT
GAT
TAT

ACA
CCA
GCA
TCA

ACC
CCC
GCC
TCC

ACG
CCG
GCG
TCG

ACT
CCT
GCT
TCT

AGA
CGA
GGA
TGA

AGC
CGC
GGC
TGC

AGG
CGG
GGG
TGG

AGT
CGT
GGT
TGT

ATA
CTA
GTA
TTA

ATC
CTC
GTC
TTC

ATG
CTG
GTG
TTG

ATT
CTT
GTT
TTT

[0356]

17

TABLE 17

Representative Examples of Seven Base Pair att

Site Overlap Regions Suitable for use in the

recombination sites of the Invention.

AAAATAC
CAAATAC
GAAATAC
TAAATAC

AACATAC
CACATAC
GACATAC
TACATAC

AAGATAC
CAGATAC
GAGATAC
TAGATAC

AATATAC
CATATAC
GATATAC
TATATAC

ACAATAC
CCAATAC
GCAATAC
TCAATAC

ACCATAC
CCCATAC
GCCATAC
TCCATAC

ACGATAC
CCGATAC
GCGATAC
TCGATAC

ACTATAC
CCTATAC
GCTATAC
TCTATAC

AGAATAC
CGAATAC
GGAATAC
TGAATAC

AGCATAC
CGCATAC
GGCATAC
TGCATAC

AGGATAC
CGGATAC
GGGATAC
TGGATAC

AGTATAC
CGTATAC
GGTATAC
TGTATAC

ATAATAC
CTAATAC
GTAATAC
TTAATAC

ATCATAC
CTCATAC
GTCATAC
TTCATAC

ATGATAC
CTGATAC
GTGATAC
TTGATAC

ATTATAC
CTTATAC
GTTATAC
TTTATAC

[0357]

18

TABLE 18

Nucleotide sequences of att sites.

attB0
AGCCTGCTTT TTTATACTAA CTTGAGC
(SEQ ID NO:)

attP0
GTTCAGCTTT TTTATACTAA GTTGGCA
(SEQ ID NO:)

attL0
AGCCTGCTTT TTTATACTAA GTTGGCA
(SEQ ID NO:)

attR0
GTTCAGCTTT TTTATACTAA CTTGAGC
(SEQ ID NO:)

attB1
AGCCTGCTTT TTTGTACAAA CTTGT
(SEQ ID NO:)

attP1
GTTCAGCTTT TTTGTACAAA GTTGGCA
(SEQ ID NO:)

attL1
AGCCTGCTTT TTTGTACAAA GTTGGCA
(SEQ ID NO:)

attR1
GTTCAGCTTT TTTGTACAAA CTTGT
(SEQ ID NO:)

attB2
ACCCAGCTTT CTTGTACAAA GTGGT
(SEQ ID NO:)

attP2
GTTCAGCTTT CTTGTACAAA GTTGGCA
(SEQ ID NO:)

attL2
ACCCAGCTTT CTTGTACAAA GTTGGCA
(SEQ ID NO:)

attR2
GTTCAGCTTT CTTGTACAAA GTGGT
(SEQ ID NO:)

attB5
CAACTTTATT ATACAAAGTT GT
(SEQ ID NO:)

attP5
GTTCAACTTT ATTATACAAA GTTGGCA
(SEQ ID NO:)

attL5
CAACTTTATT ATACAAAGTT GGCA
(SEQ ID NO:)

attR5
GTTCAACTTT ATTATACAAA GTTGT
(SEQ ID NO:)

attB11
CAACTTTTCT ATACAAAGTT GT
(SEQ ID NO:)

attP11
GTTCAACTTT TCTATACAAA GTTGGCA
(SEQ ID NO:)

attL11
CAACTTTTCT ATACAAAGTT GGCA
(SEQ ID NO:)

attR11
GTTCAACTTT TCTATACAAA GTTGT
(SEQ ID NO:)

attB17
CAACTTTTGT ATACAAAGTT GT
(SEQ ID NO:)

attP17
GTTCAACTTT TGTATACAAA GTTGGCA
(SEQ ID NO:)

attL17
CAACTTTTGT ATACAAAGTT GGCA
(SEQ ID NO:)

attR17
GTTCAACTTT TGTATACAAA GTTGT
(SEQ ID NO:)

attB19
CAACTTTTTC GTACAAAGTT GT
(SEQ ID NO:)

attP19
GTTCAACTTT TTCGTACAAA GTTGGCA
(SEQ ID NO:)

attL19
CAACTTTTTC GTACAAAGTT GGCA
(SEQ ID NO:)

attR19
GTTCAACTTT TTCGTACAAA GTTGT
(SEQ ID NO:)

attB20
CAACTTTTTG GTACAAAGTT GT
(SEQ ID NO:)

attP20
GTTCAACTTT TTGGTACAAA GTTGGCA
(SEQ ID NO:)

attL20
CAACTTTTTG GTACAAAGTT GGCA
(SEQ ID NO:)

attR20
GTTCAACTTT TTGGTACAAA GTTGT
(SEQ ID NO:)

attB21
CAACTTTTTA ATACAAAGTT GT
(SEQ ID NO:)

attP21
GTTCAACTTT TTAATACAAA GTTGGCA
(SEQ ID NO:)

attL21
CAACTTTTTA ATACAAAGTT GGCA
(SEQ ID NO:)

attR21
GTTCAACTTT TTAATACAAA GTTGT
(SEQ ID NO:)

7. Conclusion

[0358] Various embodiments of the present invention have been described above. It should be understood that these embodiments have been presented by way of example only, and not limitation. It will be understood by those skilled in the relevant art that various changes in form and detail of the embodiments described above may be made without departing from the spirit and scope of the present invention as defined in the claims. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

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