AU754276B2 - Methods for producing libraries of expressible gene sequences - Google Patents

Description

WO 99/51766 PCT/US99/07270 METHODS FOR PRODUCING LIBRARIES OF EXPRESSIBLE GENE

SEQOUENCES

Field of the Invention The invention disclosed herein relates to the fields of genomics and molecular biology. More specifically the invention relates to new high through-put methods of making libraries of expressed gene sequences and the libraries made using said methods.

Background of the Invention Recent breakthroughs in nucleic acid sequencing technology have made possible the sequencing of entire genomes from a variety of organisms, including humans. The potential benefits of a complete genome sequence are many, ranging from applications in medicine to a greater understanding of evolutionary processes.

These benefits cannot be fully realized, however, without an understanding of how and where these newly sequenced genes function.

Traditionally, functional understanding started with recognizing an activity, isolating a protein associated with that activity, then identifying and isolating the gene, or genes, encoding that protein. Each gene of interest was identified, isolated and expressed separately, a relatively time consuming process.

Recently, breakthroughs in high through-put DNA sequencing technology have allowed massive amounts of gene sequence information to become available to the public. Yet methods of expressing these sequences to produce the proteins encoded by them for study have still required that each sequence be manipulated one at a time. Accordingly, a need exists for the development of methods for the rapid, simultaneous expression of large numbers of gene sequences. The invention described herein addresses this and related needs as will become apparent upon inspection of the specification and the appended claims.

ra .1 2 Brief Description of the Invention The present invention comprises a method for producing libraries of expressible gene sequences. The method of the invention allows for the simultaneous manipulation of multiple gene sequences and thus allows libraries to be created in an efficient and high through-put manner. The expression vectors containing verified gene sequences can be used to transfect cells for the production of recombinant proteins. The invention method utilizes known techniques in such a way as to create an efficient high through-put means of producing libraries of expressible gene sequences.

The invention further comprises libraries of expressible gene sequences produced 0o using the method of the invention and expression vectors used in the construction of such libraries.

Brief Description of the Figure Figure 1 shows a schematic representation of the vaccinia topoisomerase type I cloning method used in the practice of the invention.

Summary of the Invention According to a first aspect of this invention there is provided a method for producing a library of expressible open reading frames, the method comprising: a) amplifying deoxyribonucleic acid (DNA) molecules comprising a plurality of open reading frames (ORFs) using a primer pair, wherein the primer pair comprises a 5' primer, 20 which comprises a nucleotide sequence starting 5'-CACCATG, thereby producing a plurality of amplified ORFs; b) inserting amplified ORFs of the plurality into an expression vector, thereby producing expression vectors comprising the amplified ORFs; and c) verifying the size and orientation of the amplified ORFs in the expression vectors, thereby producing a library of expressible ORFs.

According to a second aspect of this invention there is provided a method for producing a library of selected expressible open reading frames (ORFs), the method comprising: a) amplifying deoxyribonucleic acid (DNA) molecules comprising a plurality of ORFs using a primer pair, wherein the primer pair comprises a 5' primer, which comprises a nucleotide sequence starting 5'-CACCATG, and a 3' primer, which causes the 30 amplification product to end just prior to a stop codon, thereby producing a plurality of amplified ORFs; b) purifying amplified ORFs of the plurality, thereby obtaining purified amplified ORFs; c) inserting the purified amplified ORFs into expression vectors using a vaccinia DNA topoisomerase, thereby producing expression vectors comprising the [I:\DayLib\LIBUU]52780.doc:MCC amplified ORFs; d) transforming cells with the expression vectors comprising the amplified ORFs; and e) selecting transformed cells containing expression vectors comprising ORFs in an orientation for expression of a polypeptide encoded by the ORR.

Detailed Description of the Invention The present invention comprises a method for producing libraries of expressible gene sequences. The invention method comprises the following steps: amplifying a plurality of gene sequences, purifying the amplified gene sequences, inserting each of the purified gene sequences into an expression vector, and verifying the size and orientation of the inserted gene sequence.

In the first step, the gene sequences that are to be expressed are amplified. By "6amplification" it is meant that the copy number of the gene sequence(s) is increased.

One commonly used method of amplification is the polymnerase chain reaction (PCR). In brief, starter DNA is heat-denatured into single strands. Two synthetic oligonucleotides, one complementary to sequence at the 3' end of the sense strand of DNA segment of interest and the other complementary to the sequence at the 3' end of .0o %0:0• 7 [I:DayLibL1IBU]52780.doc; MCC WO 99/51766 PCT/US99/07270 3 the anti-sense strand of a DNA segment of interest, are added in great excess to the DNA sequence to be amplified and the temperature is lowered to 50 600 C. The specific oligonucleotides hybridize with the complementary sequences in the DNA and then serve as primers of DNA chain synthesis, which requires the addition of a supply of deoxynucleotides and a temperature-resistant DNA polymerase, such as Taq polymerase, which can extend the primers at temperatures up to 720 C. When synthesis is complete, the whole mixture is heated further (up to 950 C) to melt the newly formed DNA duplexes. When the temperature is lowered again, another round of synthesis takes place, since an excess of primer is still present. Repeated cycles of synthesis and melting quickly amplify the sequence of interest. A more detailed description of PCR can be found in Erlich, Ed, PCR Technology: Principles and Applications for DNA Amplification, W.H. Freeman and Co., 1992 and Erlich, et al, Eds., Polymerase Chain Reaction, Cold Spring Harbor Laboratory, 1989, both of which are incorporated by reference herein.

Starter DNA can come from a variety of sources. It can be total genomic DNA from an organism, for example, or can be cDNA that has been synthesized from cellular mRNA using reverse transcriptase. Sources of suitable RNA include normal and diseased tissues, cellular extracts, and the like.

In practicing the method of the invention, the desired gene sequences can come from any source. The examples presented below show the amplification of all open reading frames (ORFs) from a single organism, Saccharomyces cerevisiae, for example. By "open reading frame" it is meant a segment of DNA that exists between a start codon and a stop codon and is likely to represent a gene. The examples presented below further show the amplification of a group of human genes thought to be important in the development of cancer.

Public databases exist that contain the entire or partial genome of a particular organism, for example yeast (Saccharomyces cerevisiae), prokaryotes (Bacillus subtilis, E. coli, Borrelia burgdorferi, Helicobacter pylori, Mycoplasma genitalium, and the like), fish (Fugu rubripes), mammals (human, mouse), plants (rice, cotton) WO 99/51766 PCT/US99/07270 4 and the like. Well known databases include GenBank, Unigene, EMBL, IMAGE and TIGR, for example. Public databases such as these can be used a source of gene sequences for use in the method of the invention.

The primers employed in the amplification step are specific for each desired gene sequence and include a variety of unique features. For example, the 5' "sense" primer starts with the sequence 5'-CACCAIG... (the start codon is underlined). The CACC sequence is added as a Kozak consensus that aids in translational efficiency.

When the gene sequence being amplified represents a full-length gene, the 3' "antisense" codon is preferably designed to make the amplification product end at the 3rd position of the last codon of the gene being amplified, plus a single adenine residue. This facilitates the fusion of the coding region in-frame with a heterologous peptide sequence such as an epitope tag, an affinity purification tag, and the like (see below). The gene sequence need not encode a full-length sequence, however, as the invention methods are equally suitable for any gene sequence, including Expressed Sequence Tags (ESTs). The primers can be synthesized and dried in multiwell formats, such as 96-well microtiter plates to facilitate identification and further processing.

The amplified gene products are next isolated from the other components of the amplification reaction mixture. This purification can be accomplished using a variety of methodologies such as column chromatography, gel electrophoresis, and the like. A preferred method of purification utilizes low-melt agarose gel electrophoresis.

The reaction mixture is separated and visualized by suitable means, e.g. by ethidium bromide staining. DNA bands that represent correctly sized amplification products are cut away from the rest of the gel and placed into appropriate corresponding wells of a 96-well microtiter plate. These plugs are subsequently melted and the DNA contained therein utilized as cloning inserts. The use of gel electrophoresis has the advantage that the practitioner can purify the desired amplified gene sequence while additionally verifying that the sequence is of the correct size, represents the entire desired gene sequence.

WO 99/51766 PCT/US99/07270 The purified, amplified gene sequences are next inserted into an expression vector. A variety of expression vectors are suitable for use in the method of the invention, both for prokaryotic expression and eukaryotic expression. In general, the expression vector will have one or more of the following features: a promoterenhancer sequence, a selection marker sequence, an origin of replication, an affinity purification tag sequence, an inducible element sequence, an epitope-tag sequence, and the like.

Promoter-enhancer sequences are DNA sequences to which RNA polymerase binds and initiates transcription. The promoter determines the polarity of the transcript by specifying which strand will be transcribed. Bacterial promoters consist of consensus sequences, -35 and -10 nucleotides relative to the transcriptional start, which are bound by a specific sigma factor and RNA polymerase. Eukaryotic promoters are more complex. Most promoters utilized in expression vectors are transcribed by RNA polymerase II. General transcription factors (GTFs) first bind specific sequences near the start and then recruit the binding of RNA polymerase II.

In addition to these minimal promoter elements, small sequence elements are recognized specifically by modular DNA-binding/trans-activating proteins AP-1, SP-1) which regulate the activity of a given promoter. Viral promoters serve the same function as bacterial or eukaryotic promoters and either provide a specific RNA polymerase in trans (bacteriophage T7) or recruit cellular factors and RNA polymerase (SV40, RSV, CMV). Viral promoters are preferred as they are generally particularly strong promoters.

Promoters may be, furthermore, either constitutive or, more preferably, regulatable inducible or derepressible). Inducible elements are DNA sequence elements which act in conjunction with promoters and bind either repressors (e.g.

lacO/LAC Iq repressor system in E. coli) or inducers gall/GAL4 inducer system in yeast). In either case, transcription is virtually "shut off" until the promoter is derepressed or induced, at which point transcription is "turned-on".

WO 99/51766 PCT/US99/07270 6 Examples of constitutive promoters include the int promoter of bacteriophage k, the bla promoter of the p-lactamase gene sequence of pBR322, the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.

Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage (PL and PR), the trp, reca, lacZ, LacI, AraC and gal promoters ofE. coli, the a-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and the sigma-28-specific promoters of B. subtilis (Gilman et al., Gene sequence 32:11-20(1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478, 1986), and the like.

Exemplary prokaryotic promoters are reviewed by Glick Ind. Microbiol. 1:277- 282, 1987); Cenatiempo (Biochimie 68:505-516, 1986); and Gottesman (Ann. Rev.

Genet. 18:415-442, 1984).

Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the early promoter (Benoist et al., Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (ISA) 79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984), the CMV promoter, the EF-1 promoter, Ecdysone-responsive promoter(s), and the like.

Selection marker sequences are valuable elements in expression vectors as they provide a means to select for growth only those cells which contain a vector.

Such markers are of two types: drug resistance and auxotrophic. A drug resistance marker enables cells to detoxify an exogenously added drug that would otherwise kill the cell. Auxotrophic markers allow cells to synthesize an essential component (usually an amino acid) while grown in media which lacks that essential component.

Common selectable marker gene sequences include those for resistance to antibiotics such as ampicillin, tetracycline, kanamycin, streptomycin, bleomycin, WO 99/51766 PCT/US99/07270 7 hygromycin, neomycin, Zeocin T M and the like. Selectable auxotrophic gene sequences include, for example, hisD, which allows growth in histidine free media in the presence of histidinol.

A preferred selectable marker sequence for use in yeast expression systems is URA3. Laboratory yeast strains carrying mutations in the gene which encodes decarboxylase, an enzyme essential for uracil biosynthesis, are unable to grow in the absence of exogenous uracil. A copy of the wild-type gene (ura4+ in S. pombe and URA3 in S. cerevisiae) will complement this defect in trans.

A further element useful in an expression vector is an origin of replication sequence. Replication origins are unique DNA segments that contain multiple short repeated sequences that are recognized by multimeric origin-binding proteins and which play a key role in assembling DNA replication enzymes at the origin site.

Suitable origins of replication for use in expression vectors employed herein include E. coli oriC, 2p and ARS (both useful in yeast systems), sfl, SV40 (useful in mammalian systems), and the like.

Additional elements that can be included in expression vectors employed in the invention method are sequences encoding affinity purification tags or epitope tags.

Affinity purification tags are generally peptide sequences that can interact with a binding partner immobilized on a solid support. Synthetic DNA sequences encoding multiple consecutive single amino acids, such as histidine, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high affinity binding to a resin column, such as nickel sepharose. An endopeptidase recognition sequence is often engineered between the polyamino acid tag and the protein of interest to allow subsequent removal of the leader peptide by digestion with a specific protease. Sequences encoding peptides such as the chitin binding domain (which binds to chitin), glutathione-S-transferase (which binds to glutathione), biotin (which binds to avidin or strepavidin), and the like can also be used for facilitating purification of the protein of interest. The affinity purification tag can be separated WO 99/51766 PCT/US99/07270 8 from the protein of interest by methods well known in the art, including the use of inteins (protein self-splicing elements, Chong, et al, Gene 192:271-281, 1997).

Epitope tags are short peptide sequences that are recognized by epitope specific antibodies. A fusion protein comprising a recombinant protein and an epitope tag can be simply and easily purified using an antibody bound to a chromatography resin. The presence of the epitope tag furthermore allows the recombinant protein to be detected in subsequent assays, such as Western blots, without having to produce an antibody specific for the recombinant protein itself. Examples of commonly used epitope tags include V5, glutathione-S-transferase (GST), hemaglutinin the peptide Phe-His-His-Thr-Thr, chitin binding domain, and the like.

A further useful element in an expression vector is a multiple cloning site or polylinker. Synthetic DNA encoding a series of restriction endonuclease recognition sites is inserted into a plasmid vector downstream of the promoter element. These sites are engineered for convenient cloning of DNA into the vector at a specific position.

The foregoing elements can be combined to produce expression vectors useful in the practice of the present invention. Suitable prokaryotic vectors include plasmids such as those capable of replication in E. coli (for example, pBR322, ColEl, pSC101, PACYC 184, itVX, pRSET, pBAD (Invitrogen, Carlsbad, CA) and the like). Such plasmids are disclosed by Sambrook (cf. "Molecular Cloning: A Laboratory Manual", second edition, edited by Sambrook, Fritsch, Maniatis, Cold Spring Harbor Laboratory, (1989)). Bacillus plasmids include pC194, pC221, pT127, and the like, and are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include plJIOl (Kendall et al., J. Bacteriol. 169:4177-4183,1987), and streptomyces bacteriophages such as 4C31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54).

WO 99/51766 PCT/US99/07270 9 Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).

Suitable eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2micron circle, pcDNA3.1, pCDNA3.1/GS, pYES2/GS, pMT, p IND, pIND(Spl), pVgRXR (Invitrogen), and the like, or their derivatives. Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274, 1982); Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Dilon et al., J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).

Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. PCR amplification or sitespecific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase.

DNA ligase has limitations, however, in that it is relatively slow acting and temperature sensitive.

Thus, when inserting the purified, amplified gene sequence into the expression vector the use of an enzyme that can both cleave and religate DNA in a site specific manner is preferred. Any site-specific enzyme of this type is suitable, for example, a type I topoisomerase or a site-specific recombinase. Examples of suitable sitespecific recombinases include lambda integrase, FLP recombinase, P1-Cre protein, Kw recombinase, and the like (Pan, et al, J. Biol. Chem. 268:3683-3689, 1993; Nunes-Duby, et al, EMBO J. 13:4421-4430, 1994; Hallet and Sherratt, FEMS Microbio. Revs 21:157-178, 1997; Ringrose, et al, Eur J. Biochem 248:903-912, 1997).

A particularly suitable enzyme for use in the invention method is a type I topoisomerase, particularly vaccinia DNA topoisomerase. Vaccinia DNA topoisomerase binds to duplex DNA and cleaves the phosphodiester backbone of one strand. The enzyme exhibits a high level of sequence specificity, akin to that of a restriction endonuclease. Cleavage occurs at a consensus pentapyrimidine element in the scissile strand. In the cleavage reaction, bond energy is conserved via the formation of a covalent adduct between the 3' phosphate of the incised strand and a tyrosyl residue of the protein. Vaccinia topoisomerase can religate the covalently held strand across the same bond originally cleaved (as occurs during DNA relaxation) or it can religate to a heterologous acceptor DNA and thereby create a recombinant molecule.

When the substrate is configured such that the scissile bond is situated near (within 10 basepairs of) the 3' end of a DNA duplex, cleavage is accompanied by the spontaneous dissociation of the downstream portion of the cleaved strand. The resulting topoisomerase-DNA complex, containing a 5' single-stranded tail, can religate to an acceptor DNA if the acceptor molecule has a 5' OH tail complementary to that of the activated donor complex.

In accordance with the present invention, this reaction has been optimized for joining PCR-amplified DNA fragments into plasmid vectors (See Figure

PCR

Sfragments are naturally good surrogate substrates for the topoisomerase I religation step because they generally have 5' hydroxyl residues from the primers used for the amplification reaction. The 5' hydroxyl is the substrate forthe religation reactions.

The use of vaccinia topoisomerase type I for cloning is described in detail in US Patent No. 5,766,891.

SThe gene sequence being inserted into the expression vector can insert in either the sense or antisense direction. Therefore, the invention method provides for verification of both the size and orientation of the insert to insure that the gene sequence will express the desired protein. Preferably, the insert plus vector is utilized in a standard bacterial transformation reaction and the contents of the transformation WO 99/51766 PCT/US99/07270 11 plated onto selective growth media. Bacterial transformation and growth selection procedures are well known in the art and described in detail in, for example, Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995.

Individual bacterial colonies are picked and grown in individual wells of a multiwell microtiter plate containing selective growth media. An aliquot of these cells is used directly in a diagnostic PCR reaction. Primers for this reaction are designed such that only plasmids with correctly oriented inserts give amplification product. The amplified DNA is separated and visualized by SDS-PAGE gel electrophoresis using standard protocols (see Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed. 1995).

Performing the PCR reaction directly from the cultured cell lysates, rather than first preparing DNA from the bacteria, is a particular advantage of the invention method as it significantly reduces both the time needed to generate the required data and the cost of doing so.

Once plasmids containing the gene sequence insert in the correct orientation have been identified, plasmid DNA is prepared for use in the transformation of host cells for expression. Methods of preparing plasmid DNA and transformation of cells are well known to those skilled in the art. Such methods are described, for example, in Ausubel, et al, supra.

Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system. Prokaryotes most frequently are represented by various strains of E. coli. However, other organisms may also be used, including other bacterial strains.

Recognized prokaryotic hosts include bacteria such as E. coli and those from genera such as Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated. The prokaryotic host selected for use herein must be compatible with the replicon and control sequences in the expression plasmid.

WO 99/51766 PCT/US99/07270 12 Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO, 3T3 or CHOKI, HEK 293 cells or cells of lymphoid origin (such as 32D cells) and their derivatives. Preferred mammalian host cells include nonadherent cells such as CHO, 32D, and the like. Preferred yeast host cells include S. pombe, Pichia pastoris, S. cerevisiae (such as INVScl), and the like.

In addition, plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, nopaline synthase promoter and polyadenylation signal sequences, and the like.

Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase or MT promoter can be used. Rubin, Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can be engineered to express large amounts of peptide encoded by a desire gene sequence in insects cells (Jasny, Science 238:1653, 1987); Miller et al., In: Genetic Engineering (1986), Setlow, et al., eds., Plenum, Vol. 8, pp. 277-297).

In a further embodiment of the invention, there are provided libraries of expressible gene sequences produced by the methods of the invention. As shown in more detail in the Examples presented below, such libraries comprise gene sequences from a variety of sources such as yeast, mammals (including humans), and the like.

The present invention also features the purified, isolated or enriched versions of the expressed gene products produced by the methods described above.

Kits comprising one or more containers or vials containing components for using the libraries of the present invention are also within the scope of the invention.

Kits can comprise any one or more of the following elements: one or more expressible gene sequences, cells which are or can be transfected with said gene sequences, and antibodies recognizing the expressed gene product or an epitope tag associated therewith. Cells suitable for inclusion in such a kit include bacterial cells, yeast cells (such as INVScl), insect cells or mammalian cells (such as CHO).

WO 99/51766 PCT/US99/07270 13 In one embodiment, such a kit can comprises a detergent solution, preferably the Trax® lysing reagent NP-40 and 9% Triton X-100 in IX PBS). Also included in the kit can be one or more binding partners, an antibody or antibodies, preferably a pair of antibodies to the same expressed gene product, which preferably do not compete for the same binding site on the expressed gene product.

In another embodiment, a kit can comprise more than one pair of such antibodies or other binding partners, each pair directed against a different target molecule, thus allowing the detection or measurement of a plurality of such target molecules in a sample. In a specific embodiment, one binding partner of the kit may be pre-adsorbed to a solid phase matrix, or alternatively, the binding partner and matrix are supplied separately and the attachment is performed as part of the assay procedure. The kit preferably contains the other necessary washing reagents wellknown in the art. For EIA, the kit contains the chromogenic substrate as well as a reagent for stopping the enzymatic reaction when color development has occurred.

The substrate included in the kit is one appropriate for the enzyme conjugated to one of the antibody preparations. These are well-known in the art, and some are exemplified below. The kit can optionally also comprise a target molecule standard; an amount of purified target molecule that is the target molecule being detected or measured.

In a specific embodiment, a kit of the invention comprises in one or more containers: a solid phase carrier, such as a microtiter plate coated with a first binding partner; a detectably labeled second binding partner which binds to the same expressed gene product as the first binding partner; a standard sample of the expressed gene product recognized by the first and second binding partners; (4) concentrated detergent solution; and optionally, diluent.

The invention will now be described in greater detail by reference to the following non-limiting examples.

WO 99/51766 PCT/US99/07270 14 Example 1 High-throughput Expression of Yeast ORFs The following example illustrates the creation of a library of expressible yeast gene sequences.

Amplification 6,032 yeast ORFs and a corresponding gene-specific primer of the 3' end of each were obtained from Research Genetics (Huntsville, AL) in a 96-well microtiter plate format at a concentration of 0.3 ng/tl. Each gene specific primer was designed to exclude the gene's stop codon. Since the templates each contain a common sequence immediately 5' of the start ATG GCAGTCCTGGAATTCCAGCTGACCACC) (SEQ ID NO:1), it was possible to amplify each template with a common 5' primer.

pl of ORF template was added to a fresh 96-well microtiter plate (polycarbonate Thermowell Thinwall, Model M. Cat 6511) using a 12 channel pipetter. 6 tl of specific 3' primer solution (2 tM) was added and the total volume per well brought to 30 p with PCR cocktail, immediately after which the plate was placed on ice. (PCR cocktail for 120 reactions 720 pl 5X Buffer J, 48 pl dNTPs stock), 12 upl common 5' primer (1 pg/tl stock), 48 pl Taq DNA polymerase (Boeringer-Mannheim or Promega, 5 units/gl), 1.92 pl Pfu DNA polymerase (Stratgene, cat. 600153-81, 2.5 units/il) and 1464 pl distilled water. 5X Buffer J: 300 mM Tris (pH 75 mM ammonium sulfate, 10 mM MgCl 2 The rubber Hybaid Micromat lid was washed by soaking in 0.1 M HC1, the rinsed for 2 minutes with distilled water and dried completely before applying to the 96-well plate.

The PCR reaction was performed using a Hybaid, Ltd. (Middlesex, UK) thermo-cycler according to the manufacturer's instructions. The conditions used were as follows: pre-melt step: 940 C x 4 min; melt step: 940 C x 30 sec, anneal step: 580 C x 45 sec, extend step: 72' C x 3 min repeated for 25 cycles; final extension: 720 C x 4 min; final block temperature set to room temp (approx. 220 The plates were stored at 40 C.

WO 99/51766 PCT/US99/07270 Purification The plates were spun briefly at 1000 rpm, then 10 pl of 6X gel loading dye was added to each well (6X gel loading dye: 6 mM Tris (pH 6 mM EDTA, 0.03% Bromphenol Blue, 30% glycerol). The entire contents of each well were loaded onto a 1% low melt agarose (Invitrogen 46-0150) gel (plus ethidium bromide at 20 Al of a 10 mg/ml solution added to 400 mis of agarose) in IX TAE (50X TAE 242g Tris base, 57.1 ml glacial acetic acid, 100 ml 0.5 M EDTA, pH 8.0 per liter (water)) and run at 110 120 volts for 1.25 to 1.5 hours. A UV light box was used to visualize the amplification products and ensure that only correct-sized PCR products are used in the insertion step.

Insertion into expression vector(s) The portion of each lane containing the amplified gene sequence was cut from the gel and transferred to a well in a 96-well microtiter plate, melted on a heat block (750 and a portion of the melt multi-channel pipetted into a 96-well microtiter plate (7 tl/well) containing one of two expression vectors: TOPO-adapted pcDNA3.1/GS or pYES2/GS (Invitrogen, Carlsbad, CA) previously digested with HindIII. The plate was covered with parafilm and incubated at 370 C for 7 minutes.

Top 10 Chemically Competent Cells (Invitrogen) were added to each well (45 pl/well, whereupon the plate was re-covered and incubated on ice for 5 minutes.

The cells were then heat shocked on a 42° C block for 1 minute and returned to ice for 1 minute. An aliquot of SOC medium was added to each well (150 gl, 20g tryptone, yeast extract, 0.5g NaC1, 250 mM KC1, 20 ml 1M glucose/liter), and the plate was incubated at 370 C for 90 to 120 minutes.

The contents of each well were plated onto a LB(10g tryptone, 5g yeast extract, 10g NaCI per liter)/1.5% agar petrie plate containing the appropriate selection marker (ampicillin (50 ug/ml) for pYES2/GS and Zeocin T M (25 ug/ml) for pcDNA3.1/GS). The petrie plates were grown overnight at 370 C.

Verification of size and orientation WO 99/51766 PCT/US99/07270 16 Contamination is a potentially serious problem in this step. Care should be taken to guard against contaminating the process through airborne contamination, unsterile reagents or equipment, or well-to-well contamination.

Eight colonies were picked from each petrie plate and placed in eight individual wells of a 96-well microtiter plate. Each well contained 100 p of 2X LB plus 100 jpg/ml ampicillin or 50 g.g/ml Zeocin M as appropriate for the expression vector used. The plates were incubated overnight at 370 C.

The plates were spun briefly at 1000 rpm. The cells were stirred by pipetting up and down in a pipetter, then 2 ul from each well was transferred to a corresponding well in a PCR reaction plate containing 28 .l/well PCR cocktail (PCR cocktail for 840 reactions 5040 pl 5X Buffer J, 336 gl dNTPs (50mM stock), 84 pl common primer (1 pg/pl stock, Dalton Chemical Lab. Inc, Ont. CAN), 84 l 3' H6stopprevu primer (1 p.g/p.l, Dalton Chemical Lab. Inc, Ont. CAN), 336 pl Taq DNA polymerase (Boeringer-Mannheim or Promega, 5 units/pl), and 17.64 mls distilled water.

H6stopprevu primer has the sequence 5' AAA CTC AAT GGT GAT GGT GAT GAT GACC (SEQ ID NO:2).

The PCR reaction was run essentially as described above with the following cycle: pre-melt step: 940 C x 10 min; melt step: 940 C x 1 min, anneal step: 670 C x 1 min, extend step: 720 C x 3 min 35 cycles; final extension: 720 C x 4 min; final block temp set to room temp (approximately 220 The plates were spun briefly at 100 rpm and 6 p.l of 6X gel loading dye added to each well. Samples were run on a 1% agarose gel which was subsequently stained with ethidium bromide. Only plasmids with correctly oriented inserts give an amplification product in this step.

The location of the positive clones was entered into a database and a spreadsheet of positive clones generated. The spreadsheet was downloaded onto a Qiagen BioRobot 9 6 0 0TM to direct the re-racking of the positive cultures into deep- WO 99/51766 PCT/US99/07270 17 well culture blocks. Essentially, a single positive culture for each clone was grown and used to prepare plasmid DNA according to the Quia-Prep Turbo protocol.

CHO cells were transfected with the prepared plasmid DNA using the Pfx-6 PerFect Lipid system (Invitrogen, Cat #T930-16). Yeast cells (INVScl) were transfected using the S.C. EasyComp Transformation kit (Invitrogen, Cat #K5050- 01). Expression was verified by Western blot using anti-V5 antibody to detect the epitope tag. A total of 558 clones expressing a correct protein were obtained after a single pass.

Example 2 High-throughput Expression of Human Gene Sequences The following example illustrates the construction of a library of expressible human gene sequences using the method of the invention. Primers were constructed based on sequences of human genes available from GenBank.

Fetal human heart tissue was obtained from the International Institute for the Advancement of Medicine (IIAM). Poly A+ mRNA was isolated using the FastTrackTM 2.0 Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The mRNA was converted to first-strand cDNA using a cDNA Cycle® Kit (Invitrogen) using the oligo dT primer provided and the protocols suggested. A single cDNA synthesis reaction was split into 12 separate wells of a 96-well PCR amplification plate, and PCR amplifications were performed using specific primer sets, essentially as described above, with the exception that the ratio of Taq to Pfu was 50:1 in the initial amplification (final cone. 2 U Taq:0.04 U Pfu/well).

Primers were synthesized using a Primerstation 960 (Intelligent Automation Systems, Inc.) used according to the manufacturer's instructions and were designed from sequences downloaded from Unigene and sent directly to the synthesizer.

Approximately 15 nMoles of each primer, having an average length of 25 basepairs, was synthesized in a 96-well format. After synthesis, the primers were cleaved from the supports, deprotected and dried in the same 96-well format (see manufacturer's instructions).

WO 99/51766 WO 9951766PCT/US99/07270 18 The amplified gene sequences were purified and inserted into the pcDNA3. 1 /GS expression vector essentially as described above. The expression vectors containing sequences verified to be in the correct orientation were transfected into CHO cells in 96-well deep-well blocks using the Pfx-6 PerFect Lipid system (Invitrogen, Cat #T930-16). Cell lysates were made 48 hours alter transfection, and the lysates were separated by SDS-PAGE and analyzed by Western blot according to standard protocols using an anti-VS epitope tag Mab/horseradish peroxidase conjugate. Table I lists the human proteins successfuilly expressed using this methodology. A total of 66 clones expressing a correct protein, out of 118, were obtained after a single pass.

Table 1 Human ORFs Plate Number Accession Number Description Predicted Actual Size M235 C7 H-A06977 albumin 67.1 67.OkDa El H-AB002391 Human mRNA for KIAA0393 68.09 68 complete cds H3 H-AB006969 Homo sapiens hGAA1I mRNA, 68.42 cds E2 H-AB007875_ Homo sapiens KIAAO415 mRINA, 51.48 51 cds Dl H-AB007887 Homo sapiens KDa0427 mRNA, 66.55 cds MK521 D6 H-ABO10710 Homo sapiens mRINA for lectin- 30.14 like oxidized LDL receptor, cds G3 H-AD00 1528 Homo sapiens spermidine 40.37 aminopropyltransferase mRNA, complete cds H-AE000659 Homo sapiens T-cell receptor 12.39 16 alpha delta locus from bases 250472 to 501670 (section 2 of of the C E2 H-AF004022 Homo sapiens protein kinase 38.28 44 complete cds MW428 C1 H--AF00423 1 Homo sapiens 65.78 monocyte/macrophage Ig-related receptor MIR-l10 (MIR cl- complete cds AS -AF0427 Homo sapiens angiopoietin-2 5.760 mRNA, complete cds546 -77,065 1 oospescroooe2 H-AF006501 cosmid clone ci 155, RNA polymerase Il subunit 14.4 kDa (POLRF) gene, complete cds; 14.08 I I L_ WO 99/51766 WO 9951766PCTIUS99/07270 H4 H-AF008936 Homo sapiens syntaxin- I6B 35.75 47 complete cds H-AF009243 Homo sapiens proline-rich Gla 22.33 36 protein 2 (PRGP2) mRNA, M462 D6 H-AFO 1 3249 Homo sapiens leukocyte- 31.68 associated Ig-like receptor- I (LAIR-i1) mRNA, complete cds AlI H-AFO 13512 untitled 53.02 53 A3 H-AF0 13970 Homo sapiens MTG8-like protein 66.55 (MTGR1) mRNA, complete cds M467 A7 H-AFO 14807 Honmo sapiens 23.54 29.OkDa phosphatidylinositol synthase mRNA, complete cds D2 H-AF015257 Homo sapiens flow-induced 41.36 endothelial G protein-coupled receptor (FEG- I) mRNA, cds M422 B5 H-AF01 7307 Homo sapiens Ets-related 40.92 49.OkDa transcription factor (ERT) mRNA, complete cds A6 H-AF0 17656 Homo sapiens G protein beta 5 38.94 48 subunit mRNA, complete cds El- H-AF0 17995 Homo sapiens 3-phosphoinositide 61.27 52 dependent protein kinase- I (PDK I) mRNA, complete cds G I H-AF019612 Homo sapiens S21? mRINA, 57.2 57 complete cds D3 H-AF020591 Homo sapiens zinc finger protein 78.76 74 mRNA, complete A7 H-AF022385 Homo sapiens apoptosis-related 23.43 33 protein TFAR15 mRNA, complete H6 H-AF024714 Homo sapiens interferon- 37.84 48 inducible protein (AIM2) mRNA, complete cds BIH-AF025527 Homo sapiens leucocyte 48.4 47 inimunoglobulin-like receptor-4 (LTR-4) mRNA, complete M424 B4 H-AF025532 Homo sapiens leucocyte, 49.39 59.OkDa imxnunoglobulin-like receptor-S mRNA, complete cds H-AF026071F Homo sapiens soluble death 30.58 receptor 3 beta (DR3) mRNA, cds M428 Al H-AF026273 Homo sapiens interleukmn-I 65.01 68.OkDa receptor-associated kinase-2 complete cds B6 H-AF026293 Homo sapiens chaperonin 58.96 58 containing t-complex polypeptide 1, beta subunit (Cctb) nRINA, cds WO 99/51766 WO 99/1 766PCTIUS99/07270 H-AF026548 Homo sapiens branched chain 45.43 alpha-ketoacid dehydrogenase kinase precursor, mRNA, nuclear gene encoding mitochondrial complete cds B2 H-AF027204 Homo sapiens putative tetraspan 21.78 27 transmembrane protein L6H mRNA, complete cds M426 D3 H-AF0286o08 Homo sapiens SP I-Uie zinc 56.43 64.OkDa finger transcription factor SLP mRNA, complete cds B I H-AF029232 Homo sapiens calpamodulin 70.62 (CalpM) mRNA, complete cds M422 A7 H-AF029761 Homo sapiens decoy receptor 2 42.57 complete cds M477 F3 H-AF029893 Homo sapiens i-beta-1,3-N- 45.76 acetyiglucosaminyltransferase mRNA, complete cds H-AF032437 Homo sapiens mitogen activated 51.92 protein kinase activated protein kinase gene, complete cds M416 F3 H-AF035824 Homo sapiens vesicle soluble 25.63 36.OkDa NSF attachment protein receptor (VTI 1) mRNA, complete cds F3 H-AF037335 Homo sapiens carbonic anhydrase 39.05 39 precursor (CA 12) mRNA, complete cds GI H-AF039019 Homo sapiens zinc finger DNA 87.45 87 binding protein 89 kDa (ZBP-89) mRNA, complete cds GI H-AF039136 Homo sapiens Fas binding protein 81.51 98 (hDaxx) mRNA, complete cds A7 H-AF040705 Homo sapiens putative tumor 31.57 41 suppressor protein unspliced form (Fus-2) mRNA, complete cds M469 FlI H-AF040958 Homo sapiens lysosomal 45.76 46.OkDa neuraminidase precursor, mRNA, complete cds G2 H-AF043 472 Homo sapiens Shab-related 54.12 64 delayed-rectifier K+ channel alpha subunit (Kv9.3) mRNA, cds E2 H-AJO0 1340 Homo sapiens mRNA for U3 52.36 snoRNP associated 55 kDa protein______ GI H-DO0096 Transtyretin (prealbumin) 16.28 C4 H-DO0408 Cytochrome P450 111A7 (P450- 55.44 64 M302 E7 H-DO0682 cofilin 18.37 M383 G2 H-DO0726 ferrochelatase 46.64 50.0kDa M383 C3 H-D00760 proteasome, subunit HC3 25.85 34.OkDa M305 B4 H-D00761 proteasome, subunit HC5 26.62 33 M266 F7 H-DO0763 proteasome, subunit HC9 28.82 33 WO 99/51766 WO 9951766PCTIUS99/07270 E2 H-D00860 Phosphoribosyl pyrophosphate 35.09 47 subunit I 2 15-13 H-D 10522 human mRNA for 80K-L protein 35 36.59 M423 F5 H-D 1086 Interleukin 2 receptor gamma 40.7 45.0 kDa, M248 D2 H-D 11094 positive modulator of HIV tat- 47.74 mediated transactivation G3 1--DI 1428 Peripheral myelin protein 22 17.71 17 M424 D3 H-D13 168 Human gene for endothelin-B 48.73 48.OkDa.

M271 B8 H-D13315 glyoxalase 1, 20.35 34.OkDa

LACTOYLGLUTATHIONE

LYASE. CATALYZES THE CONVERSION OF HEMIMERCAPTAL, FORMED FROM METHYLGLYOXAL AND GLUTATHIONE, TO S-

_________LACTOYLGLUTATHIQNE.

M306 F1 H-D13627 hypothetical protein 60.39 (GB:D13627)_____ M248 DI H-D 13630 hypothetical protein 46.2 49 (GB:D 13630) Human mRNA for gene, complete cds M270 D5 H-D13634 hypothetical protein 34.65 42.OkDa 3634) M250 D2 H-D1 3642 hypothetical protein 44 48.OkDa (GB:D I3642),Human mRNA for 17 gene, complete cds M250 E6 H-DI3748 translation initiation factor 4A 44.77 49.OkDa M305 C3 H-D 13 892 carboxyl methyltransferase, 25.19 34 DI H-D13900 enoyl-Coenzyme A hydratase, 32.01 58 chain, mitochondrial El H-D 14446 Human HFREP-1 mRNA for 34.43 protein, complete cds 167-14 H-D14497 H-.sapiens (Ewing's sarcoma cell 51.44 64 line) mRNA encoding open frame M266 D2 H-D14520 basic transcription element- 24.2 33.OkDa protein 2 M318 D2 H-D14658 hypothetical protein 13.64 17 D2 H-1314661 Human mRNA for KIAAO 105 16.72 28 complete cds M236 E2 H-D 14662 HYPOTHETICAL 29.5 KD 24.75 36.OkDa PROTEIN IN UBP13-KIPI INTERGENIC REGION cerevisiae] M271 G6 H-D14695 hypothetical protein 43.12 (GB:D14695), Human mRNA for gene, complete cds.

M311I A3 H-D14696 hypothetical protein 25.74 14696) WO 99/51766 WO 9951766PCT/US99/07270 H3 H-D 14697 Farnesyl diphosphate synthase 46.2 (farnesyl pyrophosphate synthetase, dimethylallyltranstransferase, geranyltranstransferase) M271 E7 H-D 14705 catenmn, alpha Catenin 99.77 110 (cadherin-associated protein), alpha 1 (lO2kD). ASSOCIATES WITH THE CYTOPLASMIC DOMAIN OF A VARIETY OF CADHERINS. M236 A6 H-D 14811 hypothetical protein 30.25 42 (GB:D1481 1) M250 A3 H-D 14 812 hypothetical protein (GB:D148 12), Human mRNA for KIAA0026 gene, complete cds H-D314874 Human mRNA for 20.46 33 ___________adrenomedullmn, complete cds F3 H-D 14887 Human mRNA for TFIIA-42, 41.47 cds M250 H6 H-D 16234 phospholipase C, alpha, 55.66 56.OkDa PROBABLE PROTEIN DISULFIDE ISOMERASE ER- 60 PRECURSOR [Homo sapiens]_____ M305 131 H-D 16480 enoyl-CoA hydratase/3- 84.04 84 hydroxyacyl-CoA dehydrogenase triflinctional protein, alphasubunit, mitochobdrial______ M271 G2 H-D16481 3-ketoacyl-CoA thiolase, beta subunit, mitochodrial, Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl- Coenzyme Athiolase/enoyl- Coenzyme A hydratase (trifunctional protein), beta HI H-D 16626 Histidine ammonia-lyase 72.38 64 A2 H-D17532 Human mRNA for RCK, 52.03 53 complete cds M266 F4 H-D]7554 DNA-binding protein TAX 31.79 38 M248 A3 H-1321235 xeroderma pigmentosum group C 40.04 repair complementing protein M235 El H-1321261 SM22-ALPHA HOMOLOG, 22 31 hypothetical protein M3 11 El H-D2 1262 hypothetical protein 77.950 63 1262) M466 B34 H-D21 853 Human mRNA for KIAAO I 11 45.32 49.OkDa complete cds M3 11 H3 H-1323 660 ribosomal protein L4 47.08 47 M419 ElI H-1326309 human mRNA for LIMK (LIM 7 1.240 WO 99/51766 WO 9951766PCT/US99/07270 M27 1 B9 H-1326362 hypothetical protein 79.97 (GB:D26362), Human mRNA for gene, complete cds M361 H2 H-1326598 proteasome, subunit HsC 10-1I 22.66 33.OkDa M302 G4 H-D26599 proteasome, subunit HsC7-l 22.22 34 GI1 H-D26600 Human mRNA for proteasome 29.15 36 subunit HsN3, complete cds G9 H-D28540 hypothetical protein, CDCIO 44.77 homolog M266 AS H-D2901 1 proteasome, subunit X 22.99 23 M236 F3 H-D29012 Proteasome (prosome, macropain) 26.4 32.OkDa delta subunit, beta type, 6 Cl H-D30037 Human mRNA for 29.92 38 phosphatidylinositol transfer protein (PI-TPbeta), complete cds M250 H4 H-1330655 translation initiation factor 4AII, 44.88 and ribosomal binding protein 167-26 H-D30742 human mRNA for calmodulin- 52.10 dependent protein kinase IV M236 A4 H-D3 1767 hypothetical protein 18.59 (GB:D3 1767), Human mRNA for KIAA0058 gene, complete cds El H-D331883 Human mRNA for KIAA0059 50.93 64 gene, complete cds; G2 H-1332 129 MHC class I protein HLA-A 40.26 M422 A6 H-D3 7965 Human mRNA for PDGF receptor 41.36 beta-like tumor suppressor complete M305 H4 H-D3 8047 26S proteasome regulatory 28.340 34.OkDa P31 M423 B2 H-D38081 Thromboxane A2 receptor 37.84 M317 D3 H-D38305 ErbB-2 transducer 38.06 49 M270 A8 H-D38583 calgizzarin, Human mRNA for 11.66 12 calgizzarin, complete M270 A6 H-D42038 hypothetical protein 15.29 27 (GB:D42038), Human mRNA for KIAA0087 gene, complete cds M318 F3 H-D42085 hypothetical protein 90.2 100 (GB M311 C2 H-1343 642 YL- 1 protein homolog 40.15 36 El H-1345213 Human mRNA for zinc finger 12.87 complete cds M236 B2 H-D45248 proteasome activator hPA28, 26.4 38 subunit beta, may be cell adhesion protein H3 H-D45887 Human mRNA for cahnodulin, 16.5 cds; 166-3 H-D45906 human mRNA for LIMK-2 70 70.25 A7 H-D49357 Human nRINA for S- 43.56 51 adenosylimethionine synthetase, complete cds CS H-D49489 Human mRNA for protein 48.51 54 disulfide isomerase-related PS, complete WO 99151766 WO 9951766PCTIUS99/07270 M482 E2 H-1349958 Human fetus brain mRNA for 30.69 32.OkDa membrane glycoprotein M6, cds M305 G5 H-D350063 proteasome, subunit p40 35.75 39 M250 B6 H-13503 10 cyclin 1, Human mRNA for cyclin 41.58 47 1, complete cds E3 H-D50419 Homo sapiens mRNA for OTKI18, 78.32 64 cds M298 B 1 H-D5 0495 transcription elongation factor h- 33 33.OkDa SIT-TI (GB:D50495) M302 A3 H-1350840 ceranide glucosyltransferase 43.45 44 167-40 H-1350863 human mRNA for TESK 1 68.9 3 166-28 H-D50927 human myeloblast niRNA for 60.46 64 KLAA0137 gene DI H-D63521 Homo sapiens mRNA for LECT2 16.72 16 precursor, complete cds M302 A5 H-1378 134 glycine-rich binding protein CIRP 19.03 M313 E5 H-1378275 proteasome subunit p 4 2 42.9 48.OkDa B3 H-1379205 Human mRNA for ribosomal 5.72 protein L39, complete cds A4 H-D379206 Human gene for ryudocan core 21.89 33 exonl-5, complete cds Al H-1380008 Human mRNA for K1AA0 186 21.67 32 complete cds M298 H4 H-D83004 ubiquitin-conjugating enzyme E2 16.83 32.OkDa similar to Drosophila bendless product C3 H-1383 702 Human brain mRNA for 64.57 64 photolyase homolog, complete M306 A] H-1383 735 neutral calponin 34.1 34.0kDa H2 H-D386322 Homo sapiens mRNA for 67.21 64 calniegin, complete cds B] H-1386979 Human mRNA for KIAA0226 82.72 82 complete cds 169-16 H-D387 116 dual specificity mitogen-activated 38.24 42 kinase kinase 3 166-27 H-D387 119 human cancellous bone osteoblast 37.80 for GS3955 E2 H-D88 308 Homo sapiens mRNA for very- 68.31 64 long-chain acyl-CoA synthetase, cds 166-26 H-D89077 human mRINA for Src-like 30.43 38 protein M440 H2 H-1389479 Homo sapiens mRNA for STIB2, 32.67 38.0kDa cds HI H-D390086 Human pyruvate dehydrogenase 39.6 (EC 1.2.4. 1) beta subunit gene, exons 1-10 M362 Fl H-1390209 DNA-binding protein 38.72 48.OkDa TAXREB67 M316 B2 H-J00068 actin, alpha 1, skeletal muscle 41.58 M250 B2 H-JOO 194 major histocompatibility complex, 28.0536Oa HC class 11, DR alpha WO 99/51766 WO 9951766PCTILIS99/07270 G2 H-J00212 Interferon, alpha 21 20.9 G I H-JO0287 Human pepsinogen gene 42.79 48 M298 C2 H-J0261 1 apoipoprotein D 20.9 31 .OkDa M266 C4 H-J02683 ADP/ATP carrier protein 32.89 36 M383 H2 H-JO2685 plasminogen activator inhibitor, 45.76 placenta 167-3 H-J02853 "casein kinase 11, alpha chain" 43.08 E3 H-J02854 Human 20-kDa myosin light 19.03 31 chain (MLC-2) mRNA, complete M248 F3 H-J02 874 fatty-acid-binding protein 4, 14.63 17 adipocyte, LIPID TRANSPORT PROTEIN IN ADIPOCYTES M235 D5 H-J02939 antigen 4F2, heavy chain 58.3 58 C3 H-J02943 Corticosteroid binding globulin 44.66 M248 F2 H-J02966 adenine nucleotide translocator 1 32.78 33 (skeletal muscle) [ANTI], CATALYZES THE EXCHANGE OF ADP AND ATP ACROSS THE MITOCHONDRIAL INNER MEMBRANE. El H-J02982 Glycophorin B 10.12 167-91 H-J03075 "protein kinase c substrate, 80 kD 58.04 98 protein heavy M266 A3 H-J03191 profilin 1 15.51 17.OkDa M248 H4 H-J03231I glucose-6-phosphate 56.76 51 ~dehydrogenase [G6PD] M266 F2 H-J03459 LEUKOTRIENE A-4 67.32 64 HYDROLASE [Homo sapiens] A2 H-J03460 Prolactin-induced protein 16.17 26 M271 E5 H-J03799 laminin receptor 1, Lamninin 32.56 receptor (2H5 epitope). RIBOSOMAL PROTEIN SA ~~[Homo sapiens]. M440 A4 H-J03 890 Human pulmonary surfactant 21.78 protein C (SP-C) and pulmonary surfactant protein ClI (SP-C 1) complete cds M271I D8 H-J03 934 NAD(P)H menadione 30.25 38 oxidoreductase 1, dioxininducible. INVOLVED IN __________DETOXICATION PATHWAYS.____ M27 1 A8 H-J04031I trifunctional enzyme 102.96 11 7.OkDa (GB:J0403 C-I

TETRAHYDROFOLATE

SYNTHASE, CYTOPLASMIC sapiens] M305 F6 H-J04046 calmodulin 3 [CALM3] 16.5 M305 G7 H-J04071 cytotoxic T-lymphocyte- 27.28 38 associated serine esterase 1 (cathepsin G-like 1, granzyme B) M31 D2 HJ418 lysosomal-associated membrane 44.99 47 protein 2 WO 99/51766 WO 9951766PCT/US99/07270 M300 F4 H-J04205 Sjogren syndrome antigen B 44.99 51 .OkDa M416 G8 H-J04430 Acid phosphatase 5, tartrate 35.64 BI H-J04501 Glycogen synthase 1 (muscle) 81.18 81 M313 B5 H-J04543 synexin 51.37 51 BI H-J04605 Peptidase D 54.34 M250 C6 H-J04615S small nuclear ribonucleoprotein 26.51 34.OkDa SM-D, ROLE IN THE PREmRNA SPLICING OR IN

__________SNRNPSTRUCTUIRE.____

M248 E2 H-J04964 steroid sulfatase (microsomal) 64.24 M250 A7 H-J05249 replication protein A, 32 kDa 29.81 36.OkDa subunit, REQUIRED FOR SV DNA REPLICATION IN VITRO, RP-A IS SINGLE- STRANDED DNA-BINDING FlI H-JO5272 IMP (inosmne monophosphate) 56.65 51 1 169-15 H-JO5401 "creatine kinase, sarcomeric 50 46.16 mitochondrial precursor'"______ M266 E4 H-JO5448 RINA polymerase 11, subunit B33 30.36 M305 C2 H-K00558 tubulin, alpha kI [TUBA*] 49.72 52.OkDa M416 H7 H-KO 15 71 Human T-cell receptor active 34.43 36.OkDa beta-chain, mRNA from cell line complete cds M31 1 E4 H-KO 1763 haptoglobin 38.28 47.OkDa H-K02 100 Human omnithine 39.05 47 transcarbamylase (OTC) mRNA, coding sequence______ M302 D5 H-K02574 purine nucleoside phosphorylase 31.9 36.OkDa 169-39 H-K02581 "thymidine kinase, cytosolic" 34 25.81 M248 E4 H-K03020 phenylalanine hydroxylase [PAH] 49.83 M556 B3 H-K03 191 Cytochrome P450, subfamily 1 56.43 53.OkDa (aromatic compound-inducible), H12 H-1,00 190 Antithrombin 111 51.15 169-62 H-LO 1087 "protein kinase c, theta type" 80 77.7 M318 C2 H-LO 1124 ribosomal protein 513 16.72 28 M313 Fl1 H-L02321 glutathione S-transferase M5 24.09 28 M305 E5 H-L02426 protease 26S, regulatory subunit 4 48.51 53 M302 D4 H-L02547 cleavage stimulation factor, 50 47.52 subunit M266 H7 H-L02648 transcobalamin 11 47.08 48.OkDa E2 H-L02932 Human peroxisome proliferator 51.59 59 activated receptor mRNA, cds M270 Al1 H-L03380 gonadotropin-releasing hormone 36.19 36 receptor [GRHR], THS RECEPTOR MEDIATES ITS ACTION BY ASSOCIATION WITH G PROTEINS____ WO 99/51766 WO 9951766PCT/US99/07270 M270 HI1 H-L0341 1 RD protein [RDBP], Radin blood 41.91 59.0 kDa group D3 H-L03426 Human XE7 mRNA, complete 42.46 coding regions BI1 H-L03785 Myosin, light polypeptide 5, 19.14 32 A7 H-L04483 ribosomal protein S!21 9.24 34 M416 B2. H-L05 147 Human dual specificity 20.46 phosphatase tyrosine/serine mRNA, complete cds 215-38 H-L05624 dual specificity mitogen-activated 50 43.30 protein kinase kinase M271 D4 H-L06132 anion channel, voltage-gated, 31.24 37 isoform 1. FORMS A CHANNEL THROUGH THE CELL MEMBRANE, THAT ALLOWS DIFFUSION FROM SMALL HYDROPHYLIC MOLECULES.____ 169-27 H-L06 139 tyrosine-protein kinase receptor 125 123.7 TIE-2 precursor______ HI H-L06 147 Human (clone SY 11) golgin-95 68.31 68 complete cds M250 Al1 H-L06419 procollagen-lysine, 2-oxoglutarate 80.08 (lysine ~~hydroxylase) M236 F6 H-L06498 ribosomal protein S20 13.2 23.OkDa M3 18 Dl1 H-L06499 ribosomal protein L37a 10.23 27 M270 Dl H-L07414 CD40 antigen ligand [CD4OLGI, 28.82 36 NVOLVED IN IMMUNOGLOBULIN CLASS

~SWITCHING.________

M298 A6 H-L07548 aminoacylase 1 44.99 52.OkDa M424 C3 H-L07592 Human peroxisome proliferator 48.62 48.OkDa activated receptor mRNA, complete cds M298 G6 H-L07633 proteasome (prosome, macropain) 27.5 33.OkDa activator subunit I (PA28 alpha) [PSMEII M3 18 B 1 H-L08096 CD70 antigen (CD27 ligand) 21.34 28 D2 H-L08 187 cytokine receptor EB13 25.3 42 M313 F4 H-L08850 amyloid, non-A beta component, 15.51 3 1.OkDa Alzheimer's disease M426 El1 H-L08895 MADS box transcription enhancer 52.14 factor 2, polypeptide C (myocyte enhancer factor M266 A8 H-L09235 ATPase, vacuolar 67.98 64.OkDa M266 D 1 H-L09604 differentiation-dependent 16.83 17.OkDa intestinal membrane A4 protein (Homo sapiens) M3 17 ClI H-LI0338 sodium channel, voltage-gated, 24.09 24 I, beta polypeptide [SCN1B I M317 ElI H-L 10717 tyrosine-protein kinase ITKITSK 68.270 _68.OkDa M300 B5 H-L 10820 formyl peptide receptor I [FPR1] 38.61 37 WO 99/51766 WO 99/1 766PCTIUS99/07270 M3 12 A4 H-LI 0838 pre-mRNA splicing factor SRp2O 18.15 -31 .OkDa M300 A5 H-LI0918 chemokine receptor 1 39.16 M3 11 F2 H-Li 11245 complement component 4-binding 27.83 beta M266 B7 H-LI 1353 neurofibromatosis 2 (bilateral 65.56 63.OkDa acoustic neuroma) M31 1 B3 H-L 11667 cyclophilin 40 40.81 215-49 H-L 11695 serine/threonine-protein kinase 64 55.40 R4 precursor M466 C2 H-Li 11931 Human cytosolic serine 53.24 56.OkDa hydroxymethyltransferase mRNA, complete cds M271 87 H-L12 168 ADENYLYL CYCLASE- 52.36 ASSOCIATED PROTEIN 1 sapiens]______ M416 D4 H-L 12964 Interleukin-activated receptor, 28.16 38.OkDa homolog of mouse Ly63 B3 H-1,13203 Human HNF-3/fork-head 38.72 49 homolog-3 HFH-3 mRNA, cds D2 H-L 13 744 Human AF-9 mRNA, complete 62.59 63 167-8 H-L13943 glycerol kinase 60 57.71 M311 G3 H-L13974 leucine zipper protein 41.14 51 (GB:L13974) M271 H5 H-L13977 LYSOSOMAL PRO-X 54.67 57

CARBOXYPEPTIDASE

PRECURSOR [Homo sapiens]. M270 G2 H-L 14283 protein kinase C, zeta [PRKCZ], 65.23 98 SERINE- AND THREONINE- SPECIFIC ENZYME._____ M235 A3 H-L 14286 antioxidant protein, thiol-specific 21.89 32.OkDa M426 H3 H-L14778 Protein phosphatase 3 (formerly 57.42 2B3), catalytic subunit, alpha isoform (calcineurin A alpha){f alternative products) 84 H-L 15702 complement factor B 84.15 100 M426 A4 H-L16794 Human transcription factor 57.42 (MEF2) mRNA, complete cds 215-25 H-L 16862 g protein-coupled receptor kinase 70 63.4 GRK6 167-74 H-L16991 thymidylate kinase 36 23.39 169-3 H-LI 8964 "protein kinase c, iota type" 80 64.64 M305 E2 H-L18972 hypothetical protein (GB:L 18972) 75.24 78 M426 D4 H-L 19067 Human NF-kappa-B transcription 59.18 63.OkDa factor p65 subunit mRNA, cds 2 15-26 H-L19268 Homo sapiens myotonic 70 68.71 dystrophy associated protein mRNA____ WO 99/51766 WO 99/1 766PCT/US99/07270 M271 El H-L 19297 carbonic anhydrase V [CA5J, 33.66 42 Mitochondrial carbonic anhydrase. REVERSIBLE HYDRATATION OF CARBON M298 G4 H-L19437 transaldolase 37.18 39.OkDa M423 C4 H-L 19593 Interleukin 8 receptor, beta 39.71 4 1.OkDa G I H-L 19686 Homo sapiens macrophage 12.76 13 migration inhibitory factor (MIF) complete cds; G2 H-L 19739 metallopanstimulin 1 9.35 32 M302 E3 H-L 19871 activating transcription factor 3 20.02 36.OkDa 167-86 H-L20422 14-3-3 protein eta 34 27.1 3 M440 B2 H-L20492 Human gamma-glutamyl 24.86 35.0 kDa transpeptidase mRNA, complete cds M3 15 B 1 H-L20688 GDP-dissociation inhibitor 22.22 32 protein rhoA M271 H3 H-1,20941 ferritin, heavy polypeptide. 20.24 32 FERRITIN IS AN

INTRACELLULAR

MOLECULE THAT STORES IRON IN A SOLUBLE, NONTOXIC, READILY _________AVAILABLE FORM.

M235 B7 H-1,21893 Na+/taurocholate cotransporter, STRICTLY DEPENDENT ON THE_ FlI H-L,21934 Sterol 0-acyltransferase (acyl- 60.61 Coenzyme A: cholesterol C2 H-L22075 Human guanine nucleotide 41.58 regulatory protein (G 13) mRINA, ~complete cds 169-18 H-L22206 vasopressin v2 receptor 60 58.00 M421 A10 H-L22214 Human adenosine AlI receptor 35.97 38.OkDa (ADORAI1) mRNA exons 1-6, cds M424 F1 H-1,23959 Homo sapiens E2F-related 45.21 53.OkDa transcription factor (DP- 1) complete cds C2 H-1,24498 Human gadd45 gene, complete 18.26 28 ~cds M302 E2 H-L25080 proto-oncogene rhoA, multidrug 21.34 31 resistance protein M270 B8 H-L25081 guanine nucleotide-binding and 21.34 transforming protein rhoC, ras-related homolog 9 M236 E3 H-1,25085 Sec6l complex, beta subunit, 10.67 19 PROTEIN TRANSLOCATION IN THE ENDOPLASMIC RETICULUM_____ 167-85 H-L256 10 cyclin-dependent kinase inhibitor 32 18.11 1 WO 99/51766 WO 99/1 766PCTIUS99/07270 B2 H-1,2561 0 cyclin-dependent kinase inhibitor 18.110 M297 H2 H-L26232 cathepsin Alphospholipid transfer 54.34 64.OkDa protein 42.31__ 167-4 H-1,263 18 stress-activated protein kinase 5 23

JNKI

M428 Fl H-L27586 Human TR4 orphan receptor 67.76 67.OkDa complete cds M302 E5 H-L2771 1 protein phosphatase KAPi1 23.43 28 M250 A6 H-L28010 Homo sapiens HnRNP F protein complete Fl H-1,28821 Alpha mannosidase 11 isozyme 87.67 87 167-89 H-L28824 tyrosine-protein kinase SYK 70 _69.92 M298 E6 H-L28997 ADP-ribosylation factor-like gene 20.02 33.OkDa

I

D4 H-L29219 Homo sapiens cik I mRNA, 53.35 complete cds 169-63 H-1,29222 Homo sapiens clki mRNA 25 15.0 3 M429 B3 H-1,29277 Signal transducer and activator of 84.81 88.OkDa transcription 3 (acute-phase factor)______ Cl H-L29433 Human factor X (blood 53.79 64 factor) gene G3 H-1,31860 Glycophorin A 16.61 26 DI H-L3 1881 Nuclear factor I/X (CCAAT- 48.62 48 transcription factor) 169-13 H-L31951 human protein kinase (JNK2) 55 46.71 AlI H-L32 179 Arylacetamide deacetylase 44 B2 H-L3 3404 Human stratum corneum 27.94 36 chymotryptic enzyme mRNA, cds M3 12 D3 H-1,33799 procollagen C-proteinase 49.5 51 .OkDa enhancer 169-77 H-L33801 human protein kinase mRNA 55 46.27 M305 D6 H-L3404 1 L-glycerol-3-phosphate:NAD-' 38.5 42.OkDa B4 H-1,34355 Homo sapiens (clone p4) 50 kD 42.68 47 dystrophin-associated glycoprotein mRINA, complete M297 B3 H-1,35013 spliceosomal protein SAP 49 46.75 52.OkDa 167-32 H-L35253 human CSaids binding protein 52 39.67 mRNA____ M266 D6 H-1,35545 C/activated protein C receptor, 26.29 38.OkDa endothelial M300 Fl H-L35594 autotaxin 100.76 91 .OkDa M318 E2 H-L36720 bystin 33.77 .29 M305 H2 H-1,37 127 RINA polymerase Il 12.98 16 M300 Dl H-L38490 ADP-ribosylation factor 22.22 32 WO 99/51766 WO 9951766PCT/US99/07270 M318 ElI H-1,38941 ribosomal protein L34 12.98 18 C2 H-1,38969 Homo sapiens thrombospondin 3 105.27 110 (THBS3) gene, complete cds M476 F4 H-1,39060 Homo sapiens transcription factor 49.61 53.OkDa 1 mRNA, complete K4300 E4 H-1,40399 hypothetical protein (GB:L40399) 29.26 36 E3 H-1,40802 Homo sapiens 17-beta- 42.68 hydroxysteroid debydrogenase 7-HSD) gene M478 Fl H-1,40904 H. sapiens peroxisoine 52.69 proliferator activated receptor complete cds M306 C2 H-1,41268 natural killer associated transcript 37.62 M306 E2 H-1,41270 natural killer associated transcript 50.16 M306 F2 H-L,41347 natural killer associated transcript 33.55 M468 C3 H-1,41351 Homo sapiens prostasmn mRNA, 37.84 cds 169-53 H-1,41816 Homo sapiens cam kinase 1 48 40.77 167-25 H-L,41939 tyrosine-protein kinase receptor 108 108.6 EPH-3 precursor C3 H-1,42374 Homo sapiens protein 54.78 64 phosphatase 2A B56-beta (PP2A) complete M306 B I H-1,42531 glutathione synthetase 52.25 54.OkDa M302 F6 H-1,42856 RNA polymerase 11 transcription 13.09 factor SIll, p 18 subunit M313 C7 H-1,76200 guanylate kinase (GUKI) 21.78 32.OkDa M428 ElI H-1,76702 Homo sapiens protein 66.33 68.OkDa phosphatase 2A B56-delta (PP2A) complete cds M478 AlI H-L76703 Homo sapiens protein 51.48 phosphatase 2A B56-epsilon mRNA, complete cds 166-52 H-L,77213 H.sapiens phosphomevalonate 34 21.19 ___kinase mRNA _T_.38 169-64 H-L77964 H.sapiens ERK3 mRNA 100793 M360 C3 H-MI10050 fatty-acid-binding protein 2, 14.08 H-MI10050 fatty-acid-binding protein 2, 14.08 36 intestinal M421 E7 H-M 10058 Asialoglycoprotein receptor 1 32.12 48.Okla M429 D3 H-MI0901 Glucocorticoid receptor 85.58 M3 12 G I H-Mi 11025 asialoglycoprotein receptor 2 34.32 34.OkDa 167-44 H-M 1026 interferon alpha-4 precursor 33 20.86 F2 H-MI 1321 Human group-specific component 52.25 56 vitamin D-binding protein complete cds M236 B5 H-MI 1354 histone H3.2, CENTRAL ROLE IN NUCLEOSOME

FORMATION.

15.07 WO 99/51766 WO 9951766PCT/US99/07270 M236 G2 H-M 1143 3 retinol-binding protein 1, cellular 14.96 28 transport protein______ M270 G7 H-MI 11560 aldolase A FRUCTOSE- 40.15 BISPHOSPHATE ALDOLASE A [Homo sapiens]______ H3 H-MI 1117 Human heat shock protein (hsp '70.51 gene, complete cds El H-M12523 Human serum albumin (ALB) 67.1 complete cds H-M12963 Alcohol dehydrogenase I (class 41.36 48 alpha polypeptide D6 H-M13228 D4 H-M13981 Inhibin, alpha 40.37 M236 G4 H-M13982 interleukin 4 [IL4] precursor, B- 16.94 activator M271 B6 H-M 14043 lipocortin 11, Annexin 11 37.4 45.0kDa (lipocortin 11). CALCIUM- REGULATED MEMBRANE-

PROTEIN

M271 F4 H-M14218 arginiosuccinate lyase 51.04 56 M297 A3 H-M14221 cathepsin B 37.4 32.OkDa M305 B2 H-M14328 enolase, alpha 47.85 167-54 H-M 14333 human c-syn protooncogene 60 59.14 167-5 1 H-M 14505 H.sapiens mRNA (open reading 36 33.40 patient 215-74 H-M 14676 human src-like kinase (slc) 60 59.14 167-55 H-M14780 "creatine kinase, m chain" 52 41.98 M416 F8 H-MI5059 Fc frgment of IgE, low affinity 35.42 11, receptor for (CD23A) M271 Fl H-M15182 glucuronidase, beta [GUSB], 71.72 72 PLAYS AN IMPORTANT ROLE IN THE DEGRADATION OF DERMATAN AND KERATAN

SULFATES.

2 15-37 H-M15465- human pyruvate kinase type L 64 59.80 M298 A4 H-M 15796 cyclin 28.82 43.OkDa C3 H-M 15800 Mal, T-cell differentiation protein 16.94 17 M440 El1 H-M 15841 Human U2 small nuclear RINA- 24.86 34.OkDa associated B" antigen mRNA, cds M248 C3 H-M 15887 endozepine 9.68 M463 A2 H-M 15990 human c-yes- I RNA 59.800 M418 E2 H-M16038 tyrosmne-protein kinase LYN 56.390 64.0kDa M266 D3 H-M 16342 HETEROGENEOUS NUCLEAR 32.01 49

RIBONUCLEOPROTEINS

C1/C2 [Homo sapiens]; small nuclear ribonucleoprotein, C 167-20 H-M 16591 tyrosine-protein kinase HCK 60 55.62 C7 H-M16591 tyrosine-protein kinase HCK 55.620 WO 99/51766 WO 9951766PCTIUS99/07270 M305 E7 H-M 16660 heat shock 90kD protein 1, beta 79.75 167-65 H-M16750 PIM-lI proto-oncogene 38 34.50 ______________serine/threonine-protein kinase M3 11 Al H-M 16827 acyl-Coenzyme A dehydrogenase, 46.42 to C- 12 straight-chain D3 H-M 16961 Alpha-2-HS-glycoprotein alpha 40.48 beta chain D3 H-M 16974 Complement component 8, alpha 64.35 M248 C2 H-M17017 INTERLEUKJN-8 PRECURSOR 11 I1 sapiens] M305 E4 H-M17885 ribosomal phosphoprotein P0, 34.98 37.OkDa M339 E2 H-M17887 ribosomal phosphoprotemn P2 12.76 19.0kDa M248 D5 H-MI18731 galactose- I -phosphate 41.91 42 ______________uridylyltransferase [GALT] _F2 H-M19309 Troponin TI, skeletal, slow 30.69 M385 E2 H-M19713 tropomyosin, alpha, muscle 31.35 41I.OkDa 167-79 H-M 19722 proto-oncogene tyrosine-protein 64 58.26

FGR

M248 H I H-M20560 Annexin III (lipocortin 111), 35.64 37 INHIBITOR OF ___________PHOSPHOLIPASE A2 M235 H I H-M20681 GLUCOSE TRANSPORTER 54.67 3, BRAIN 167-29 H-M2 1616 beta platelet-derived growth 121 121.7 factor receptor precursor M305 A3 H-M21812 myosin light chain 2 18.81 167-30 H-M22146' "40S ribosomal protein S4, x 34 26.91 isoform" M302 D6 H-M22430 phospholipase A2 RASF-A 15.95 31I.OkDa E2 H-M2249 I Bone morphogenetic protein 3 52.03 M340 A2 H-M22538 NADH-ubiquinone reductase, 24 27.5 33 subunit, mitochondrial B2H-M22632 Glutamic-oxaloacetic 47.41 47 transaminase 2, mitochondrial aniinotransferase 2) B4 H-M22960 Protective protein for beta- 52.91 (galactosialidosis) Mi250 C4 H-M22995 ras-related protein RAPIA, member of RAS oncogene family B3 H-M23254 Calpain, large polypeptide L2 77.11 77 M266 B4 H-M236 13 Nucleophosmin (nucleolar 32.45 42 phosphoprotein B23, numatrin), BELIEVED TO BIND SINGLE- NUCLEIC ACIDS M469 D2 H-M23668 Homo sapiens adrenodoxin gene 20.35 M478 H3 H-M24439 Human liver/bone/kidney-type 57.75 64.OkDa alkaline phosphatase (ALPL) F5H-M24470 Glucose-6-phosphate 38.06 44 dehydrogenase WO 99/51766 WO 9951766PCT/US99/07270 M270 E5 H-M24898 thyroid hormone triiodothyronine 67.65 receptor c-erbA, ear-i1, Thyroid hormone receptor, alpha (avian erythroblastic leukemia viral (verb-a) oncogene homolog) D3 H-M24902 Acid phosphatase, prostate 42.57 54 D6 H-M25809 ATPase, H+ transporting, 56.32 57 lysosoinal (vacuolar proton pump), beta polypeptide, isoform 1 167-77 H-M26252 "pyruvate kinase, M2 isozyme" 60 58.48 M271 F8 H-M26326 keratin 18 47.41 50.0kDa B I H-M2690 1 Human renin. gene 44.44 M-771 G4 H-M27396 asparagine synthetase 61.82 62 M338 133 H-M27542 globulin, sex hormone-binding 39.200 M512 B6 H-M27602 Protease, serine, 2 (trypsin 2) 27.28 36.OkDa M270 B6 H-M27691 DNA-binding protein CREB, 36.08 cAMP-responsive ClI H-M27878 Zinc finger protein 84 (HPF2) 81.29 81 M270 F6 H-M28209 guanine nucleotide-binding 22.66 protein rabi MS512 H5 H-M28210 RAB3A, member RAS oncogene 24.31 36.OkDa B33 H-M28214 Homo sapiens GTP-binding 24.2 34 protein (RAB3B) mRNA, cds M300 C5 H-M28249 integrin, alpha 2 (CD49B, alpha 2 130.02 130.OkDa subunit of VLA-2 receptor) [ITGA21 M248 136 H-M28372 zinc finger protein 9 (a cellular 19.58 28.OkDa retroviral nucleic acid binding protein) M248 CS H-M28983 interleukin 1, alpha [ILIA] 29.92 42 M298 Cl H-M29536 translation initiation factor 2, beta 36.74 subunit M425 AS H-M29696 Interleukin 7 receptor 50.6 63.OkDa El H-M29960 Human steroid receptor (TR2- 11) 66.44 mRNA, complete cds M361 D3 H-M29971 6-O-methylguanine-DNA 22.88 33.OkDa methyltransferase [MGMT] 167-67 H-M30448 "casein kinase 11, beta chain" 34 23.72 M250 E2 H-M31211 MYOSIN LIGHT CHAIN 1, 22.99 SLOW-TWITCH MUSCLE A ISOFORM [Homo sapiens] M31 1 C4 H-M3 1452 proline-rich protein 65.78 68 M3 12 H3 H-M3 1469 ras-like protein TC4 23.87 32.OkDa.

167-41 H-M3 1606 "phosphorylase B kinase gamma s0 44.7 catalytic chain, testis isoform" B34 H-M3 1642 Hypoxanthine 24.09 36 phosphoribosyltransferase I syndrome) M416 D8 H 3192Fc fragment of IgG, low affinity 3.845.OkDa.

a, receptor for (CD32) WO 99/51766 WO 9951766PCTIUS99/07270 M305 A8 H-M3201 I neutrophil cytosolic factor 2 57.97 58 chronic granulomatous autosomal 2) [NCF2] B2 H-M323 15 Human tumor necrosis factor 50.82 mRNA, complete cds M266 C2 H-M33374 cell adhesion protein SQMI 14.96 l8.OkDa M431IFFl H-M33375 dihydrodiol dehydrogenase 4 .33.99 G6 H-M33680 Human 26-kDa cell surface 26.07 24 protein TAPA- 1 mRNA, complete Fl H-M33772 Human fast skeletal muscle 17.71 29 troponin C gene 167-15 H-M34065 in-phase inducer phosphatase 3 55 52.10 F4 H-M34079 Human immunodeficiency virus 44.55 52 tat transactivator binding protein- (tbp- 1) mRNA, complete cds 169-86 H-M34181 "cAMP-dependent protein kinase, 50 38.68 beta-catalytic subunit" DI H-M34379 Elastatse 2, neutrophil 29.48 M3 14 El H-M3467 I CD59 glycoprotein precursor 14.150 M266 C3 H-M35252 CO-029 (GB:M35252) 26.18 M315 A4 H-M36035 benzodiazapine receptor 18.7 19 (peripheral) M300 CI H-M36340 ADP-ribosylation factor 1 20.02 M3 12 C3 H-M36341 ADP-ribosylation factor 2 19.91 29 D6 H-M36634 Vasoactive intestinal peptide 18.81 28 169-26 H-M36881 proto-oncogene tyrosine-protein 60 56.06 kinase LCK 167-76 H-M36981 nucleoside diphosphate kinase B 26 16.79 M298 D6 H-M37400 aspartate aminotransferase, 45.54 cytosolic 167-88 H-M377 12 galactosyltransferase associated 55 48.36 kinase P58/GTA M424 F4 H-M3 8258 Retinoic acid receptor, gamma 1 50.05 58.OkDa M266 H3 H-M38690 CD9 antigen, INVOLVED IN 25.19 26.OkDa PLATELET ACTIVATION AND AGGREGATION. M270 AS H-M55265 casein kinase 11, alpha catalytic 43.12 subunit 169-74 H-M55284 human protein kinase C-L 80 75.09 (PRKCL) mRNA M512 B3 H-M55514 Potassium voltage-gated channel, 71.94 100.OkDa shaker-related subfamily, member 4 M271 F5 H-M57567 ADP-ribosylation factor 5 [ARS]. 19.91 32.OkDa INVOLVED IN PROTEIN TRAFFICKING AND ACTS AS AN ALLOSTERIC ACTIVATOR OF CHOLERA TOXIN.____ Mv250 Dl H-M57627 interleukin 10 [IL,10], 19.69 27 SUPPRESSOR FACTOR FOR THl IMMUNE RESPONSES SIMILARITY). WO 99/51766 WO 9951766PCTIUS99/07270 M302 D3 H-M57730 EPH-related receptor tyrosine 22.620 36.OkDa.

kinase ligand I precursor M248 B5 H-M58458 ribosomal protein S4, X-linked 29.04 36.OkDa.

M248 A5 H-M58459 ribosomal protein S4, Y-Iinked 29.04 36 [RPS4Y] M248 G5 H-M58525 CATECHOL 0- 29.92 36

METHYLTRANSFERASE,

MEMBRANE-BOUND FORM ~[Homo sapiens], COMT M482 B2 H-MS 99 16 Sphingomyelin phosphodiesterase 69.3 69.0kDa.

1, acid lysosomal (acid ___________sphingomyelinase) M390 Cl H-M60091 galactose-1I-phosphate 41.8 uridylyltransferase M3 16 B I H-M603 14 bone morphogenetic protein 5 50.05 B4 H-M60459 Erythropoietin receptor 55.99 C7 H-M60483 Human protein phosphatase 2A 34.1 56 catalytic subunit-alpha gene, cds M462 D7 H-M60484 Human protein phosphatase 2A 34.1 44.OkDa catalytic subunit-beta gene, cds A12 H-M60527 deoxycytidine kinase 28.670 167-5 H-M60724 human p70 ribosomal S6 kinase 66 57.82 mRNA_____ 167-17 H-M60725 human p70 ribosomal S6 kinase 62 55.29 mRNA M271 A4 H-M61 199 cleavage signal 1, ESTs, Highly 27.5 36.OkDa similar to CLEAVAGE SIGNAL- 1 PROTEIN [Homo sapiens]_____ B I H-M61733 Homo sapiens erythroid 70.62 71 membrane protein 4.1 mRNA, cds M298 AlI H-M6 1764 tubulin, gamma 49.72 M422 E2 H-M62505 Complement component 5 38.61 38.OkDa I (C5a ligand) M313 G5 H-M62810 transcription factor 1, 27.17 mitochondrial C9 H-M62839 apolipoprotein H 38.06 H-M63 154 Gastric intrinsic factor (vitamin B 45.98 52 synthesis) 167-6 H-M63 167 RAC-alpha serine/threonine 64 52.87 BI H-M63573 Peptidylprolyl isomerase B 23.87 33 B) M302 H2 H-M63603 phospholamban 5.83 6 M306 DI H-M63838 interferon, gamma-inducible 80.3 108 protein 16 M423 H3 H-M63959 Low density lipoprotein-related 39.38 48.OkDa protein-associated protein 1 (alpha-2-macroglobulin receptorassociated protein WO 99/51766 WO 99/1 766PCTIUS99/07270 G3 H-M64099 Human ganuna-glutmnyl 64.57 52 transpeptidase-related protein mRNA, complete cds M475 B8 H-M64673 Human heat shock factor 1 58.3 65.0 kDa (TCF5) mRNA, complete cds M266 D5 H-M64716 ribosomal protein S25 13.86 17.OkDa M248 C6 H-M64752 glutamnate receptor, ionotropic, 99.88 100 AMPA 1 [GRIAl]____ M3 12 G3 H-M64925 palm itoylated membrane protein, 51.37 51 .OkDa 55 M302 C7 H-M65292 complement factor H-related 36.41 (GB:M65292) D3 H-M68516 Human protein C inhibitor gene, 44.77 54 cds 167-27 H-M68520 cell division protein kinase 2 38 32.85 M236 D5 H-M68867 Cellular retinoic acid-binding 15.29 19.0kDa protein 2, MAY REGULATE THE ACCESS OF RETINOIC ACID TO THE NUCLEAR RETIhJOIC ACID RECEPTORS.____ M441 El H-M69226 monoamine oxidase A [MAQA] 58.08 64.OkDa M298 D5 H-M72393 calcium-dependent phospholipid- 82.5 11 7.OkDa protein M422 D5 H-M73238 Ciliary neurotrophic factor 41.03 51I.OkDa ClI H-M73255 Human vascular cell adhesion 81.4 81 molecule- I (VCAMl1) gene, M422 G6 H-M73481 Human gastrin releasing peptide 42.35 receptor (GRPR) mRNA, cds M235 G6 H-M73499 carboxylesterase, INVOLVED IN' 62.48 THE DETOXIFICATION OF XENOBIOTICS AND THE ACTIVATION OF ESTER AND

PRODRUGS.____

M302 DlI H-M73547 polyposis locus DPI1 20.46 28 M300 H4 H-M73969 interleukin 8 receptor, beta 39.71 36 [IL8RB] G I H-M7449 1 ADP-ribosylation factor 3 20.02 31 B4 H-M74816 B2 H-M751 10 H,K-ATPase, beta subunit 32.12 37 M4 16 B8 H-M76766 General transcription factor 111B 34.87 44.OkDa 167-18 H-M77 198 RAC-beta serine/threonine kinase 64 57.27 167-87 H-M77348 PMEL 17 protein precursor 74 73.55 C4 H-M77698_ YYI transcription factor 45.65 48 M248 G6 H-M80261 apurinic/apyrimidinic (abasic) 35.09 37.OkDa endonuclease [APE], REPAIRS OXIDATIVE DNA DAMAGES

VITRO

169-50 H-M80359 putative serine/threonine-protein 80 78.50 P78 M330 H I H-M80461 ixnxunoglobulin-associated beta 25.370 27.OkDa [IGB] WO 99/51766 WO 9951766PCT/US99/07270 169-1 H-M80613 ring3 protein 100 83.01 M298 A2 H-M80783 B 12 protein 34.87 43.0kDa 217-1 H-M81457 calpactin I light chain 10 10.74 M422 C6 H-M8 1589 Homo sapiens serotonin ID 41.58 41I.OkDa receptor (5-HT1D') mRNA, M424 AlI H-M8 1590 Homo sapiens serotonin ID 43.01 48.OkDa receptor (5-HT1D-) mRNA, cds; M250 HI H-M8 1592 gammua-glutaniyl carboxylase 83.49 [GGCXI, CONVERTS GLUTAMATE RESIDUES TO

GAMMA-

_________CARBOXYGLUTAMATE

M250 F2 H-M81601 TRANSCRIPTION 33.22 36.OkDa ELONGATION FACTOR S-Il sapiens] C2 H-M8 1650 Human semenogeim I (SEMGI) 50.93 52 complete cds M266 A4 H-M81757 ribosomal protein S 19 16.06 18 169-6 1 H-M8 1933 rn-phase inducer phosphatase 1 57 57.60 M302 H I H-M82809 annexin IV 35.42 38.OkDa M300 C4 H-M83653 cytoplasmic phosphotyrosyl 17.49 28.OkDa phosphatase, type I 169-14 H-M83941 tyrosne-protemn kinase receptor 108 108.2 ETK I precursor FlI H-M84443 Galactokinase 2 50.49 52 M305 H6 H-M84747 interleukin 9 receptor [IL9R] 57.53 58 167-53 H-M86400 14-3-3 protein zeta/delta 33 27.02 M271 C8 H-M86521 transketolase 68.64 68.OkDa 169-51 H-M86699 human kinase (TTK) mRNA 92 92.58 M3 16 F2 H-M86752 transformation-sensitive protein 59.84 M270 C8 H-M86921 membrane glycoprotein mb-i1, 24.97 34 Immunoglobulin-associated alpha, ASSOCIATED TO SURFACE 1GM-RECEPTOR; MAY BE INVOLVED IN

TRANSDUCTION

H-M87507 Homo sapien interleukin- I beta 44.55 convertase (ILI BCE) mRNA, cds M305 B7 H-M8801 I glucokinase [GCKI 51.26 M305 HI H-M88279 immunophilin FKBP52 50.6 64.OkDa M420 FlI H-M88468 mnevalonate kinase 43 .600 47.OkDa M305 A7 H-M89913 dUTP pyrophosphatase 15.62 19 M3 16 E2 H-M90657 tumor-associated antigen L6 22.33 28 167-31 H-M908 13 human D-type cyclin (CCND2) 36 31.86 Al H-M91036 H.sapiens G-gamma globin and 16.28 18 A-gamma globin genes, complete cds's WO 99/51766 WO 9951766PCTIUS99/07270 G2 H-M9 1463 Human glucose transporter 55.66 52 (GLUT4) gene, complete cds AlI H-M9 1670 Human ubiquitin carrier protein 24.86 36 (E2-EPF) mRNA, complete cds E4 H-M92444 Homo sapiens 35.09 apurinic/apyrimidinic endonuclease (HAP 1) gene, cds M305 C4 H-M94556 single-stranded DNA-binding 16.39 mitochondrial G12 H-M94856 fatty-acid-binding protein 14.96 36 M453 C3 H-M95623 Homo sapiens 39.82 50.0kDa hydroxymethylbilane synthase complete cds M302 F2 H-M95787 smooth muscle protein SM22 22.22 _33.OkDa Al H-M95809 Human basic transcription factor 60.39 64 62kD subunit (BTF2), complete cds M271 E8 H-M96982 small nuclear ribonucleoprotein 26.51 39.OkDa U2 auxiliary factor, 35 kDa, SPLICING FACTOR U2AF KD SUBUNIT. NECESSARY FOR THE SPLICING OF PRE- M416 B3 H-M96995 Growthff factor receptor-bound 23.98 32.OkDa 2 G2 H-M96995 Growth factor receptor-bound 23.98 49 protein 2 H4 H-M970 16 Bone morphogenetic protein 8 44.33 61 (osteogenic protein 2) M271 DI H-M97 190 Sp2 transcription factor [SP21, 54.56 BINDS TO GC BOX PROMOTERS ELEMENTS AND SELECTIVELY ACTIVATES mRNA SYNTHESIS FROM GENES THAT CONTAIN FUNCTIONAL RECOGNITION

SITES.

M271 ClI H-M97191 Sp 3 transcription factor [SP3], 71.94 72 BINDS TO GT AND GC BOXES PROMOTERS ELEMENTS.

PROBABLE

TRANSCTRIPTIONAL

ACTIVATOR.

M305 C7 H-M973 88 transcription repressor (interacting 19.47 with the TATA-binding protein) [DR1 217-13 H-M97675 human transmembrane receptor 100 103.1 mRNA B3 H-M97856 Nuclear autoantigenic sperm 86.68 87 protein (histone-binding) M429 _G2 H-M9793s Homo sapiens transcription factor 82.61 89.OkDa mRNA, complete cds WO 99/51766 WO 99/1 766PCT/US99/07270 DI H-M99487 Human prostate-specific 82.61 92 membrane antigen (PSM) mRNA, cds M363 AlI H-P0002 riboflavin synthase beta chain 17.27 M363 BI1 H-POO04 carbonic anhydrase, (icfA) 24.42 M363 Cl H-POO05 orotidine 5'-phosphate 25.08f M363 Dl H-P0006 pantoate-beta-alanine ligase, 30.47 M3 79 Al H-POO 10-2 chaperone and heat shock protein 60.17 M363 El H-POO 11I co-chaperone (groES) 13.09 M363 Fl H-POO12 DNA primase (dnaG) 61.6 M363 GI H-P00 13 hypothetical protein 38.61 M363 HI H-P00 14 hypothetical protein 30.36 M363 A2 H-POO 15 hypothetical protein 10.34 M363 B2 H-P00 16 hypothetical protein 9.68 M363 C2 H-P00 17 virB4 homolog (virB4) 86.68 M363 D2 H-POO 18 hypothetical protein 51.7 M363 E2 H-P0021I hypothetical protein 21.01 M363 F2 H-P0022 conserved hypothetical integral 57.42 protein______ M363 G2 H-P0026 citrate synthase (gitA) 46.97 M363 H2 H-P0027 isocitrate dehydrogenase (icd) 46.86 M363 A3 H-P0028 conserved hypothetical secreted 19.58 protein______ M363 B3 H-P0030 hypothetical protein 65.34 M363 C3 H-P0031I hypothetical protein 15.18 M363 D3 H-P0034 aspartate 1-decarboxylase (panD) 12.98 M363 E3 H-P0035 conserved hypothetical protein 10.78 M363 F3 H-P0037 NADH-ubiquinone 38.72 ___________oxidoreductase subunit M363 G3 H-P0044 GDP-D-mannose dehydratase 42.02 M363 H3 H-P0047 hydrogenase expression/formation 36.63 (hypE) M363 A4 H-P0048 transcriptional regulator (hypF) 84.7 M363 B4 H-P0052 hypothetical protein 36.41 M363 C4 Hl-POO55 proline permease (putP) 54.67 M363 D4 H-P0056 delta- I -pyrroline-5-carboxylate 130.46 ______________dehydrogenase M363 E4 H-P0057 hypothetical protein 7.7 M363 F4 H-P0063 hypothetical protein 54.67 M363 G4 H-P0064 hypothetical protein 15.4 M363 H4 H-P0066 conserved hypothetical ATP- 91.52 binding protein_____ M363 AS H-P0067 urease accessory protein (ureH) 29.26 M363 B5 H-P0068 urease accessory protein (ureG) 22 M363 C5 H-P0075 urease protein (ureC) 49.06 M363 D5 H-P0077 peptide chain release factor RE-I 38.83 WO 99/51766 PCT/US99/07270 M363 E5 H-P0082 methyl-accepting chemotaxis 74.14 transducer (tlpC) M363 F5 H-P0086 conserved hypothetical protein 49.61 M363 G5 H-P0087 hypothetical protein 50.38 M363 H5 H-P0088 RNA polymerase sigma-70 factor 73.92 (rpoD) M363 A6 H-P0089 pfs protein (pfs) 25.52 M363 B6 H-P0090 malonyl coenzyme A-acyl carrier 34.1 protein transacylase (fabD) M363 C6 H-P0093 hypothetical protein 12.21 M363 D6 H-P0096 phosphoglycerate dehydrogenase 34.65 M304 Al H-P0099 methyl-accepting chemotaxis 74.36 protein (tlpA) M304 BI H-PO 100 conserved hypothetical protein 40.59 M304 Cl H-POO 101 hypothetical protein 27.94 M304 DI H-PO 104 2',3'-cyclic-nucleotide 64.02 phosphodiesterase (cpdB) M304 El H-PO 105 conserved hypothetical protein 17.16 M304 Fl H-PO 106 cystathionine gamma-synthase 41.91 (metB) M304 GI H-P0107 cysteine synthetase (cysK) 33.77 M304 HI H-PO 108 hypothetical protein 20.57 M304 A2 H-PO 109 chaperone and heat shock protein 68.31 (dnaK) M304 B2 H-PO 110 co-chaperone and heat shock 20.9 protein (grpE) M304 C2 H-PO 111I hypothetical protein 30.47 M304 D2 H-PO 113 hypothetical protein 10.89 M304 E2 H-PO 114 hypothetical protein 69.19 M304 F2 H-PO 115 flagellin B (flaB) 56.65 M304 G2 H-PO 116 DNA topoisomerase I (topA) 81.07 M304 H2 H-P0117 conserved hypothetical protein 33.99 M304 A3 H-PO 118 hypothetical protein 43.56 M304 B3 H-PO 119 hypothetical protein 50.82 M304 C3 H-P0120 hypothetical protein 43.89 M304 D3 H-PO 121 phosphoenolpyruvate synthase 89.43 (ppsA) M304 E3 H-PO 122 hypothetical protein 4.84 M304 F3 H-PO 123 threonyl-tRNA synthetase (thrS) 67.43 M304 G3 H-PO 124 translation initiation factor IF-3 22.44 (infC) M304 H3 H-P0125 ribosomal protein L35 (rpl35) 7.15 M304 A4 H-P0126 ribosomal protein L20 (rpl20) 12.87 M304 B4 H-PO 127 outer membrane protein (omp4) 31.57 M304 C4 H-P0128 hypothetical protein 4.62 M304 D4 H-PO 129 hypothetical protein 15.62 M304 E4 H-PO 130 hypothetical protein 31.57 M304 F4 H-P0 131 hypothetical protein 3.74 M304 G4 H-P0132 L-serine deaminase (sdaA) 50.16 M304 H4 H-PO 133 serine transporter (sdaC) 45.54 M304 A5 H-PO 134 3-deoxy-D-arabino-heptulosonate 49.5 7-phosphate synthase (dhsl) M304 B5 H-PO 135 hypothetical protein 4.95 WO 99/51766 PCT/US99/07270 M304 C5 H-PO 136 bacterioferritin comigratory 16.83 protein (bcp) M304 D5 H-PO 137 hypothetical protein 23.32 M304 E5 H-P0138 conserved hypothetical iron-sulfur 53.02 protein M304 F5 H-P0139 conserved hypothetical secreted 26.73 protein M304 GS H-P0140 L-lactate permease (IctP) 60.5 M304 H5 H-P0141 L-lactate permease (IctP) 60.72 M304 A6 H-PO 142 A/G-specific adenine glycosylase 36.19 (mutY) M304 B6 H-PO 144 cytochrome c oxidase, heme b 53.79 and copper-binding subunit, membrane-bound (fixN) M304 C6 H-PO 145 cytochrome c oxidase, monoheme 25.63 subunit, membrane-bound (fixO) M304 D6 H-PO 146 cbb3-type cytochrome c oxidase 8.14 subunit Q (CcoQ) M304 E6 H-P0147 cytochrome c oxidase, diheme 31.57 subunit, membrane-bound (fixP) M304 F6 H-P0148 hypothetical protein 7.59 M304 G6 H-P0150 hypothetical protein 21.67 M304 H6 H-P0152 hypothetical protein 31.68 M304 A7 H-PO 153 recombinase (recA) 38.28 M304 B7 H-PO 154 enolase (eno) 46.97 M304 C7 H-P0155 hypothetical protein 10.12 M304 D7 H-PO 157 shikimic acid kinase I (aroK) 17.93 M304 E7 H-P0158 hypothetical protein 35.09 M304 F7 H-P0159 lipopolysaccharide 1,2- 41.03 glucosyltransferase (rfaJ) M304 G7 H-P0161 hypothetical protein 4.07 M304 H7 H-P0162 conserved hypothetical protein 26.51 M304 A8 H-P0163 delta-aminolevulinic acid 35.64 dehydratase (hemB) M304 B8 H-P0164 signal-transducing protein, 28.05 histidine kinase M304 C8 H-P0165 hypothetical protein 19.14 M304 D8 H-PO 166 response regulator (ompR) 24.86 M304 E8 H-PO167 hypothetical protein 17.38 M304 F8 H-PO 168 hypothetical protein 9.68 M304 G8 H-PO 170 hypothetical protein 27.94 M304 H8 H-PO 171 peptide chain release factor RF-2 40.04 (prfB) M304 A9 H-PO 172 molybdopterin biosynthesis 43.12 protein (moeA) M304 B9 H-P0173 flagellar biosynthetic protein 28.16 (fliR) M304 C9 H-PO 174 hypothetical protein 28.49 M304 D9 H-PO 175 cell binding factor 2 33 M304 E9 H-P0176 fructose-bisphosphate aldolase 33.88 (tsr) M304 F9 H-PO 177 translation elongation factor EF-P 20.68 (efp) WO 99/51766 WO 9951766PCT/US99/07270 M304 G9 H-PO 178 spore coat polysaccharide 37.51 protein E M304 H9 1--POI 79 ABC transporter, ATP-binding 23.54 M304 AlO H-PO 180 apolipoprotein N-acyltransferase 46.86 M304 BlO H-PO 182 lysyl-tRNA synthetase (lysS) 55.22 M304 CIO H-PO 183 serine hydroxymethyltransferase 45.87 M304 D1O H-PO 184 hypothetical protein 19.91 M304 EIO H-P0185 hypothetical protein 29.48 M304 FlO H-POI 186 hypothetical protein 44.55 M304 GlO H-P0187 hypothetical protein 10.56 M304 H1O H-POI188 hypothetical protein 3.74 M304 All1 H-PO 189 conserved hypothetical integral 19.58 protein M304 B 11 H-PO 190 conserved hypothetical secreted 55.33 protein M304 CII1 H-P0191 fumarate reductase, iron-sulfur 27.06 subunit (frdB) M304 DlI1 H-P0 192 fumarate reductase, flavoprotein 78.65 subunit (frdA) M304 El I H-PO 193 fumarate reductase, cytochromne b 28.16 subunit M304 1l11 H-PO 194 triosephosphate isomnerase (tpi) 25.85 M304 GIl1 H-P0195 enoyl-(acyl-carrier-protein) 30.36 reductase (NADH) (fabl) M3U65 Al H-PO 197 S-adenosylmethionine synthetase 42.46 2 M365 B1 H-P0203 hypothetical protein 10.12 M365 CI H-P0209 hypothetical protein M365 Dl H-P0213 glucose inhibited division protein 68.42 (gidA)_ M38 1 El H-P02 18 hypothetical protein 20.24 M365 El H-P0221 nifUJ-like protein 35.97 M365 Fl H-P0227 outer membrane protein (omnp5) 76.12 M365 GI1 H-PO228 conserved hypothetical integral 43.01 membrane protein M365 HI H-P0230 CTP:CMP-3-deoxy-D-manno- 26.84 octulosonate-cytidylyl-transferase M9365 A2 H-P0233 conserved hypothetical protein 43.01 M365 B2 H-P0235 conserved hypothetical secreted 39.16 protein M365 C2 H-P0236 hypothetical protein 13.64 M365 D2 H-P0238 prolyl-tRNA synthetase (proS) 63.58 M381 E2 H--P0243 neutrophil activating protein 15.95 (napA) (bacteriofernitin)______ M365 E2 H-P0244 signal-transducing protein, 42.02 histidine kinase M36 F2 H-P246flagellar basal-body P-ring protein 37.73 (figl) WO 99/51766 WO 9951766PCT/US99/07270 M365 G2 H-P0247 ATP-dependent RINA helicase, 54.23 DEAD-box family M365 H12 H-P0248 conserved hypothetical protein 39.93 M379 BI H-P0249-2 hypothetical protein 19.8 M379 CI H-P0250-2 oligopeptide ABC transporter, 56.87 ATP-binding protein M38 1 A3 H-P0251 oligopeptide ABC transporter, 37.29 permease protein M379 El H-P0252-2 outer membrane protein (omp7) 53.68 M365 A3 H-P0254 outer membrane protein (omp8) 47.52 M365 B3 H-P0255 adenylosuccinate synthetase 45.32 M365 C3 H-P0257 conserved hypothetical secreted 24.2 M365 D3 H-P0259 exonuclease VII, large subunit 46.31 (xseA) M381 D3 H-P0260 adenine specific DNA 42.35 _______________methyltransferase (mod) M365 E3 H-P0263 adenine specific DNA 27.83 ______________methyltransferase (hpaim) M365 F3 H-P0264 ATP-dependent protease binding 94.27 (clpB) M365 G3 H-P0266 dihydroorotase (pyrC) 41.69 M365 H3 H-P0267 chlorohydrolase 45.1 M365 A4 H-P0271I hypothetical protein 36.08 M365 B4 H-P0275 ATP-dependent nuclease (addB) 47.41 M3 81 G3 H-P0276 hypothetical protein 20.46 M365 C4 H-P0278 guanosine pentaphosphate 53.35 ______________phosphohydrolase (gppA) M365 D4 H-P0279 lipopolysaccharide 37.51 ________________heptosyltransferase- 1 (rfaC) M365 E4 H-P0280 heat shock protein B (ibpB) 36.19 M365 F4 H-P0282 hypothetical protein 52.91 M365 G4 H-P0283 3-dehydroquinate synthase (aroB) 37.84 M365 H4 H-P0284 conserved hypothetical integral 57.64 protein M365 A5 H-P0285 conserved hypothetical protein 46.09 M38 1 A4 H-P0287 hypothetical protein 19.03 M381 C4 H-P0288 hypothetical protein 17.38 M366 Al H-P0389 superoxide dismutase (sodB) 23.54 M366 BI H-P0390 adhesin-thiol peroxidase (tagD) 18.37 M366 CI H-P0391 purine-binding chemotaxis 18.26 M366 Dl H-P0392 histidine kinase (cheA) 88.44 M366 El H-P0393 chemotaxis protein (cheV) 34.32 M366 Fl1 H-P0394 hypothetical protein 27.83 M366 GI H-P0395 conserved hypothetical protein 24.53 M366 HI H-P0396 conserved hypothetical protein 67.87 M366 A2 H-P0397 phosphoglycerate dehydrogenase 57.75 M366 B2 H-P0398 hypothetical protein 20.13 M3665 C2 H-P0399-- ribosomal protein S I (rpsl1) 61.27 WO 99/51766 PCT/US99/07270 M366 D2 H-P0403 phenylalanyl-tRNA synthetase, 36.19 alpha subunit (pheS) M366 E2 H-P0404 protein kinase C inhibitor 11.55 (SP:PI6436) M366 F2 H-P0405 nifS-like protein 48.51 M366 G2 H-P0406 hypothetical protein 21.67 M366 H2 H-P0407 biotin sulfoxide reductase (bisC) 87.67 M381 DI H-P0409 GMP synthase (guaA) 55.99 M381 Fl H-P0410 putative neuraminyllactose- 27.5 binding hemagglutinin homolog (hpaA) M366 A3 H-P0411 hypothetical protein 11.66 M366 B3 H-P0412 hypothetical protein 3.63 M366 C3 H-P0413 transposase-like protein, PS3IS 29.59 M366 D3 H-P0414 IS200 insertion sequence from 15.29 SARA17 M366 E3 H-P0415 conserved hypothetical integral 68.64 membrane protein M366 F3 H-P0416 cyclopropane fatty acid synthase 42.9 (cfa) M366 G3 H-P0417 methionyl-tRNA synthetase 71.61 (metS) M366 H3 H-P0418 hypothetical protein 36.96 M366 A4 H-P0419 conserved hypothetical protein 28.82 M366 B4 H-P0420 hypothetical protein 15.73 M366 C4 H-P0421 type 1 capsular polysaccharide 42.9 biosynthesis protein J (capJ) M366 D4 H-P0422 arginine decarboxylase (speA) 67.76 M366 E4 H-P0424 hypothetical protein 68.2 M366 F4 H-P0425 hypothetical protein 45.98 M366 G4 H-P0427 hypothetical protein 12.32 M366 H4 H-P0433 hypothetical protein 16.28 M366 A5 H-P0436 hypothetical protein 13.42 M366 B5 H-P0437 IS605 transposase (tnpA) 15.73 M366 C5 H-P0438 IS605 transposase (tnpB) 47.08 M366 D5 H-P0442 hypothetical protein 9.79 M366 E5 H-P0445 hypothetical protein 6.82 M366 F5 H-P0452 hypothetical protein 57.09 M366 G5 H-P0455 hypothetical protein 11.44 M366 H5 H-P0457 hypothetical protein 9.68 M366 A6 H-P0463 type I restriction enzyme M 53.68 protein (hsdM) M366 B6 H-P0464 type I restriction enzyme R 116.16 protein (hsdR) M366 C6 H-P0465 conserved hypothetical protein 69.52 M366 D6 H-P0466 conserved hypothetical protein 28.16 M366 E6 H-P0467 conserved hypothetical integral 12.76 membrane protein M366 F6 H-P0468 conserved hypothetical protein 54.56 M366 G6 H-P0469 conserved hypothetical protein 17.93 M366 H6 H-P0471 glutathione-regulated potassium- 45.87 efflux system protein (kefB) M366 A7 H-P0472 outer membrane protein (ompl 1) 20.57 WO 99/51766 WO 9951766PCTIUS99/07270 M366 B7 H-P0473 molybdenum ABC transporter, 27.17 periplasmic molybdate-binding protein (modA) M366 C7 H-P0474 molybdenum ABC transporter, 24.75 protein (modB) M366 D7 H-P0475 molybdenum ACtransporter, 29.26 protein M366 E7 H-P0476 glutamyl-tRNA synthetase (gltX) 51.04 M366 F7 H-P0477 outer membrane protein (ompl12) 40.48 M366 G7 H-P0478 adenine specific DNA 60.06 ______________methyltransferase (VSPIM) M366 H7 H-P0479 hypothetical protein 31.13 M366 A8 H-PO481 adenine specific DNA 2.32 methyltransferase (MFOKI) M366 B8 H-P0482 hypothetical protein 18.81 M366 C8 H-P0483 cytosine specific DNA 36.3 _________methyltransferase

(H-PHIMC)

M367 Al H-PO486 hypothetical protein 58.19___ M367 B 1 H-P0487 hypothetical protein M367 CI H-P0489 hypothetical protein 32.56 M367 Dl H-P0490 putative potassium channel 41.69 putative M367 El H-P049 1 ribosomal protein L28 (rpL28) 6.93 M367 Fl H-P0492 hypothetical protein 30.69 M367 G 1 H-P0494 UTDP-N-acetylmnuramnoylalanine- 46.53 ligase (murD) M367 HI H-P0495 hypothetical protein 9.57 M367 A2 H-P0496 conserved hypothetical protein 14.74 M367 B2 H-P0498 sodium- and chloride-dependent 48.73 M367 C2 H-P0499 phospholipase AlI precursor (DR- 39.16 phospholipase A) M367 D2 H-P0500 DNA polymerase III beta-subunit 41.25 M367 E2 H-P0501 DNA gyrase, sub B (gyrB) 85.14 M367 F2 H-P0503 hypothetical protein 27.17 M367 G2 H-P0504 hypothetical protein M 367 H2 H-P0505 hypothetical protein 17.05 M367 A3 H-P0507 conserved hypothetical protein 23.43 M367 B3 H-P0509 glycolate oxidase subunit (glcD) 50.6 M367 C3 H-P05 10 dihydrodipicolinate reductase 28.05 M367 D3 H-P05 12 glutamine synthetase (glnA) 53.02 M367 E3 H-P0514 ribosomal protein L9 (rp9) 16.61 M367 F3 H-P0515 heat shock protein (hsV) 19.91 M367 G3 H-P0516 heat shock protein (hsU) ORFI1 48.84 M367 H3 H-P05 17 GTP-binding protein (era) 33.33 M367 A4 H-POS519 conserved hypothetical protein 30.47 M367 B4 H-P0520 cag pathogenicity island protein 12.76 M367 C4 H-P0522 [cag pathogenicity island protein 53.02 I WO 99/51766 PCT/US99/07270 M367 D4 H-P0523 cag pathogenicity island protein 18.7 (cag4) M367 E4 H-P0525 virB 1 homolog 36.41 M367 F4 H-P0526 cag pathogenicity island protein 22 (cag6) M367 G4 H-P0528 cag pathogenicity island protein 57.53 (cag8) M379 HI H-P0531-2 cag pathogenicity island protein 24.09 (cagl 1) M367 H4 H-P0S32 cag pathogenicity island protein 30.91 (cagl2) M367 AS H-P0534 cag pathogenicity island protein 21.67 (cagl3) M367 B5 H-P0541 cag pathogenicity island protein 40.81 M367 CS H-P0542 cag pathogenicity island protein 15.73 (cag21) M367 D5 H-P0545 cag pathogenicity island protein 22.88 (cag24) M367 E5 H-P0549 glutamate racemase (glr) 28.16 M367 FS H-P0550 transcription termination factor 48.29 Rho (rho) M367 GS H-P0551 ribosomal protein L31 (rpl31) 7.48 M367 H5 H-P0552 conserved hypothetical protein 31.68 M367 A6 H-P0553 conserved hypothetical protein 25.08 M367 B6 H-P0554 hypothetical protein 35.42 M367 C6 H-P0555 hypothetical protein 30.14 M367 D6 H-P0556 hypothetical protein 16.06 M367 E6 H-P0557 acetyl-coenzyme A carboxylase 34.43 (accA) M367 F6 H-P0558 beta ketoacyl-acyl carrier protein 45.43 synthase II (fabF) M367 G6 H-P0561 3-ketoacyl-acyl carrier protein 27.28 reductase (fabG) M367 H6 H-P0562 ribosomal protein S21 (rps21) 7.81 M367 A7 H-P0S63 hypothetical protein 45.87 M367 B7 H-P0566 diaminopimelate epimerase 30.14 (dapF) M367 C7 H-P0568 hypothetical protein 28.16 M367 D7 H-P0570 aminopeptidase a/i (pepA) 54.67 M367 E7 H-P0571 conserved hypothetical integral 21.23 membrane protein M379 A2 H-P0572-2 adenine 19.8 phosphoribosyltransferase (apt) M379 B2 H-P0573-2 hypothetical protein 12.21 M379 C2 H-P0574-2 galactosidase acetyltransferase 16.72 (lacA) M379 D2 H-P0575-2 conserved hypothetical membrane 25.63 protein M379 E2 H-P0576-2 signal peptidase I (lepB) 32.01 M367 F7 H-P0577 methylene-tetrahydrofolate 32.23 Sdehydrogenase (folD) M367 G7 H-P0579 hypothetical protein 20.35 WO 99/51766 WO 9951766PCT/US99/07270 M367 117 H-P0580 hypothetical protein 41.03 M367 A8 H-P058 1 dihydroorotase (pyrC) 37.4 M367 138 H.-P0582 hypothetical protein 35.75 M367 C8 H-P0583 hypothetical protein 32.34 M368 Al H-P0584 flagellar switch protein (Mi) 13.64 M368 Bi1 H-P0585 endonuclease III (nth) 24.09 M368 C I H-PO587 ammnodeoxychorismate lyase 36.3 M368 Dl H-P0591 ferredoxin oxidoreductase, 20.57 gamma subunit M368 El H-P0593 adenine specific DNA 65.89 methyltransferase M368 Fl H-P0594 hypothetical protein 6.05 M368 GI H-P0596 hypothetical protein 21.23 M368 HI H-PO597 penicillin-binding protein IA 72.6 (PBP- lA) M368 A2 H-P0599 hemolysin secretion protein 47.74 precursor (hyiB) M368 B2 H-P0601 flagellin A (flaA) 56.21 M368 C2 H-P0602 endonuclease 111 24.09 M368 D2 H-P0603 hypothetical protein 20.9 M379 F2 H-P0608-2 hypothetical protein 17.71 M368 E2 H-P0614 hypothetical protein 12.32 M368 F2 H--P0616 chemotaxis protein (cheV) M368 G2 H-P06 17 aspartyl-tRNA synthetase (aspS) 63.58 M368 H2 H-P0621 DNA mismatch repair protein 83.93 (MutS) M368 A3 H-P0622 hypothetical protein 13.31 M368 B3 H-P0623 UDP-N-acetylmuraniate-alanine 49.5 ligase M368 C3 H-P0625 protein E (gcpE) 39.6 M368 D3 H-P0626 tetrahydrodipicolinate N- 44.22 succinyltransferase (dapD) M368 E3 H-P0627 hypothetical protein 12.21 M368 F3 H-P0629 hypothetical protein M368 G3 H-P0630 modulator of drug activity 21.45 (mda.66) M368 113 H-P063 1 quinone-reactive Ni/Fe 42.35 hydrogenase, small subunit M368 A4 H-P0632 quinone-reactive Ni/Fe 63.69 hydrogenase, large subunit M368 B4 H-P0633 quinone-reactive Ni/Fe 24.75 hydrogenase, cytocbrome b subunit (hydC) M368 C4 H-P0634 quinone-reactive Ni/Fe 19.69 (hydD) M368 D4 H-P0635 hypothetical protein 56.43 M368 E4 H-P0636 hypothetical protein 10.23 M368 F4 H-P0637 hypothetical protein 16.61 M368 G4 H-P0638 outer membrane protein (ompl 3) 33.66 M368 H4 H-P0643 glutaniyl-tRNA synthetase (gItX) _48.4 WO 99/51766 WO 99/1 766PCT/US99/07270 M368 A5 H-P0644 conserved hypothetical integral 10.78 membrane protein M368 B5 H-P0645 soluble lytic murein 61.71 transglycosylase M368 C5 H-P0646 UJDP-glucose pyrophosphorylase 30.14 (gaIU) M368 D5 H-P0647 hypothetical protein 14.96 M368 E5 H-P0648 UJDP-N-acetylglucosamnine 46.53 enolpyruvyl transferase (murZ) M368 F5 H-P0649 aspartate ammonia-lyase (aspA) 51.59 M368 G5 H-P0650 hypothetical protein 21.67 M379 A3 H-P0651-2 flicosyltransferase 52.47 M38 1 E3 H-P0652 phosphoserine phosphatase (serB) 22.88 M368 H5 H-P0653 nonheme iron-containing ferritin 18.48 M379 G2 H-P0654-2 conserved hypothetical protein 39.71 M379 H2 H-P0655-2 protective surface antigen D 15 100.87 M368 A6 H-P0656 conserved hypothetical protein 42.24 M368 B6 H-P0657 processing protease (ymxG) 47.63 M368 C6 H-P0658 PETI 12-like protein 52.36 M368 D6 H-P0659 hypothetical protein 45.65 M368 E6 H-P0660 hypothetical protein 37.29 M368 F6 H-P0661 ribonuclease H (rnhA) 15.84 M368 G6 H-P0662 ribonuclease III (mc) 26.51 M368 H6 H-P0663 chorismate synthase (aroC) 40.26 M368 A7 H-P0665 oxygen-independent 50.38 coproporphyrinogen III oxidase ~(hemN) M368 B7 H-P0667 hypothetical protein 9.46 M368 C7 H-P0668 hypothetical protein 66.88 M368 D7 H-P067 1 outer membrane protein (omp 14) 29.81 M368 E7 H-P0672 solute-binding signature and 43.01 mitochondrial signature protein M379 B3 H-P0673-2 hypothetical protein 46.97 M381 H3 H-P0674 hypothetical protein 25.19 M368 F7 H-P0676 methylated-DNA--protein- 18.59 ~~cysteine methyltransferase (dati) M368 G7 H-P0677 conserved hypothetical integral 28.16 ~membrane M368 H7 H-P0679 lipopolysaccharide biosynthesis 31.9 ~protein M369 Al H-P0681 hypothetical protein 18.59 M369 BI H-P0682 hypothetical protein 13.97 M369 Cl H-P0683 UJDP-N-acetylglucosamine 47.74 ______________pyrophosphorylase M369 Dl1 H-P0685 flagellar biosynthetic protein 19.03 M369 El H-P0687 iron(I1) transport protein (feoB) 70.73 M369 Fl 1-lP068 8 hypothetical protein 18.37 M369 GI1 H-P0690 acetyl coenzyme A 43.12 _______________acetyltransferase (thiolase) WO 99/51766 WO 9951766PCT/US99/07270 M381 Al H-P0691 3-oxoadipate coA-transferase 2T5.63 A (yxjD) M381I B I H-P0692 3-oxoadipate coA-transferase 22.88 subunit B M369 HI H-P0694 hypothetical protein 28.38 M369 A2 H-P0695 hydantoin utilization protein A 78.54 M369 B2 H-P0697 hypothetical protein 18.59 M369 C2 H-P0699 hypothetical protein 37.73 M369 D2 H-P0700 diacylglycerol kinase (dgkA) 14.19 M369 E2 H-P0701 DNA gyrase, sub A (gyrA) 91.08 M369 F2 H-P0703 response regulator 42.02 M369 G2 H-P0707 conserved hypothetical protein 33.99 M369 H2 H-P0711I hypothetical protein 44.77 M369 A3 H-P0715 ABC transporter, ATP-binding 26.51 M369 B3 H-P0716 conserved hypothetical protein 14.74 M369 C3 H-P07 18 conserved hypothetical integral 23.21 membrane protein______ M369 D3 H-P0719 hypothetical protein M369 E3 H-P0723 L-asparaginase Il (ansB) 36.41 M369 F3 H-P0724 anaerobic C4-dicarboxylate 48.84 protein (dcuA) M369 G3 H-P0727 transcriptional regulator, putative 36.19 M369 H3 H-P0728 conserved hypothetical protein 37.07 M369 A4 H-P0730 hypothetical protein 11.22 M369 B4 H-P0732 hypothetical protein 13.09 M369 C4 H-P0734 conserved hypothetical protein 48.4 M369 D4 H-P0735 xanthine guanine phosphoribosyl 16.94 transferase (gpt) M369 E4 H-P0737 conserved hypothetical integral 17.49 protein______ M381 H-2 H-P0738 D-alanine:D-alanine ligase A 38.28 M369 F4 H-P0739 2-hydroxy-6-oxohepta-2,4- 26.62 hydrolase M369 G4 H-P0741 conserved hypothetical protein 17.82 M369 H4 H-P0745 conserved hypothetical protein 36.08 M369 AS H-P0747 conserved hypothetical protein 43.34 M369 B5 H-P0748 cell division protein (fsE) 24.64 M369 CS H-P0749 cell division membrane protein 29.59 M369 D5 H-P0750 hypothetical protein 44.11 M369 ES H-P0752 flagellar hook-associated protein 74.25 2 (fliD)__ M38 1 F3 H-P0755 molybdopterin biosynthesis 23.21 (ioeB) M379 C3 H-P0757-2 beta-alanine synthetase homolog 32.23 M369 F5 H-P075S8 conserved hypothetical integral 48.18 membrane protein M369 G5 H-P0759 conserved hypothetical integral 45.98 protein M36) 115 H-P0761I hypothetical protein 122.11___ WO 99/51766 WO 9951766PCT/US99/07270 M369 A6 H-P0762 hypothetical protein 20.46 M369 B6 H-P0767 hypothetical protein 2.75 M369 C6 H-P0768 molybdenum cofactor 35.42 biosynthesis protein A (moaA) M369 D6 H-P0769 molybdopterin-guanine 22.22 dinucleotide biosynthesis protein A (mobA) M369 E6 H-P0771I hypothetical protein 27.06 M369 F6 H-P0772 N-acetylmuramoyl-L-alanine 48.51 (amiA) M369 G6 H-P0773 hypothetical protein 40.04 M369 H6 H--P0777 uridine 5 t -monophosphate (UMP) 26.51 (pyrH) M370 Al H-P0782 hypothetical protein M370 BI H-P0783 hypothetical protein 18.26 M370 Cl1 H-P0792 sigma-54 interacting protein 55.77 M370 DI H-P0793 polypeptide deformylase (def) 19.25 M370 El H-P0794 ATP-dependent cip protease 21.67 proteolytic component (clpP) M370 Fl -H-P0796 outer membrane protein (omnp18) 30.69 M379 G3 H-P0797-2 flagellar sheath adhesin hpaA 28.71 M379 H3 H-P0798-2 molybdenum cofactor 17.49 biosynthesis protein C M370 G I H-P0799 molybdopterin biosynthesis 19.47 protein (mog) M370 HI1 H-P0800 molybdopterin converting factor, 16.06 2 (moaE) M379 A4 H-P080 1-2 molybdopterin converting factor, 8.25 subunit 1 (moaD) M379 B4 -H-P0802-2 GTP cyclohydrolase Il (nibA) 21.23 M379 D3 H-P0803-2 hypothetical protein 30.8 M379 E3 H-P0804-2 GTP cyclohydrolase 11/3,4- 37.95 dihydroxy-2-butanone 4phosphate synthase (ribA, ribB) M379 F3 H-P0805-2 Iipooligosaccharide 5G8 epitope 31.35 biosynthesis-associated protein M370 A2 H-P0806 hypothetical protein 22.77 M379 C4 H-P0807-2 iron(I1I) dicitrate transport protein 86.68 (fecA) M370 B2 H-P0808 holo-acp synthase (acpS) 13.2 M370 C2 H-P0809 hypothetical protein 20.24 M370 D2 H-P08 10 conserved hypothetical protein 22.11 M370 E2 H-P08 11 hypothetical protein 11.99 M370 F2 H-P08 12 hypothetical protein 37.07 M370 G2 H-P08 13 conserved hypothetical protein 22.66 M370 H2 H-P0814 thiamnin biosynthesis protein 28.16 (thiF)_ M370 A3 H-P0815 flagellar motor rotation protein 28.38 M370 B3 H-P0831 conserved hypothetical ATP 21.67 M379 D4 H-P0832-2 spermidine synthase (speE) 28.93 WO 99/51766 PCT/US99/07270 M379 E4 H-P0833-2 hypothetical protein 32.23 M370 C3 H-P0834 GTP-binding protein homologue 50.49 (yphC) M370 D3 H-P0835 histone-like DNA-binding protein 10.45 HU (hup) M370 E3 H-P0836 hypothetical protein 13.2 M370 F3 H-P0837 hypothetical protein 11.33 M370 G3 H-P0838 hypothetical protein 22.66 M370 H3 H-P0839 outer membrane protein P1 64.68 (ompPl) M370 A4 H-P0840 flaAl protein 36.74 M370 B4 H-P0841 pantothenate metabolism 46.86 flavoprotein (dfp) M370 C4 H-P0843 thiamin phosphate 24.2 pyrophosphorylase/hyroxyethylthi azole kinase (thiB) M370 D4 H-P0845 thiamin phosphate 30.14 pyrophosphorylase/hyroxyethylthi azole kinase (thiM) M370 E4 H-P0850 type I restriction enzyme M 58.08 protein (hsdM) M370 F4 H-P0851 conserved hypothetical integral 25.08 membrane protein M370 G4 H-P0854 GMP reductase (guaC) 36.08 M370 H4 H-P0858 ADP-heptose synthase (rfaE) 50.82 M370 A5 H-P0859 ADP-L-glycero-D-mannoheptose- 36.41 6-epimerase (rfaD) M370 B5 H-P0861 hypothetical protein 27.17 M370 C5 H-P0862 hypothetical protein 24.64 M379 F4 H-P0863-2 hypothetical protein 59.73 M370 D5 H-P0865 deoxyuridine 5'-triphosphate 16.06 nucleotidohydrolase (dut) M370 E5 H-P0866 transcription elongation factor 18.15 GreA (greA) M379 G4 H-P0867-2 lipid A disaccharide synthetase 39.71 (lpxB) M379 H4 H-P0870-2 flagellar hook (flgE) 79.09 M370 F5 H-P0871 CDP-diglyceride hydrolase (cdh) 26.95 M370 G5 H-P0872 alkylphosphonate uptake protein 12.1 (phnA) M370 HS H-P0873 hypothetical protein 7.92 M371 Al H-P0879 hypothetical protein 22.33 M371 BI H-P0883 Holliday junction DNA helicase 20.24 (ruvA) M371 Cl H-P0885 virulence factor mviN protein 50.82 (mviN) M371 DI H-P0886 cysteinyl-tRNA synthetase (cysS) 51.26 M371 El H-P0889 iron(III) dicitrate ABC 35.97 transporter, permease protein (fecD) M371 Fl H-P0890 conserved hypothetical protein 28.27 M371 G 1 H-P0891 conserved hypothetical protein 19.25 M371 HI H-P0892 conserved hypothetical protein 10.01 WO 99/51766 WO 99/1 766PCT/US99/07270 M37 1 A2 H-P0894 conserved hypothetical protein 9.79 M37 1 B2 H-P0895 hypothetical protein 13.86 M37 1 C2 H-P0896 outer membrane protein (omp 19) M37 1 D2 H-PO897 hypothetical protein 22.99 M371 E2 H-PO898 hydrogenase expression/formation 40.81 M37 1 F2 H-P0899 hydrogenase expression/formation 8.58 M371 G2 H-P0900 hydrogenase expression/formation 26.73 M37 1 H2 H-P0905 phosphotransacetylase (pta) 24.64 M371 A3 5H-P0906 hypothetical protein 58.08 M37 1 B3 H-P0907 hook assembly protein, flagella 33.22 (flgD) M37 1 C3 H--P0909 hypothetical protein 22.22 M371 D3 H-P0912 outer membrane protein (ornp20) 56.76 M371 E3 H-P0913 outer membrane protein (omnp2l1) 58.3 M371 F3 H-P0914 hypothetical protein 56.65 M371 G3 H-P0915 iron-regulated outer membrane 61.93 protein M371I H3 H-P09 16 iron-regulated outer membrane 27.5 protein M380 Al H-P0917-2 hypothetical protein 2.64 M371I A4 H-P09 18 hypothetical protein 15.84 M371 B4 H-P0920 conserved hypothetical integral 25.41 membrane protein M37 1 C4 H-P0921I glyceraldehyde-3 -phosphate 36.63 dehydrogenase (gap) M37 1 D4 H-P0923 outer membrane protein (omp22) 40.7 M37 1 E4 H-P0925 recombinational DNA repair 21.34 ~protein M371I F4 H-P0927 heat shock protein (htpX) 35.97 M371 G4 H-P0928 GTP cyclohydrolase I (folE) 19.91 M37 1 H4 H-P0929 geranyltranstransferase (ispA) 33.44 M37 1 AS H-P0930 stationary-phase survival protein 29.48 (surE) M371 B5 H-P0931 hypothetical protein 16.17 M371 CS H-P0932 hypothetical protein 11.11 M37 1 D5 H-P0933 hypothetical protein 22.11 M37 1 ES H-P0934 conserved hypothetical protein 27.72 M371 F5E H-P0935 hypothetical protein 17.82 M37 1 G5 H-P0936 proline/betaine transporter (proP) 42.9 M371 H5 H-P0938 hypothetical protein 12.76 M371 A6 H-P0939 amino acid ABC transporter, 26.18 protein M37 1 B6 H-P0940 amino acid ABC transporter, 28.27 periplasmic binding protein M371 C6 H-P0941 alanine racemase, biosynthetic 41.58 (air)_ Mi/l 16 H-P0942 D-alanmne glycmne permease (dagA) 49.61 WO 99/51766 WO 9951766PCT/US99/07270 M371 E6 H-P0943 D-amino acid dehydrogenase 452 ~(dadA) M371 F6 H-P0944 translation initiation inhibitor, 13-.86 putative M371I G6 H-P0946 conserved hypothetical integral 54.67 protein______ M371 H6 H-PO947 hypothetical protein 13.31 M371 A7 H-P0949 conserved hypothetical secreted 16.61 M37 1 B7 H-P0950 acetyl-CoA carboxylase beta 31.9 subunit M371 C7 H-P0951 hypothetical protein 22.66 M37 1 D7 H-P0952 conserved hypothetical integral 24.09 protein______ M37 1 E7 H-P0953 hypothetical protein 20.79 M37 1 F7 H-P0955 prolipoprotein diacylglyceryl 31.35 M37 1 G7 H-P0956 conserved hypothetical protein 26.73 M37 1 H7 H-P0957 3-deoxy-d-manno-octulosonic- 43.34 transferase M371 A8 H-P0958 hypothetical protein 28.05 M371I B8 H-P0960 glycyl-tRNA synthetase, alpha 33.44 M37 1 C8 H-P096 1 glycerol-3 -phosphate 34.43 M380BI1 H-P0965-2 hypothetical protein 48.84 M37 1 D8 H-P0966 conserved hypothetical protein 60.5 M380 Fl H-P0968-2 hypothetical protein 2.42 M371 E8 H-P0969 cation efflux system protein 112.31 M37 1 F8 H-P0970 nickel-cobalt-cadmium resistance 39.6 protein_(nccB)_______ M371 G8 H-P0971 hypothetical protein M371 H8 H-P0972 glycyl-tRNA synthetase, beta 77.22 (glyS) M371I A9 H-P0973 hypothetical protein 38.94 M380 CI H-P0974-2 phosphoglycerate mutase (pgm) 54.12 M380 Dl H-P0975-2 conserved hypothetical protein 10.34 M380 El H-P0976-2 adenosylmethionine-8-amino-7- 48.07 oxononanoate aminotransferase M380 HI H-P0994-2 hypothetical protein 29.48 M380 GI H-PlOOO-2 PARA protein 24.09 M380 A2 H-PIOO1-2 hypothetical protein 10.45 M380 B2 H-PI002-2 hypothetical protein 43.45 M380 C2 H-PI003-2 hypothetical protein 40.81 M380 D2 H-PI004-2 hypothetical protein 30.14 M380 E2 H-PI005-2 hypothetical protein 11.55 M380 F2 H-P1006-2 conjugal transfer protein (traG) 19.58 M380 G2 H-Plo 17-2 amino acid permease (rocE) 57.2 M380 H2 H-P 1042-2 hypothetical protein 38.39 M380 A3 H-PI1056-2 hypothetical protein 31.35___ WO 99/51766 WO 9951766PCTIUS99/07270 M380 B3 H-PI1075-2 conserved hypothetical secreted 48.29 protein______ M373 Al H-P1076 hypothetical protein 18.92 M373 B I H-PI 077 nickel transport protein (nixA) 36.52 M373 CI H-PI1080 conserved hypothetical integral 20.9 protein M373 DI H-PI081 hypothetical protein 22.88 M373 El H-PI082 multidrug resistance protein 60.72 M373 Fl H-PI083 hypothetical protein 52.8 M373 GI H-PI084 aspartate transcarbanioylase 33.88 M373 HI H-P 1085 hypothetical protein 18.92 M373 A2 H-P 1086 hemolysin (tly) 25.96 M373 B2 H-P1087 riboflavin biosynthesis regulatory 30.91 (ibC) M373 C2 H-PI1088 transketolase A (tktA) 70.62 M373 D2 H-P1 091 alpha-ketoglutarate permease 46.97 M373 E2 H-P 1092 flagellar basal-body rod protein 29.7 M373 F2 H-PI1096 IS605 transposase (tnpA) 15.73 M373 G2 H-PI1098 conserved hypothetical secreted 32.01 protein M373 H2 H-PI 101 glucose-6-phosphate 46.86 ~dehydrogenase (g6pD) M373 A3 H-P 102 glucose-6-phosphate 1- 25.08 dehydrogenase (devB) M373 B3 H-PI 103 glucokinase (glk) 37.07 M373 C3 H-Pl 108 pyruvate ferredoxin 20.57 gamma subunit M373 D3 H-PlI 109 pyruvate ferredoxin 14.41 delta subunit M373 E3 H-PI 110 pyruvate ferredoxin 44.88 alpha subunit M373 F3 H-Pl 111 pyruvate ferredoxin 34.65 beta subunit M373 G3 H-PI 112 adenylosuccinate lyase (purB) 48.51 M380 C3 H-Pl 113-2 outer membrane protein (omp24) 30.58 M373 H3 H-Pl 117 conserved hypothetical secreted 28.27 ~~protein M373 A4 H-PI 1120 hypothetical protein 15.95 M373 B4 H-PI 1121 cytosine specific DNA 34.43 ~methyltransferase (BSP6IM) M380 D3 H-PI 1122-2 hypothetical protein 8.47 M373 C4 H-P 1123 peptidyl-prolyl cis-trans 20.46 isomerase, FKBP-type rotainase (slyD) M373 D4 H-Pl 1124 hypothetical protein 36.52 M373 E4 H-P1 125 peptidoglycan associated 19.8 ~~lipoprotein precursor (omp 18) M373 F4 H-P 1126 colicin tolerance-like protein 45.98 (totB) I_ WO 99/51766 WO 9951766PCTIUJS99/07270 M373 G4 H-PI 128 hypothetical protein 9.35 M373 H4 H-Pl 1129 biopolymer transport protein 14.74 (exbD) M373 A5 H-Pl 131 ATP synthase Fl, subunit epsilon 13.75 ~~(atpC) M373 B5 H-PI 134 ATP synthase FlI, subunit alpha 55.44 M373 C5 H-PI 135 AT? synthase Fl, subunit delta 19.91 M373 D5 H-P1 137 ATP synthase FO, subunit b' 15.95 M373 E5 H-PI 138 plasmid replication-partition 32.01 protein M373 F5 H-PI 139 SpoOJ regulator (soj) 29.15 M373 G5 H-Pl 1140 biotin operon repressor/biotin 23.43 acetyl coenzyme A carboxylase synthetase M373 H5 H-Pl 1141 inethionyl-tRNA 33.44 ________________formyltransferase (tint) M373 A6 H-PI 1144 hypothetical protein 9.46 M373 B6 H-Pl 1145 hypothetical protein 11.44 M373 C6 H-Pl 1147 ribosomal protein L 19 (rpll19) 13.09 M373 D6 H-PI 148 tRNA (guanine-Ni)- 25.3 methyltransferase (trmD) M373 E6 H-Pl 1149 conserved hypothetical protein 20.35 M380 F3 H-PI 150-2 hypothetical protein 12.76 M373 F6 H-P1 1152 signal recognition particle protein 49.39 (ffh) M380 G3 H-Pl 1153-2 valyl-tRNA synthetase (valS) 96.25 M380 E3 H-Pl 157-2 outer membrane protein (omp26) 135.41 M373 G6 H-Pl 158 pyrroline-5-carboxylate reductase 28.38 M373 H6 H-P1 159 cell filamentation protein (fic) 19.58 M373 A7 H-P 1160 conserved hypothetical protein 15.51 M380 A4 H-P 1163-2 hypothetical protein 7.04 M373 B7 H-PI 165 tetracycline resistance protein 42.57 tetA(P), putative M373 C7 H-Pl 1168 carbon starvation protein (cstA) 75.68 M373 D7 H-P 1169 glutamine ABC transporter, 23.98 protein (glnP) M380 H3 H-PI 1169-2 glutamine ABC transporter, 23.98 protein (glnP) M374 AlI H-P 170 glutamine ABC transporter, 24.64 protein (glnP) M374 BI H-PI 171 glutamine ABC transporter, AT?- 27.39 binding protein (gtnQ) M374 ClI H-PI 172 glutamine ABC transporter, 30.58 periplasmic glutamine-binding protein (glnH) M374 DI H-PI 173 hypothetical protein 20.24 M374 El H-P 1174 glucose/galactose transporter 4' 4.88 (gluP) WO 99/51766 WO 99/1 766PCT/US99/07270 M374 F1 H-P 1175 conserved hypothetical integral 47.96 protein M374 GI H-P 1177 outer membrane protein (omp,27) 70.62 M374 HI H-PI 178 purine-nucleoside phosphorylase 25.74 M374 A2 H-PI 179 phosphopentomnutase (deoB) 45.54 M374 B2 Hi-P 1180 pyrimidine nucleoside transport 46.09 (nupC) M374 C2 H-P1 183 NA+/H+ antiporter (napA) 42.24 M374 D2 H-PI 1184 conserved hypothetical integral 50.6 membrane protein M374 E2 H-PI 1185 conserved hypothetical integral 43.12 protein M374 F2 H-Pl 1186 carbonic anhydrase 22.33 M374 G2 H-P 1187 hypothetical protein 42.46 M374 H2 H-PI 188 hypothetical protein 29.7 M374 A3 H-PI 189 aspartate-semialdehyde 38.17 M374 B3 H-PI 191 ADP-heptose-Ips 38.5 ______________heptosyltransferase 11 (rfaF) M374 C3 H-P 1196 ribosomnal protein S7 (rps7) 17.16 M374 D3 H-P 1200 ribosomal protein L 10 (rpll10) 18.15 M374 E3 H-P1201 ribosomal protein L I (rpll1) 25.85 M374 F3 H-P 1202 ribosomnal protein L I I (rpll 11) 15.62 M374 G3 H-P 1203 transcription termination factor 19.47 (nusG) M380 B4 H-P1205-2 translation elongation factor EF- 44 Tu (tufB) M374 H3 H-P 1206 multidrug resistance protein 63.69 M374 A4 H-P1207 hypothetical protein 24.53 M374 B4 H-P 1210 seine acetyltransferase (cysE) 18.92 M380 F4 H-P12 13-2 polynucleotide phosphorylase 75.79 M380 G4 H-P 1214-2 conserved hypothetical protein M380 C4 H-P1215-2 hypothetical protein 8.91 M380 D4 H-P1216-2 conserved hypothetical secreted 72.71 M3 80 E4 H-P1 217-2 hypothetical protein 17.6 M374 C4 H-P 1220 ABC transporter, ATP-binding 25.19 protein M374 D4 H-P1221I conserved hypothetical protein 25.85 M374 E4 H-P 1222 D-lactate dehydrogenase (dld) 104.39 M374 F4 H-P 1224 uroporphyrinogen III cosynthase 24.97 M374 G4 H-P 1225 conserved hypothetical integral 14.41 membrane protein M374 H4 H-P 1226 oxygen-independent 38.83 coproporphyrinogen III oxidase M380 H4 H-P 1227-2 cytochrome c553 10.67 M380 A5 H-P1228-2 invasion protein (invA) 1.16 M380 B5 H-P1229-2 aspartokinase (lysC)4.6 WO 99/51766 WO 9951766PCT[US99/07270 M374 AS H-P1230 hypothetical protein 19.91 M374 B5 H-P 1231 DNA polymerase III delta prime 24.09 ~~subunit (hoIB) M374 C5 H-P1232 dihydropteroate synthase (foiP) 41.91 M380 D5 H-P1233-2 hypothetical protein 16.94 M374 D5 H-P1234 conserved hypothetical integral 32.89 ~membrane protein M374 ES H-P1235 conserved hypothetical integral 45.76 membrane protein M374 F5 H-P1236 hypotheical protein 20.24 M374 G5 H-P1237 carbamoyl-phosphate synthetase 41.36 M374 HS H-PI240 conserved hypothetical protein 21.01 M380 CS H-P1241-2 alanyl-tRNA synthetase (alaS) 93.28 M374 A6 H-P1242 conserved hypothetical protein 8.47 M380 H5 H-P1243-2 outer membrane protein (omp28) 80.74 M374 B6 H-P1244 ribosomal protein S 18 (rpsl18) 9.46 M374 C6 H-P1245 single-strand DNA-binding 19.8 M374 D6 H-P1246 ribosomal protein S6 (rps6) 15.73 M380 A6 H-P1247-2 hypothetical protein 37.51 M374 E6 H-P1248 virulence associated protein 70.95 M380 B6 H-P 1249-2 shikiniate S-dehydrogenase (aroE) 29.04 M380 ES H-P1251-2 oligopeptide ABC transporter, 38.39 protein (oppB) M380 F5 H-P 1252-2 oligopeptide ABC transporter, 65.45 periplasmic oligopeptide-binding protein M380 G5 H-P1253-2 tryptophanyl-tRNA synthetase 37.4 M374 F6 H-P1254 biotin synthesis protein (bioC) 26.51 M374 G6 H-P1255 protein translocation protein, low 2 2.22 (secG) M374 H6 H-P1256 ribosome releasing factor (frr) 20.46 M374 A7 H-P1257 orotate phosphoribosyltransferase 22.22 M374 B7 H-P1258 conserved hypothetical 17.05 ___________mitochondrial protein 4 M374 C7 H-P1260 NADH-ubiquinone 14.74 oxidoreductase, NQ07 subunit (NQO7) M374 D7 H-P 1262 NADH-ubiquinone 29.37 oxidoreductase, NQ05 subunit M374 E7 H-P1263 NADH-ubiquinone 45.1 oxidoreductase, NQ04 subunit M380 C6 H-P1264-2 hypothetical protein 8.47 M374 F7 H-P 1265 hypothetical protein 36.19 M375 Al H-P1268 NADH-ubiquinone 24.31 oxidoreductase, NQ09 subunit WO 99/51766 WO 9951766PCTIUS99/07270 M375 BI H-P1275 phosphomannomnutase (algC) 50.6 M375 CI H-P1277 tryptophan synthase, alpha 28.93 M375 Dl H-P1278 tryptophan synthase, beta subunit 43.34 M375 El H-P1279 anthranilate isomerase (trpC) 49.83 M375 Fl H-P1282 anthranilate synthase component 1 55.11 M375 GI H-P 1285 conserved hypothetical secreted 25.41 M375 HI H-P1286 conserved hypothetical secreted 20.13 M375 A2 H-P1287 transcriptional regulator (tenA) 23.98 M375 B2 H-P1288 hypothetical protein 14.63 M375 C2 H-P 1289 hypothetical protein 17.82 M375 D2 H-P1290 nicotinamide mononucleotide 24.31 ___________transporter (pnuC) M375 E2 H-P1291 conserved hypothetical protein 22.55 M375 F2 H-P1292 ribosomal protein L 17 (rpll17) 12.87 M375 G2 H-P1293 DNA-directed RNA polymerase, 37.95 subunit (rpoA) M375 H2 H-P 1294 ribosomal protein S4 (rps4) 22.99 M375 A3 H-P 1295 ribosomal protein S I 1 (rps 11) 14.52 M375 B3 H-P1296 ribosomal protein S13 (rpsl3) 13.31 M380 D6 H-P 1298-2 translation initiation factor EF-I 8.03 M375 C3 H-P1299 methionine amino peptidase 27.94 M375 D3 H-P 1302 ribosomal protein S5 (rps5) 16.94 M375 E3 H-P1303 ribosomal protein L 18 (rpll18) 13.2 M375 F3 H-P1305 ribosomal protein S8 (rps8) 14.52 M375 G3 H-P1307 ribosomal protein L5 (rpl5) 20.02 M375 H3 H-P1308 ribosomnal. protein L24 (rp124) 8.14 M375 A4 H-P1309 ribosomal protein L14 (rpll4) 13.53 M375 B4 H-P1310 ribosomal protein S 17 (rpsl17) 9.57 M375 C4 H-P1 312 ribosomal protein L16 (rpl 16) 15.62 M375 D4 H-P1314 ribosomal protein L22 (rp122) 13.53 M375 E4 H-P1315 ribosomal protein S519 (rpsl19) 10.34 M375 F4 H-P1318 ribosomnal protein L4 (rpl4) 23.76 M375 G4 H-P1319 ribosomal protein L3 (rpl3) 21.12 M375 H4 H-P1320 ribosomal protein Sl10 (rpsl10) 11.55 M375 AS H-P1321 conserved hypothetical ATP- 41.58 binding protein M375 B5 H-P1322 hypothetical protein 22.22 M375 C5 H-P1323 ribonuclease HIT (rnhB) 23.1 M375 D5 H-P 1324 hypothetical protein 9.24 M375 ES H-P1326 hypothetical protein 13.86 M375 F5 H-P1327 hypothetical protein 45.43 M375 G5 H-P1328 cation efflux system protein 37.29 M375 H5 H-P 1330 conserved hypothetical integral 12.76 111membrane protein______ WO 99/51766 WO 99/1 766PCT/US99/07270 M375 A6 H-P1331 conserved hypothetical integral 25.19 membrane protein______ M375 B6 H-P1332 co-chaperone and heat shock 4.

(dnai) M375 C6 H-P1333 hypothetical protein 42.13 M375 D6 H-P1335 conserved hypothetical protein 39.71 M375 E6 H-P1 336 hypothetical protein 27.94 M375 F6 H-P1337 conserved hypothetical protein 19.25 M375 G6 H-P1338 conserved hypothetical protein 16.39 M375 H6 H-P1 340 biopolymer transport protein 14.3 M375 A7 H-P 1341 siderophore-mediated iron 31.46 protein (tonB) M375 B7 H-P1 342 outer membrane protein (omp29) 76.12 M375 C7 H-P 1343 conserved hypothetical integral 26.73 protein_____ M375 D7 H-P 1344 magnesium and cobalt transport 35.09 M375 E7 H-P 1345 phosphoglycerate kinase M375 F7 H-P1 346 glyceraldehyde-3-phosphate 36.41 M375 G7 H-P 1347 uracil-DNA glycosylase (ung) 25.74 M375 H7 H-P1349 hypothetical protein 42.68 M375 A8 H-P1350 protease 50.6 M375 B8 H-P1355 nicotinate-nucleotide 30.14 pyrophosphorylase M375 C8 H-P1356 quinolinate synthetase A (nadA) 37.07 M375 D8 H-P 1357 phosphatidylserine decarboxylase 29.48 proenzyme (psd) M375 E8 H-P1358 hypothetical protein 18.59 M375 F8 H-P1 360 4-hydroxybenzoate 32.45 octaprenyltransferase (ubiA) M375 G8 H-P1361 competence locus E (comE3) 45.98 M375 H8 H-P1 362 replicative DNA helicase (dnaB) 53.79 M375 A9 H-P 1363 conserved hypothetical integral 51.37 membrane protein_____ M376 Al H-P1364 signal-transducing protein, 43.78 histidine kinase M376 BI H-P1365 response regulator 23.54 M376 CI H-P1371 type III restriction enzyme R 106.59 M376 Dl H-P1372 rod shape-determining protein 27.39 M376 El H-P 1373 rod shape-determining protein 38.28 M376 F l H-P 1374 ATP-dependent protease ATPase 49.17 (clpX) M376 G 1 H-P1375 UDP-N-acetylglucosamine 29.81 ______________acyltransferase (lpxA) M376 HI1 H-P 1376 (3R)-hydroxymyristoyl.(acyl 17.6 carrier protein) dehydratase M376 A2 H-P1377 hypothetical protein 16.17 WO 99/51766 PCT/US99/07270 M376 B2 H-P1378 competence lipoprotein (comL) 24.31 M376 C2 H-P1379 ATP-dependent protease (Ion) 91.96 M376 D2 H-P1380 prephenate dehydrogenase (tyrA) 29.26 M381 Cl H-P1381 hypothetical protein 8.58 M376 E2 H-P1382 hypothetical protein 14.41 M376 F2 H-P1383 restriction modification system S 17.71 subunit M376 G2 H-P1384 hypothetical protein 7.59 M376 H2 H-P1385 fructose- 1,6-bisphosphatase 32.01 M376 A3 H-P1386 D-ribulose-5-phosphate 3 23.98 epimerase (rpe) M376 B3 H-P1388 hypothetical protein 16.5 M376 C3 H-P1389 hypothetical protein 6.71 M376 D3 H-P1390 hypothetical protein 18.37 M376 E3 H-P1391 hypothetical protein 10.89 M376 F3 H-P1392 fibronectin/fibrinogen-binding 47.96 protein M376 G3 H-P1393 DNA repair protein (recN) 57.75 M376 H3 H-P1394 conserved hypothetical protein 31.35 M376 A4 H-P1395 outer membrane protein (omp30) 26.73 M376 B4 H-P1396 hypothetical protein 31.79 M376 C4 H-P1398 alanine dehydrogenase (aid) 41.91 M376 D4 H-P1399 arginase (rocF) 35.53 M376 E4 H-P1400 iron(III) dicitrate transport protein 92.73 (fecA) M376 F4 H-P1401 conserved hypothetical protein 25.96 M381 A2 H-P1402 type I restriction enzyme R 109.34 protein (hsdR) M381 B2 H-P1403 type I restriction enzyme M 89.98 protein (hsdM) M376 G4 H-P1405 hypothetical protein 3.85 M376 H4 H-P1406 biotin synthetase (bioB) 31.13 M376 A5 H-P1407 conserved hypothetical integral 32.23 membrane protein M381 C2 H-P1408 hypothetical protein 12.32 M381 D2 H-P1409 hypothetical protein 63.69 M376 B5 H-P1410 hypothetical protein 43.45 M376 C5 H-P1411 hypothetical protein 68.2 M376 D5 H-P1412 hypothetical protein 33.99 M376 E5 H-P1413 conserved hypothetical protein 16.39 M376 F5 H-P1414 conserved hypothetical protein 12.54 M376 G5 H-P1415 tRNA delta(2)- 29.37 isopentenylpyrophosphate transferase (miaA) M376 H5 H-P1418 UDP-N- 28.6 acetylenolpyruvoylglucosamine reductase (murB) M376 A6 H-P1419 flagellar biosynthetic protein 9.79 (fliQ) M376 B6 H-P1420 flagellar export protein ATP 47.85 synthase (flil) M376 C6 H-P1421 conjugative transfer regulon 33.55 protein (trbB) WO 99/51766 WO 9/51766PCTILJS99/07270 M376 D6 H-P 1423 conserved hypothetical protein 9.35 M376 E6 H-P1424 hypothetical protein 22.77 M376 F6 H-P1425 hypothetical protein 8.36 M376 G6 H-P1427 histidine-rich, metal binding 6.71 M376 H6 H-P1428 conserved hypothetical protein 398 M376 A7 H-P1429 polysialic acid capsule expression 36.3 (kpsF) M376 B7 H-P 1430 conserved hypothetical ATP- 75.9 protein M376 C7 H-P1431 16S rRNA (adenosine-N6,N6-)- 29.92 ___________dimethyltransferase (ksgA) M376 D7 H-P1432 histidine and glutamine-rich 8.03 M376 E7 H-P1433 hypothetical protein 94.27 M376 F7 H-P1434 formyltetrahydrofolate hydrolase 32.34 M376 G7 H-P1435 protease IV (PspA) 32.23 M376 H7 H-P1436 hypothetical protein 9.13 M376 A8 H-P1438 conserved hypothetical 37.29 M376 B8 H-P1439 hypothetical protein 9.02 M376 C8 H-P1440 hypothetical protein 28.6 M376 D8 H-P144 1 peptidyl-prolyl cis-trans 18.04 isomerase B, cyclosporin-type rotamase (ppi) M376 E8 H-P1442 carbon storage regulator (csrA) 8.47 M376 F8 H-P1443 conserved hypothetical protein 29.59 M376 G8 H-P 1444 small protein (smpB) 16.83 M376 H8 H-P 1445 biopolymer transport protein 16.61 (exbB) M376 A9 H-P 1446 biopolymer transport protein 14.74 M376 B9 H-P1447 ribosomal protein L34 (rp134) 4.95 M376 C9 H-P1448 ribonuclease P, protein 17.82 component M376 D9 H-P1449 conserved hypothetical protein 12.98 M376 E9 H-P 1450 60 kDa inner-membrane protein 60.28 M376 F9 H-P1451 hypothetical protein 29.15 M376 G9 H-P 1452 thiophene and furan oxidizer 50.82 M376 H9 H-P 1453 conserved hypothetical protein 82.17 M376 AlO H-P1454 hypothetical protein M376 BlO H-P1455 hypothetical protein 14.41 M376 CIO H-P1456 membrane-associated lipoprotein 19.36 M376 DIO H-P1457 hypothetical protein 23.21 M376 EI0 H-P1458 thioredoxi 11.55 M376 FIO H-P1461 cytochrome c551 peroxidase 38.61 M377 Al1 H-P1462 secreted protein involved in 19.03 motility M371B1 H-P1463 hypothetical protein 24.86 WO 99/51766 WO 9951766PCT/US99/07270 M377 CI H-P1464 conserved hypothetical secreted 29.92 M377 Dl H-P1465 ABC transporter, ATP-bindmng 28.82 (HI11087) M377 El H-P 1466 conserved hypothetical integral 41.58 protein_____ MW377 Fl H-P1467 hypothetical protein 25.52 M377 G I H-P 1468 branched-chain-amino-acid 37.51 _______________aminotransferase (ilvE) M377 HI H-P1469 outer membrane protein (omp3l1) 27.39 M377 A2 H-P1473 hypothetical protein 21.12 M377 B2 H-P 1474 thymidylate kinase (tmnk) 21.12 M377 C2 H-P1 475 lipopolysaccharide core 17.38 biosynthesis protein (kdtB) M377 D2 H-P 1476 phenylacrylic acid decarboxylase 20.68 M377 E2 H-P1479 hypothetical protein 92.95 M377 F2 H-P1480 seryl-tRNA synthetase (serS) 45.76 M377 G2 H-P1481 hypothetical protein 29.26 M377 H2 H-P 1482 hypothetical protein 9.57 M377 A3 H-P1483 gerC2 protein (gerC2) 27.17 M377 B3 H-P 1484 conserved hypothetical integral 16.39 protein M377 C3 H-P1485 proline dipeptidase (pepQ) 21.01 M377 D3 H-P 1486 conserved hypothetical integral 41.47 membrane protein M377 E3 H-P1487 conserved hypothetical integral 40.26 membrane protein M377 F3 H-P 1488 conserved hypothetical secreted 36.3 protein M377 G3 H-P1489 lipase-like protein 56.21 M381 GI H-P 1490 hemolysin 49.5 M377 H3 H-P1491 phosphate perniease 58.74 M377 A4 H-P1492 conserved hypothetical niflJ-like 9.9 protein______ M377 B4 H-P 1493 hypothetical protein 22.44 M377 C4 H-P1494 UDP-MurNac-tripeptide 49.28 M377 D4 H-P1495 transaldolase (tal) 34.87 M377 E4 H-P1496 general stress protein (ctc) 19.69 M377 F4 H-P1497 peptidyl-tRNA hydrolase (pth) 20.57 M377 G4 H-P1499 hypothetical protein 30.03 M377 H4 H-P1501 outer membrane protein (omp32) 42.79 M377 AS H-P1502 hypothetical protein 16.06 M377 B5 H-P1503 cation-transporting ATPase, P- 86.79 (copA) M377 C5 H-PI504 conserved hypothetical protein 26.29 M377 D5 H-PI505 riboflavin biosynthesis protein 37.95 M377 ES H-PI1506 glutamnate permease (gltS) 44.99 M377 F5 H-P 1507 conserved hypothetical ATh- 42.46 rM381 F2 H-P 1508 ferrodoxin-like protein WO 99/51766 WO 9951766PCT/US99/07270 T

T

M37 G5 U H-PI 509 conserved hypothetical integral membrane protein 28.93 M377 H5 H-P1510 conserved hypothetical protein 12.98 M377 A6 H-P1511I hypothetical protein 11.99 M377 B6 H-P1512 iron-regulated outer membrane 96.58 M377 C6 H-P1513 selenocystein. synthase (selA) 42.57 M377 D6 H-P15 14 transcription termination factor 43.56 NusA (nusA) M377 E6 H-P1518 hypothetical protein 10.56 M381 B3 H-P1521 type III restriction enzyme R 106.48 protein (res) M381 C3 H-P1523 DNA recombinase (recG) 68.64 M377 F6 H-P1524 hypothetical protein 12.76 M377 G6 H-PI525 hypothetical protein 23.32 M377 H6 H-P1526 exodeoxyribonuclease (lexA) 27.61 M377 A7 H-P1527 hypothetical protein 52.8 M377 B7 H-P1530 purine nucleoside phosphorylase 19.91 M377 C7 H-PI 531 hypothetical protein 8.8 M377 D7 H-P1 532 glucosamine fructose-6-phosphate 65.78 aminotransferase (isomerizing) M377 E7 H-P1533 conserved hypothetical protein 25.52 M377 F7 H-P1 534 IS605 transposase (tnpB) 47.08 M377 G7 H-P 1535 1S605 transposase (tnpA) 15.73 M377 H7 H-P1 541 transcription-repair coupling 110 factor (trcF) M377 A8 H-P 1548 conserved hypothetical integral 12.43 membrane protein M377 B8 H-P1551I conserved hypothetical secreted 14.08 protein M377 C8 H-P1552 Na+/H+ antiporter (nhaA) 48.29 M381 B4 H-P1554 ribosomal protein 52 (rps2) 29.15 M381 D4 H-P1555 translation elongation factor EF- 39.16 Ts M377 D8 H-P 1556 cell division protein (ftsl) 67.76 M381 E4 H-P1557 flagellar basal-body protein (flu) 12.1 M38 1 F4 H-PI 558 flagellar basal-body rod protein 17.82 ~(flgC) (proximal rod pin) M381 G4 H-P1559 flagellar basal-body rod protein 15.51 (flgB) (proximal rod protein)_____ M378 Al HI-PI560 cell division protein (ftsW) 42.79 M378 BI H-P1561 iron(III) ABC transporter, 36.96 periplasmic iron-binding protein M378 CI H-P1562 iron(III) ABC transporter, 36.74 periplasmic iron-binding protein M378 DI H-P 1563 alkyl hydroperoxide reductase 21.89 (tsaA) M378 El H-P 1564 outer membrane protein 29.92 WO 99/51766 WO 9951766PCT/US99/07270 M378 Fl H-P 1565 penicillin-binding protein 2 64.79 M378 GI1 H-P 1566 hypothetical protein 16.28 M378 HI H-P 1567 conserved hypothetical ATP- 22.99 binding protein M378 A2 H-P1568 hypothetical protein 20.24 M378 B2 H-PI569 hypothetical protein 21.78 M378 C2 H-P1 570 conserved hypothetical protein 18.15 M378 D2 H-P1 571 rare lipoprotein A (rIpA) 34.76 M378 E2 H-PI1572 regulatory protein DniR 41.03 M378 F2 H-PI 573 conserved hypothetical protein 28.05 M378 G2 H-PI1576 ABC transporter, ATP-binding 36.08 (abc) M378 H2 H-P1577 ABC transporter, permease 23.76 (yaeE) M378 A3 H-P1580 hypothetical protein 24.31 M378 B3 H-P1581 methicillmn resistance protein 37.07 M378 C3 H-P 1582 pyridoxal phosphate biosynthetic 28.93 protein J (pdxl) M378 D3 H-P 1583 pyridoxal phosphate biosynthetic 33.88 protein A (pdxA) M378 E3 H-P 1584 sialoglycoprotease (gcp) 37.51 M378 F3 H-P1 585 flagellar basal-body rod protein 28.93 (flgG) M378 G3 H-P 1587 conserved hypothetical protein 17.16 M378 H3 H-P1 588 conserved hypothetical protein 27.94 M381 I H-P 1590 hypothetical protein M318 G2 H-S38729 autoimmune antigen Ku, p 7 0 67.1 67 HI H-S39329 Kallikrein 1 24.64 (renal/pancreas/salivary) products) M270 G4 H-S43855 Recoverin, photoreceptor protein 22.11 32.OkDa M300 C2 H-S56151 milk fat globule protein HMFG 24.09 M318 C1 H-S57153 retinoblastoma-binding protein 1, 101.31 101 isoform I [RBBP 1] M271 B2 H-S57162 retinoblastoma-binding protein 1, 93.72 110 isoform III [RBBPI], INTERACTS WITH THE VIRAL PROTEIN-BINDING DOMAIN OF THE RETINOBLASTOMA

PROTEIN.

M317 H3 H-S62027 transducin, gamma subunit 8.25 11 M270 G6 H-S66793 arstin, X-arrestin=S-antigen 42.79 homolog [human, retina, mRNA, 1314 nt], MAY PLAY A ROLE IN AN AS YET UNDEFINED RETINA-SPECIFIC SIGNAL

TRANSDUCTION.

M419 C2 H-S67859 "transcription initiation factor Ile, 48.3604.OkDa alpha subunit" WO 99/51766 WO 9951766PCT/US99/07270 M302 D7 H-S69022 myosin, light polypeptide 2, 18.26 3 ventricular H-S69272 cytoplasmic antiproteinase=38 41.47 kda intracellular serine proteinase inhibitor [human, placenta, ~mRNA, 1465 nt] Dl H-S72043 GIF=growtb inhibitory factor 7.59 19 (~~human, brain, Genomic, 2015 ntj M266 B3 H-S74221 cytokine 1K factor 17.93 36.OkDa DI H-S74445 cellular retinoic acid-binding 15.18 23 protein [human, skin, mRNA, 735 E3 H-S74728 antiquitin=26g turgor protein 56.32 53 homolog [human, kidney, mRNA, ~~1809 nt] D4 H-S75 174 E2F transcription factor 4, 45.87 58 ~p1 07/pl130-binding 166-61 H-S76474 "trkB (altemnately spliced) 55 52.54 brain, mRNA]" 169-40 H-S76617 "B Ilk=protein tyrosine kinase 60 55.62 (human, B lymphocytes, mRNA, ~~2608 M250 D3 H-S79522 ubiquitin carboxyl-terminal 17.27 1 7.OkDa extension protein, Ubiquitin A-52 residue ribosomal protein fusion

I

M236 B4 H-S80562 calponin, acidic 36.3 49 G I H-S82470 BB Il=malignant cell expression- 37.73 34 enhanced gene/tumor progression-enhanced gene [human, UM-UC-9 bladder carcinoma cell line, mRNA, 1897 nt] M313 ElI H-S85655 prohibitin [PHBI 30.03 M465 A6 H-S87759 protein phosphatase 2C alpha 42.13 52.OkDa [human, teratocarcinoma, mRNA, nt] M472 B I H-U00803 tyrosine-protein kinase ERK 55.620 64.OkDa B2 H-U02390 Human adenylyl cyclase- 52.58 associated protein homolog CAP2 mRNA, complete cds 167-2 H-U02680 human protein tyrosine kinase 36 38.57 G2 H-U03056 Human tumor suppressor (LUCA- 47.96 47 1) mRNA, complete M512 E3 H-U03 100 Human alpha2(E)-catenin mRNA, 102.52 102.OkDa cds M306 G3 H-U03 187 H3 H-U03398 Human receptor 4-l1BB ligand 28.05 51 mRNA, complete cds D3 H-U03486 Human connexin40 gene, 39.49 cds M300 C3; H-U03643 leukophysin 25.96 34 L I WO 99/51766 WO 9951766PCTIUS99/07270 H-U03749 Human chromogranmn A (CHGA) 50.38 promoter and M3 14 C3 H-U03886 GS2 (GB:U03886) 27.94 32.OkDa M306 E3 H-U04343 CD86 antigen (CD28 antigen 35.64 47 2, B7-2 antigen) [CD86] 167-61 H-U05012 TrkC 92 90.82 M302 G5 H-U05340 cell division cycle protein p55 55 A4 H-U05659 Hydroxysteroid (1 7-beta) 34.21 36 3 Fl H-U05861 Human hepatic dihydrodiol 35.64 gene M302 B2 H-U06452 antigen MART-i, melanoma 13.09 2( OkDa 169-52 H-U06454 human AMP-activated protein 70 60.79 (hAMPK) mR1NA____ M315 A3 H-U06643 lectin, epidermal 15.07 18 HI H-U06715 Cytochrome B561 27.06 M476 E5 H-U07 132 Human steroid hormone receptor 50.82 mRNA, complete cds M236 D3 H-U07151 guanine nucleotide-binding 20.13 34 protein ADP-ribosylation factor gene 3 M317 G3 H-U07559 homeotic protein Islet-i 38.17 38 M266 Hi1 H-U07681 Human NAD(H)-specific 40.37 isocitrate dehydrogenase alpha subunit precursor mRNA, cds E3 H-U079 19 Aldehyde dehydrogenase 6 6.43 53 M298 A3 H-U08021I nicotinamide N-methyltransferase 29.15 36.OkDa M297 B 1 H-U08024 alcohollhydroxysteroid 31.46 A2 H-U08336 Human basic helix-loop-helix 21.89 42 transcription factor mRNA, cds E2 H-U09303 Human T cell leukemia LERK-2 38.17 mRNA, complete cds M250 H5 H-U09559 RCHI, RAG (recombination 58.3 58.OkDa gene) cohort 1 167-50 H-U09564 human seine kinase mRNA 72 72.12 166-74 H-U09578 human MAPKAP kinase (3pK) 50 42.09 M302 C4 H-U098 13 ATP synthase, subunit 9, 15.73 Al H-U09850 Zinc finger protein 143 (clone 68.97 68 M423 El H-U09937 Human urokinase-type 36.96 49.OkDa receptor M450 H4 H-UIOI 17 Human endothelial-monocyte 34.43 38.OkDa activating polypeptide 11 inRNA, complete cds M314 G I H-U 10248 ribosomal protein L29 17.6 27 M298 H I H-U10323 nuclear factor 45 44.77 El H-U10492 Human Mox I protein (MiOXl) 28.05 37T complete cds WO 99/51766 WO 9951766PCTIUS99/07270 F3 H-U 10686 Human MAGE-1 1 antigen 35.2 11) gene, complete cds 167-38 H-Ul 1050 human NIMA-like protein kinase 55 49.02 (NLKI) mRNA____ M266 B2 H-Ul 11292 Human Ki nuclear autoantigen 29.48 32 mRINA, complete cds, may play a in cell adhesion 167-62 H-U 11791 human cyclin Hm RNA 40 35.60 M423 D5 H-U 12255 immunoglobulin gamma heavy 40.26 48.OkDa Fc receptor RI, high affinity M302 F7 H-U12404 Csa- 19 23.98 32 M236 A2 H-U 12465 ribosomal protein L35 13.64 24 169-4 H-U12535 human epidermal growth factor 100 -90.49 receptor kinase substrate (Eps8) mRNA F3 H-U 12597 Human tumor necrosis factor type 55.22 64 2 receptor associated protein mRNA, complete cds M3 14 Dl1 H-U 12979 transcriptional coactivator PC4 14.08 23 M476 G4 H-U 13044 GA-binding protein transcription 50.05 53.OkDa alpha subunit (6OkD) M302 F3 H-U13665 cathepsin 0 (GB:U13665) 36.3 M31 1 G4 H-U13831 cellular retinol binding protein 11 14.85 A2 H-U 13991 Human TATA-binding protein 24 .09 34 associated factor 30 kDa subunit mRNA, complete cds M4 16 A4 H-U 14187 Human receptor tyrosine kinase 26.29 29.OkDa ligand LERK-3 (EPLG3) mRNA, complete cds M250 A2 H-U 14188 eph-related receptor tyrosine 22.22 27 ligand 4 [EPLG4] M302 D2 H-U14 193 human TFIIA gamma subunit 12.060 28.OkDa mRNA M416 GI1 H-U14603 Human protein-tyrosine 18.48 phosphatase (H4U-PP-1) mRNA, sequence E2 H-U14747 Visinin-like 1 21.12 M266 D4 H-U14966 ribosomal protein L5 32.78 38 M314 E2 H-U 14967 ribosomal protein L21 17.71 29 M266 F5 H-U 14968 ribosomal protein L27a 16.39 19.OkDa M248 E3 H-U14969 ribosomal protein L28 15.18 27 M266 El1 H-U14971 ribosomal protein S9 21.45 M250 C2 H-U 15009 small nuclear ribonucleprotein, 13.97 17.OkDa Sm D3 M311I D4 H-U16660 enoyl-Coenzyme A hydratase-like 36.19 38 protein, peroxisomal______ M302 H4 H-U 17074 cyclin-dependent kinase 6 18.59 29 inhibitor p 18 M306 A2 H-U 17195 A-kinase anchor protein 100 72.05 100 Dl1 H-U 17280 Steroidogenic acute regulatory 31.46 proteinII M316 Fl1 H-U19 cel dvsocycle protein 16 68.2 1.O WO 99/51766 WO 9951766PCT/US99/07270 H-U 18420 Human ras-related small GTP 23.87 33 binding protein Rab5 complete cds M3 11 A2 H-U 18423 spinal muscular atrophy gene 32.45 41 M248 D4 H-U18914 hypothetical protein, (Human 20.35 32 19.8 kDa protein mRNA, cds) M302 B5 H-U 19718 microfibril-associated 20.24 34.OkDa 2 M305 E3 H-U20240 CCAAT/enhancer-binding protein 16.61 29 M302 A8 H-U20352 malate dehydrogenase 36.85 M416 F4 H-U20391 Human folate receptor (FOLRl1) 28.38 34.OkDa gene, complete cds M311 I H-U20536 apoptotic cysteine protease Mch2 32.34 38.OkDa M431I G2 H-U20659 RNA polymerase 11, subunit B7 19.03 31 .OkDa M499 C I H-U20938 Human lymphocyte 112.86 100.OkDa dihydropyrimidine dehydrogenase mRNA, complete cds M305 F2 H-U20972 14-3-3 protein, epsilon 28.16 36 M271 D3 H-U2 1049 hypothetical protein 12.65 16 (GB:U2 1049), ESTs, Highly similar to DD96 IjH.sapiens].

M421 G5 H-U21858 Human transcriptional activation 29.15 38.OkDa factor TAF1132 mRNA, complete M424 H3 H-U22662 Human nuclear orphan receptor 49.28 49.OkDa LXR-alpha mRNA, complete cds M271 D2 H-U24074 killer cell inhibitory receptor 37.62 43 [KIR], Homo sapiens natural killer-associated transcript 3 (NKAT3), complete cds.

RECEPTOR ON NATURAL KILLER (NK) CELLS FOR ALLELES. 169-29 H-U24 153 human p21I-activated protein 60 57.82 (Pak2) gene M385 H2 H-U24 166 EBI 29.59 36.OkDa G I H-U24 169 Human JTV- I(JTV-l1) mRNA, 34.43 cds El H-U24576 Human breast tumor autoantigen 18.26 27 complete sequence G4 H-U24577 Human LDL-phospholipase A2 48.62 52 complete cds HI H-U25789 Human ribosomal protein L21 17.71 32 complete cds M416 DlI H-U25849 Human red cell-type low 17.49 28.OkDa molecular weight acid phosphatase (ACP 1) gene, region and M300 A3 H-U263 12 heterochromatin protein H-P1IHs- 19. 143 I_ gamma WO 99/51766 WO 9951766PCT/US99/07270 M4 16 D3 H-U26403 Human receptor tyrosine kmnase 25.19 ligand LERK-7 precursor (EPLG7) mRNA, complete cds M317 E2 H-U27143 human protein kinase C inhibitor- 13.900 17.Okl~a I cDNA__ H-U28249 Human 1 lkd protein mRNA, 12.32 12 complete cds F4 H-U28386 Human nuclear localization 58.3 54 sequence receptor IISRP 1 alpha mRINA, complete cds M423 E3 H-U28694 Chemokine receptor 3 3-9.16 39.OkDa M266 G6 H-U28963 Gps2 36.08 36 M306 D3 H-U30610 CD94 antigen (NKIT-cell C-type 19.8 27 lectin receptor) BI H-U31 116 Human beta-sarcoglycan A3b 35.09 33 complete cds M297 C2 H-U3 1278 mitotic feedback control protein 22.66 31I.OkDa homolog_____ M302 G2 H-U3 1384 guanine nucleotide-binding 8.14 gamma I11 subunit F4 H-U3 1986 Human cartilage-specific 35.97 47 homeodomain protein Cart- I complete cds M390 F3 H-U321 14 caveolin 2 17.93 18.OkDa E4 H-U32324 Human interleukin- I11 receptor 46.53 54 chain mRNA, complete cds Fl H-U32576 Apolipoprotein C-IV 14.08 16 M298 C4 H-U32907 p37NB protein 34.54 39 M300 D3 H-U32944 dynein, light chain 1, cytoplasmic 9.9 M297 DlI H-U32989 tryptophan 2,3-dioxygenase 44.77 166-5 1 H-U33052 "protein kinase PRK2 [human, 110 108.3 DX3 B-cell myeloma cell line, 166-64 H-U33054 "human G protein-coupled 52 63.65 receptor kinase GRK4 mRNA, splice variant" 166-88 H-U33055 "human G protein-coupled 60 60.1 receptor kinase GRK4 mRNA, beta splice variant" 166-76 H-U33056 "human G protein-coupled 58 58.59 receptor kinase GRK4 mRNA, splice variant" A2 H-U34584 17.71 31 169-87 H-U34820 human MAP kinase mRINA 55 46.49 2 15-2 H-U34822 human JNK1I alpha2 protein 55 47.04 kinase (JNK IA2) mRNA____ 169-37 H-U35002 human JNK2 betal protein kinase 50 42.09 ~(JNK2BI1) 169-25 H-U35003 human JNK2 beta2 protein kinase 55 46.71 (JNK2B2) mRNA__ 167-16 H-U35004 human JNKI betal protein kinase 52 42.31 (JNKIBI) M300 B2 H-U3 5048 TSC-22 protein 15.95 2 WO 99/51766 WO 99/1 766PCT/US99/07270 M423 E5 H-U35398 Human G protein-coupled 40.26 48.OkDa receptor mRNA, complete cds A3 H-U35735 Human RACHI (RACH 1) 42.9 78 complete M250 E5 H-U36764 Eukaryotic translation initiation 35.86 36.OkDa factor 3 (eIF-3) p36 subunit, transforming growth factor-beta receptor II interacting protein 1I M270 E4 H-U37283 rnicrofibril-associated 19.14 32 ~glycoprotein-2 (GB:U37283) M426 F3 H-U37352 Protein phosphatase 2A, 56.65 subunit B' alpha- I El H-U37529 Human substance P beta-PPT-A 14.3 22 complete cds M305 H5 H-U3 7547 apoptosis inhibitor 68.09 64 M424 D5 H-U3 8480 Human retinoid X receptor- 51.04 61 .OkDa mRNA, complete cds M270 F4 H-U38810 Human mab-21 cell fatedetermining protein homolog mRNA,____ M467 F6 H-U3 8904 Human zinc finger protein C2H2- 40.48 47.OkDa mRNA, complete cds E2 H-U393 18 Human E2 ubiquitin conjugating 16.28 22 enzyme UbcH5C complete cds 166-75 H-U39657 human MAP kinase kinase 6 40 36.81 (MKK6) mRNA____ M298 E4 H-U39945 human adenylate kinase 2 (adk2) 26.3633 38.OkDa mRNA 166-38 H-U40282 human integrin-linked kinase 55 49.68 (ILK) mRNA____ 169-65 H-U40343 human CDK inhibitor pl19INK4d 18 18. 33 E2 H-U40705 Homo sapiens telomeric repeat 48.4 52 binding factor (TRF I) mRNA, cds 166-50 H-U40989 human tat interactive protein 60 53.09 M266 H6 H-U4 1767 metargidin precursor 89.65 M270 F3 H-U4 1804 Human putative TI/ST2 receptor 25.08 binding protein precursor mRNA, cds H-U42360 Human N33 gene 38.28 38 AlI H-U43368 Vascular endothelial growth 22.88 33

B

M421 G7 H-U43901 Human 37 kD laminin receptor 32.56 58.OkDa ribosome associated protein gene, complete M392 C2 H-U43923 transcription factor SUPTH4 12.98 1 6.OkDa E2 H-U46024 Myotubular myopathy 1 66.44 58 M33 _Al 'H-U46838 pIO5MCM 90.42 97 ?.A7e,C. vIY..tur 11-U4I7I I Human transcription factor E21 I (E2FlI) gene, promoter and 48.18 53.OkDa WO 99/51766 WO 9951766PCT/US99O727O M421 H I H-U48707 Human protein phosphatase-I1 18.92 36.0kDa mRNA, complete cds M302 B7 H-U49070 peptidyl-prolyl isomerase PINI1 18.04 28.OkDa C1 H-U49188 Human placenta (Diff33) mRNA, 54.45 cds M485 H2 H-U49837 Human LIM protein MLP mRNA, 21.45 34.OkDa cds D2 H-U49897 Homo sapiens phenylalanine 49.83 64 hydroxylase (PAH) mRNA, complete cds B2 H-U49957 Human LIM protein (LPP) 67.43 67 mRNA, partial cds 166-16 H-U50196 human adenosine kinase mR!NA 50 38.02 A4 H-U50939 Human aniyloid precursor 58.85 protein-binding protein 1 mRNA, complete cds G3 H-US 1224 Human U2AFBPL gene, complete 52.8 cds M486 E3 H-US 1333 Hexokinase 3 (white cell) 101.64 100.OkDa M305 Dl H-US 1478 ATPase, Na+/K+ transporting, 30.8 36 beta 3 subunit M416 H3 H-1J521 12 Homo sapiens Xq28 genomic 25.96 36.OkDa DNA in the region of the L ICAM locus containing the genes for neural cell adhesion molecule L I (L CAM), arginine-vasopressin receptor (AVPR2), C I p 115 (Cl1), ARD 1 N-acetyltransferase related protein (TE2), renin-binding protein (RbP), host cell factor I (HCF and interleukin- 1 receptor-associated kinase (IRAK) genes, complete cds, and gene M463 El H-U53442 human p38Beta MAP kinase 40.99 49.OkDa G3 H-U53446 Human mitogen-responsive 84.81 98 phosphoprotein DOC-2 mRNA, cds M463 ClI H-U54617 human pyruvate dehydrogenase 45.28 52.OkDa isoform 4 mRNA 169-38 H-U54645 methylmalonyl-coA mutase 38 25.59 M300 H3 H-U56255 t-complex sterility protein 12.54 16 CW- I C4 H-US 6417 Human lysophosphatidic acid 31.24 46 acyltransferase-alpha inRNA, M305 A2 H-U56637 acin-capping protein alpha 31.73 subunit isoform 1 WO 99/51766 WO 9951766PCTIUS99/07270 M235 E6 H-U56814 Human DNase 1 -Like III protein 33.66 (DNASlIL3) mRNA, complete cds, involved in apoptosis Binds specifically to G-ACTIN AND BLOCKS ACTIN

__________POLYMERIZATION.____

H-U57059 31.02 36 B3 H-U57093 Human small GTP-binding 24.09 34 protein rab27b mR!NA, complete D3 H-U57099 Human APEG- I mRNA, 12.54 cds F I H-U58331 Sarcoglycan, delta (35kD 28. 2 7 24 dystrophin-associated ____________glycoprotein) M512 F4 H-U58334 Human Bcl2, p53 binding protein 110.66 108.OkDa Bbp/53BP2 (BBP/53BP2) mRNA, complete cds B3 H-U58516 Human breast epithelial antigen 42.68 BA46 mRNA, complete cds M250 E4 H-U58522 Human huntingtin interacting 22.11 protein (HIP2) mRNA, complete cds; M419 G2 H-U60207 human stress responsive 53.640 63.OkDa serine/threonine protein kinase mRNA M298 B2 H-U60276 arsA homolog (hASNA-I) 36.63 47.OkDa B2 H-U60521 Human protease proMch6 (Mch6) 45.87 52 mRNA, complete cds F3 H-U61 166 Human SH3 domain-containing 57.31 57 protein SH3P 17 mRNA, complete M250 B5 H-U6 1232 cofactor E (tubuim-folding protein), REQUIRED FOR VIABILITY IN THE ABSENCE OF THE KINESIN-RELATED CIN8 H-U62392 Homo sapiens zinc finger protein 43.45 52 complete cds G I H-U62801 Human protease M mR1'NA, 26.95 33 complete cds M266 B I H-U62962 Int-6 Human Int-6 mRNA, 49.06 52.OkDa complete M300 G I H-U63295 seven in absentia homolog 31.13 36 M306 H3 H-U64 198 98 H3 H-U64863 Human hPD-lI (hPD- 1) mRNA, 31.79 37 complete B3 H-U6558 I Human ribosomal protein L3-like 44.88 52 mRNA, complete cds M341 Dl H-U65918 DAZ homologue [DAZLA] 32.56 36.OkDa M302 El H-U65928 Jun activation domain binding 36.85 48.OkDa WO 99/51766 WO 9951766PCT/US99/07270 12 D3 H-U66347 Homo sapiens cAMP 46.97 phosphodiesterase, (PDE4C) mRNA, 4C-426 isoform, M306 F3 H-U66867 ubiquitin-conjugating enzyme E21 1 -7.49 28 M416 E2 H-U681 11 Human protein phosphatase 22.66 37.OkJ~a 2 (PPP1R2) gene F2 H-U68382 Mannosidase, alpha B, lysosomal 35.64 36 G2 H-U69141 Glutaryl-Coenzyme A 48.29 56 B2 H-U70660 Human copper transport protein 7.59 16 HAHI (HAHI) mRNA, complete M297 B2 H-U7 1374 peroxisomal. membrane protein 40.15 M306 A3 H-U75272 progastricsin [PGC] 42.79 49.OkDa A2 H-U75285 Homo sapiens apoptosis inhibitor 15.73 gene, complete cds B2 H-U77456 Human nucleosome assembly 41.36 2 mRNA, complete cds C2 H-U78294 Homo sapiens I 5S-lipoxygenase 74.47 74 complete cds F6 H-U78302 Human 2,4-dienoyl-CoA 36.96 M478 G3 H-U78798 Human TNF receptor associated 57.53 factor 6 (TRAF6) mRNA, cds G3 H-U80982 Human myeloid-specific C/EBP- 27.5 51 epsilon transcription factor gene, complete cds M468 B7 H-U82256 Homo sapiens arginase type 11 39.05 mRNA, complete cds M465 B2 H-U82812 Human scavenger receptor 38.28 48.OkDa cysteine rich Sp alpha mRNA, cds M484 D7 H-U83410 Human CUL-2 (cul-2) mRNA, 82.06 cds M467 E6 H-U83460 Human high-affinity copper 21.01 32.OkDa uptake protein (hCTRI) mRNA, cds D2 H-U84763 Homo sapiens UCP3 mRNA, 34.43 42 complete cds B2 H-U86070 Homo sapiens 28.93 36 phosphomannomutase mRNA, cds C2 H-U9044 I Human prolyl 4-hydroxylase 58.96 64 alpha (11) subunit mRNA, cds B2 H-U90543 Human butyrophilin (BTFI) 58.08 54 complete H2 H-U90545 Human sodium phosphate 44.22 36 transporter (NPT4) mRNA, cds WO 99/51766 WO 9951766PCTIUS99/07270 G2 H-U90552 Human butyrophilin (BTF5) 56.54 48 complete cds C3 H-U9 1521 Peroxisomal biogenesis factor 12 39.6 48 HI H-U9 1641 Human alpha2,8-sialyltransferase 41.47 complete cds CI H-U93869 Human RNA polymerase 111 34.98 36 subunit (RPC39) mRNA, F2 H-U94346 Human calpain-like protease 70.4 mRNA, complete cds C2 H-U94855 Human translation initiation 39.38 36 factor 3 47 kDa subunit mRNA, cds M27 1 F7 H-U95089 Epidermnal growth factor receptor. 44.66 47 M424 A5 H-U95847 Human GDNF receptor alpha 50.71 52.OkDa mRNA, complete cds D2 H-U96094 Human sarcolipin (SLN) mRNA, 3.52 cds B3 H-U96769 Homo sapiens chondroadherin 39.6 43 gene, 5'flanking region and M298 G2 H-VO0566 prolactin 25.08 M298 H2 H-V0057 1 corticotropin-releasing factor 21.67 49 217-61 H-V00572 phosphoglycerate kinase 1 50 45.94 M314 B3 H-V00597 parathyroid hormone 12.76 14 M305 B8 H-XO0 129 retmnol-binding protein 4, 22 51 F2 H-X00351 Human mRNA for beta-actin 41.36 41 A4 H-X00570 apolipoprotein C-I 9.24 M362 ElI H-XO 1057 interleukin 2 receptor, alpha 30.03 A4 H-X0 1677P Human liver mRNA for 10.45 glyceraldehyde-3 -phosphate dehydrogenase (G3PD, EC 12) M271 D6 H-X02 152 lactate dehydrogenase A [LDHA], 36.63

L-LACTATE

_________DEHYDROGENASE M CHAIN Al1 H-X02 158 Human gene for erythropoietin 21.34 32 H4 H-X02415 Human gene for fibrinogen 48.18 chain_____ H-X02750 Protein C (inactivator of 50.82 53 factors Va and VIlla) M302 B3 H-X02751I proto-oncogene N-ras 20.9 D3 H-X02812 Human mRNA for transforming 43.12 factor-beta (TGF-beta) M302 CI H-X03 124 tissue inhibitor of 22.88 36.OkDa _______________metalloproteinase M362 Bl1 H-X03342 ribosomal protein L32 14.96 24.OkDa M235 A2 H-X03484 human mRNA for raf oncogene 7 1.350 73.OkDa M318 A3 H-X03557 interferon-induced protein 56 52.69 A3 H-X03747 ATPase, Na+/K+ transporting, 33.44 1 polypeptide WO 99/51766 WO 9951766PCT/US99/07270 M305 D2 H-X04297 ATPase, Na+IK+ transporting, 112.64 99 subunit M305 A5 H-X04327 2,3-bisphosphaglycerate mutase 28.6 36 M271 G5 H-X04588 tropomyosin TM3Onm, 26.29 40.0kDa M305 C8 H-X0474 1 ubiquitin related protein 23.43 28.0kDa M236 A5 H-X0523 1 matrix metalloproteinase 1 51.7 53.OkDa (interstitial collagenase) [MMP 1],

COLLAGENS

166-53 H-XO 5246 "phosphoglycerate kinase, testis 50 45.94 M236 A] H-X05908 annexin I, REGULATES 38.17 PHOSPHOLIPASE A2 ACTIVITY, Binds CALCIUM M250 A4 H-XG6234 SI100 calcium-binding protein A8 10.34 A) M266 B6 H-X06323 ribosomal. protein L3, isoform 1 38.39 39 M313 A7 H-X06617 ribosomal protein SI 1 17.49 27 M416 E4 H-X06948 High affinity IgE receptor alpha- 28.38 36.OkDa M42 1 H7 H-X07203 Human mRNA for CD20 receptor 32.78 217-2 H-X07743 pleckstrin 38 38.57 2 17-73 H-X07767 "cAMP-dependent protein kinase, 45 38.68 subunit" M305 B3 H-X07898 troponin C, skeletal, fast 17.71 M306 El H-X07979 integrn, betal1 87.89 110 AllI H-X08004 ras-related protein rapiB 20.24 38 M235 A7 H-X12387 Cytochrome P450 111A3 55.44 oxidase chain 3) M315 F1 H-X12496 glycophorin C 14.19 24 M316 D3 H-X12517 small nuclear ribonucleoprotein 17.6 C M236 ES H-X 12534 guanine nucleotide-binding 20.24 34.OkDa rap2, ras-oncogene related M266 E3 H-X 12597 High-mobility group (nonhistone 23.76 37 chromosomal) protein 1, placenta 2 17-14 H-X12656 human mRNA for protein 40 34.06 ___________phosphatase 2A (beta type) H4 H-X12662 H.sapiens arginase gene exon 1 35.53 and flanking regions (EC 3.5.3. 1) (and joined CDS) CI H-X 12953 RAB2, member RAS oncogene 23.43 2 family H-X 13956 Human 12S RNA induced by 9.13 19 poly(rI), poly(rC) and Newcastle disease virus M297 Al1 H-X 15005 laminin receptor 1 33.11 48.OkDa M315 E3 H-X15088 guanine nucleotide binding 38.61 protein (G protein), alpha transducing (transducin) activity 1 WO 99/51766 WO 9951766PCT[US99/07270 G2 H-X 15183 Human mRNA for 90-kDa heat- 80.63 protein_____ M385 CI H-X 15422 mannose-binding lectin, soluble 27.39 27.0kDa defect) [MBL] M271 D7 H-X 15606 INTERCELLULAR ADHESION 30.36 37.OkDa MOLECULE-2 PRECURSOR sapiens]. M298 C5 H-X 15653 uracil-DNA glycosylase 33.55 37 M302 B4 H-X 15 822 cytochrome-c oxidase, VIla 9.24 liver M305 A6 H-X 15940 ribosomal protein L3 1 13.86 18 M236 G5 H-X 15949 interferon regulatory factor 2 38.5 54.OkDa BINDS AND REPRESSES REGULATORY REGION OF TYPE I IFN AND IFN- INDUCIBLE MHC CLASS I M236 C2 H-X 16064 translationally-controlled. tumor 19.03 M512 B5 H-X16323 Hepatocyte growth factor 80.19 100.OkDa A) M3 15 C3 H-X 16461 cell division cycle 2, G 1 to S and 32.78 to M M297 G2 H-X 16832 cathepsin H 36.96 M271 BI H-X 16983 integrin, alpha 4 (CD49D, alpha 4 114.29 114 subunit of VLA-4 receptor) [ITGA4], IMPORTANT FOR CELL-CELL ADHESION M270 A7 H-X 17025 plasminogen activator-inducible 25.19 34 c54, Human homolog of yeast IPP M302 C3 H-X17042 proteoglycan 1, secretory granule 17.49 26 B I H-X 17206 ribosomal protein S2 24.42 B4 H-X 17254 Transcription factor Eryfi 45.54 53 M311I H2 H-X 17610 beta-1I-glycoprotein, pregnancy- 46.97 48.OkDa (GB:X 176 10) M315 Dl1 H-X17644 G I to S phase transition protein 55 M340 G 1 H-X5 1415 lipase, hormone-sensitive [LIPE] 84.59 98.OkDa M464 A7 H-XS 1688 Cyclin A 47.63 47.0kDa M3 13 G 1 H-XS 1745 major histocompatibility complex, 40.26 class I, A M297 A2 H-XS 1804 putative receptor protein PMI 21.23 D4 H-X5 1952 Human UCP gene for uncoupling 33.88 37 exons I and 2 M300 B I H-X5201 I muscle determining factor 26.73 39 M419 G01 H-X52479 "protein kinase c, alpha type" 82.28 A2 H-X52486 Uracil-DNA glycosylase 35.97 36 E3 H-X52520 Tyrosine aminotransferase 50.05 58 BI1 H-X5263 8 6-phosphofructo-2- 51.92 47 kinase/fructose-2,6- WO 99/51766 WO 99/1 766PCTIUS99/07270 M509 C4 H-X52730 Human gene for 31.13 35.0 kDa phenylethanolammie N-methylase (PNMT) (EC M235 C5 H-X52839 ribosomal protein L17 15.51 18 M426 C2 H-X52943 Human mRNA for ATh-a 53.24 64.OkDa transcription factor M266 G5 H-X53 777 ribosomal protein L23 20.35 31 B4 H-X53961 Lactotransferrin 78.32 78 M462 C6 H-X54150 Fc fragment of IgA, receptor for 31.68 37.OkDa M302 A6 H-X54304 myosin, light polypeptide 2, 18.92 32.OkDa regulatory M311I G2 H-X54802 cytochrome-c oxidase, IV subunit 18.7 23.OkDa M270 H3 H-X54871 guanine nucleotide-binding 23.76 33.OkDa protein Rab5B, ras-oncogene related [RAB5B], PROTEIN TRANSPORT. PROBABLY INVOLVED IN VESICULAR

(BYSIMILARITY).____

M313 B6 H-X54936 placenta growth factor [PLGF*] 16.5 22.OkDa M496 B2 H-X55079 Human lysosomal alpha- 104.83 98.OkDa gene exon 1 DI H-X55330 Aspartylglucosanunidase 38.17 36 El H-X55448 H.sapiens G6PD gene for 25.41 glucose-6-phosphate M421 G6 H-X56253 Human MPR46 gene for 46kd 30. 58 52.OkDa 6-phosphate receptor 169-89 H-X56468 14-3-3 protein tau 34 27.02 M300 B4 H-X5 6549 fatty-acid-binding protein, muscle 14.74 17 M298 D2 H-X56740 guanine nucleotide-binding 23.87 31 .OkDa rablI RABI M266 E5 H-X56932 highly basic protein, 23 kDa 22.44 M318 G I H-X57025 insulin-like growth factor 1 16.94 18 M305 F5 H-X57348 protein kinase C inhibitor 27.39 M236 D6 H-X5735 1 interferon-induced protein 1-81D 14.63 24 H3 H-X57352 interferon-induced protein 1-8U 14.74 38 M305 B6 Hi-X58079 S-100 protein, alpha chain 10.45 11 E6 H-X59131 H.sapiens D13S106 mRNA for a 34.76 highly charged amino acid M248 H5 H-X59268 transcription factor IIB [TCF2B*I 34.87 49 E2 H-X59357 Epstein-Barr virus small RNA- 14.19 36 protein M236 D4 H-X59417 macropain, iota subunit, THE 27.17 36 INTERACTION OF CALPONIN WITH ACTIN INHIBITS ACTOMYOSIN MG-ATPASE M271I H4 H-X5961 8 ribonucleotide reductase, small 42.9 46 subunitII WO 99/51766 WO 9951766PCT/US99/07270 M250 G3 H-X597 10 CAAT-box DNA-binding protein, 22.66 34 subunit B, CCAAT-BINDING TRANSCRIPTION FACTOR ~SUBUNIT A [Homo sapiens]) M423 E2 H-X597 11 Nuclear transcription factor Y, 38.28 48.OkDa alpha M71 C7 H-X59798 Cyclin DI (PRADI; parathyroid 32.56 adenomatosis ESSENTIAL FOR THE CONTROL OF THE CELL CYCLE AT THE GI/S

TRANSITION._

M270 H5 H-X59834 calmodulin 41.14 53.OkDa M416 D5 H-X59871 Transcription factor 7 (T-cell 29.59 36.OkDa M485 D6 H-X60036 Phosphate carrier, mitochondrial 39.82 37.OkDa M250 D4 H-X60489 translation elongation factor 1, 24.86 33.OkDa H-X60592 Human CDw4O mRNA for nerve 30.58 46 growth factor receptor-related Bactivation molecule M312 F3 H-X61587 ras-related rhoG 21.12 21.OkDa F9 H-X6 1622 cyclin-dependent kinase 2 32.89 56 M3 13 E3 H-X6 1970 macropain, zeta subunit 26.62 M428 DlI H-X62055 tyrosine phosphatase, non- 65.78 66.OkDa type 6 M248 C4 H-X62534 high mobility group protein 2, 23.1 37 BINDS PREFERENTIALLY SINGLE-STRANDED DNA AND UNWINDS DOUBLE STRANDED DNA.__ M305 F3 H-X62753 folate-binding protein 28.38 36 M476 G2 H-X63468 H.sapiens mRNA for transcription 48.4 53.OkDa TFIIE alpha G6 H-X63469 General transcription factor TFIIE 32.12 56 subunit, 34 kD G4 H-X63522 H.sapiens mRNA DAUD16 for 58.74 54 acid X receptor b M3 16 G2 H-X63526 translation elongation factor 1, 48.18 52.OkDa gamma M305 C5 H-X63527 ribosomal protein L19 21.67 33 E2 H-X63629 Cadherin 3 (P-cadherin) 91.3 110 D4 H-X64037-2 General transcription factor IIF, 56.98 64 1 (74kD subunit) M302 C6 H-X64559 tetranectin 22.33 32.OkDa M271 H I H-X64728 choroideremnia-like [CHML], 72.27 98 CHL~ mRNA M270 ElI H-X648 10 proprotein convertase 82.94 subtilisinlkexin type 1 [PCSK 11, INVOLVED IN PROCESSING OF HORMONE AND OTHER

PRECURSORS____

M3 11 F4 H-X64877 complement factor H-related 29.81 36.OkDa I I_ protein

I_

WO 99/51766 WO 99/1 766PCT[US99/07270 M388 DI H-X65293 protein kinase C, epsilon 81.18 96.OkDa

[PRKCE]

H-X65873 kinesin, heavy polypeptide 106.04 34 F4 H-X66079 Spi-B transcription factor (Spi- 28.93 54 1/PU. 1 related) F3 H-X661 14 2-oxoglutarate carrier protein 0 37 [OGMT*] M305 C6 H-X66 141 myosin, light polypeptide 2, 18.37 31 ventricular M419 H I H-X66357 cell division protein kinase 3 33.620 44.OkDa 166-13 H-X66358 serine/threonine-protein kinase 45 39.45 KKIALRE__ 166-25 H-X66360 serine/threonine-protein kinase 60 57.60 PCTAIRE-2 M4 19 A2 H-X66363 serine/threonine-protein kinase 54.600 64.OkDa PCTAIRE-1I____ 166-37 H-X66364 H.sapiens mRNA PSSALRE for 38 32.19 sermne/threonine protein kinase M419 B2 H-X66365 cell division protein kinase 6 35.900 46.OkDa H3 H-X66839 H.sapiens MaTu MN mRNA for 50.6 54 p54/58N protein______ M266 G3 H-X67325 interferon, alpha-inducible gene 13.53 13 p27 M462 H7 H-X67594 Melanocortin I receptor (alpha 34.98 44.OkDa melanocyte stimulating hormone receptor)_______ M236 C5 H-X6795 1 Proliferation-associated gene A 2234 (natural killer-enhancing factor

PAGA____

H3 H-X68486 Adenosine receptor A2 45.43 M429 E3 H-X68561 Sp 4 transcription factor 86.35 86.OkDa M430 F2 H-X69151 ATP synthase, H+ transporting, 42.13 58.OkDa subunit C, vacuolar M236 C3 H-X69392 ribosomal protein L26 16.06 29 B3 H-X69532 H.sapiens gene for inter-alpha- 100.32 98 trypsin inhibitor heavy chain HI, exons 1-3 M236 F5 H-X69654 ribosomal protein S26 12.76 18 M42 1 GB H-X702 18 Protein phosphatase 4 (formerly 33.88 catalytic subunit M266 H5 H-X70848 protein phosphatase 1, alpha 36.41 37 subunit El H-X70940 Eukar-yotic translation elongation 51.04 1 alpha 2 M270 FlI H-X72215 [PIT I, POU domain, class 1, 32.12 transcription factor 1 (Pit 1, growth hormone factor 1) M271 A7 H-X72760 Laminin, beta 2 (lanunin S- 67.87 LAMININ IS A LAMININ-LIKE ADHESIVE PROTEIN CONCENTRATED IN THE SYNAPTIC CLEFT OF THE

NEUROMUSCULAR

WO 99/51766 WO 9951766PCT/US99/07270 M235 B I H-X7284 I Human retinoblastoma-bindmng 46.86 52.OkDa.

protein (RbAp46) mRNA, complete cds, IEF 7442 (GB:X7284 1) 217-25 H-X73428 DNA-binding protein inhibitor 20 17.08 M305 B5 H-X73459 signal recognition particle, 15.07 14 M250 D6 H-X73460 ribosomal protein L3, isoform 2, 44.44 COMPONENT OF THE LARGE SUBUNIT OF CYTOPLASMIC

__________RIBOSOMES

M462 D8 H-X74008 Protein phosphatase 1, catalytic 35.64 46.OkDa gamma isoform M266 G2 H-X74 104 Signal sequence receptor, beta; 20.24 27 translocon-associated protein, beta subunit M266 E7 H-X74262 retinoblastomna binding protein 46.86 RbAp48 Hi H-X74330 DNA primase polypeptide 1 46.31 51 (49kD) M313 F3 H-X74570 gal beta G~cNAc alpha- 36.3 46.OkDa.

2,3 sialyltransferase (GB:X74570)______ M429 H3 H-X74764 H.sapiens mRNA for receptor 94.120 98.OkDa.

protein tyrosine kinase M27 1 E6 H-X75042 V-rel avian reticuloendotheliosis 68.2 88 viral oncogene homolog M305 G2 H-X75252 phosphatidylethanolamine- 20.68 binding protein M302 G 1 H-X75593 guanine nucleotide-binding 22.44 32.OkDa protein rabl13______ 166-49 H-X75958 H.sapiens trkB mRNA for 55 52.54 ______________protein-tyrosine kinase C4 H-X76013 H.sapiens QRSHs mRNA for 85.36 _____________glutaminyl-tRNA synthetase A2 H-X76029 H.sapiens mRNA for neuromedin 19.25 M305 D5 H-X76228 ATP synthase, H+ transporting, 24.97 36 E, vacuolar M298 F6 H-X76648 glutaredoxin 11.77 11 .OkDa.

M31 1 A4 H-X76717 metallothionein 11 6.82 14 C4 H-X77533 H.sapiens mRNA for activin type 56.43 61 receptor H2 H-X77548 H. sapiens cDNA for RFG 67.65 67 169-4 1 H-X77743 H.sapiens CDK activating kinase 45 38.1 3 A4 H-X77909 H.sapiens IKBL mRNA 42.02 52 M305 C1 H-X78 136 heterogeneous nuclear 40.26 _____________ribonucleoprotein. E2 M306 G2 H-X78416 casein, alpha [CSN 1] 20.46 33 M271 C2 H-X78678 ketohexokinase (fructokinase) 32.89 39 [KHKI, H-sapiens KHK mRNA for ketohexokinase, clone WO 99/51766 WO 9951766PCTIUS99/07270 M305 D4 H-X79193 cyclin-dependent kinase 7 38.17 (homnolog of Xenopus MO 15 cdkkinase) [CDK7] M43 1 F2 H-X7 9389 glutathione S-transferase TI 26.51 34.OkDa M298 C6 H-X79537 glycogenin 30.8 34.OkDa M440 ClI H-X79865 H.sapiens Mrpl17 mnRNA 21.89 3 1.OkDa M298 F5 H-X80229 protein kinase PKN 52.8 64.OkDa 167-39 H-X80230 H.sapiens mRNA (clone C-2k) 42 40.99 mRYA for serine/threonine kinase 217-49 H-X80343 H.sapiens p35 mRNA for 40 33.84 subunit of cdk5 kinase M270 D7 H-X80695 cytochrome oxidase-assembly 47.96 protein, OXAlI, H.sapiens mRN~A M266 B5 H-X80909 nascent polypeptide-associate 23.76 37.OkDa alpha M416 D9 H-X809 10 Protein phosphatase 1, catalytic 36.08 beta isoform.

E2 H-X81 198 Archain 52.03 63 169-6 H-X81817 H.sapiens BAP31 mRNA 32 27.1 3 E4 H-X82018 H.sapiens mRNA for ZID protein 46.75 57 M313 D7 H-X82456 MLN50 28.82 33 A2 H-X82629 H.sapiens mRINA for Mox-2 33.44 42 M236 DI H-X83006 lipocalin, neutrophil gelatinase 21.89 34.OkDa associated 166-40 H-X83 107 H.sapiens Bmx mRNA for 75 74.32 cytoplasmic tyrosine kinase E3 H-X83425 H.sapiens LU gene for Lutheran 69.19 59 group glycoprotein C6 H-X83703 H.sapiens mRNA for cytokine 35.2 54 inducible nuclear protein M416 H2 H-X83928 H.sapiens mRNA for transcription 23.32 33.OkDa TMID subunit TAFI128 166-17 H-X85106 H.sapiens mRNA for ribosomal 90 80.70 S6 kinase_____ 166-39 H-X85337 H.sapiens mRNA for myosin light 110 109.0 kinase D2 H-X85750 H.sapiens mRINA for transcript 26.29 associated with monocyte to macrophage differentiation______ M266 E6 H-X87 176 1 7-beta-hydroxysteroid 81.07 type 4 M297 F2 H-X87689 CLCP 23.21 33.OkDa M300 A2 H-X87843 cyclin H assembly factor 34.1 47 M271 E3 H-X89750 homeotic protein, TGIF, 30.03 32.OkDa H.sapiens mRNA for TGIF M235 GI H-X90529 guanine nucleotide-binding 34.54 protein ragA [RAGA] M302 E6 H-X90583 translocon-associated protein, 19.14 28.OkDa M306 G I H-X90872 gp2512 23.65 33 WO 99/51766 WO 9951766PCTIUJS99/07270 M4 16 D2 H-X9 1504 Transcription factor COUP 2 22.22 32.0kDa M250 B3 H-X92098 transmembrane protein rnp24 22.22 M271 G7 H-X92106 bleomycin hydrolase. 50.16 PROTECTING NORMAL AND MALIGNANT CELLS FROM TOXICITY. F3 H-X92715 Zinc finger protein 74 (Cos52) 63.03 47 M270 H6 H-X92720 H.sapiens mRNA for 70.51 71 phosphoenolpyruvate H-X92762 H.sapiens mRNA for tafazzins 32.23 37 M298 D3 H-X93036 MAT-8 9.68 16.OkDa M476 A5 H-X93595 H.sapiens mRNA for NK receptor 50.16 56.OkDa (clone 17.I1C) M417 D2 H-X93920 protein tyrosine phosphatase 4 1.980 48.OkDa H-X95592 H.sapiens mRNA for CID protein 15.62 28 M298 B4 H-X95648 translation initiation factor 213, 33.66 34.OkDa.

alpha subunit______ F3 H-X95735 H.sapiens mRNA for zyxin 2 63.03 72 M386 B3I H-X96752 L-3-hydroxyacyl-CoA 34.65 dehydrogenase, SCHAD gene M422 B6 H-X97229 H.sapiens mRNA for NK 41.58 48.OkDa clone library 15.212 B3 H-X98 173 H.sapiens mRNA for MACH- 51.15 51 protein______ 166-14 H-X99325 H.sapiens; mRNA for Ste2O-like 55 46.9 3 C4 H-X99459 H.sapiens mRNA for sigma 3B 21.34 M424 C4 H-Y00291I Human hap mRNA encoding a 49.39 59.OkDa DNA-binding hormone receptor M386 HI H-Y00345 polyadenylate-binding protein 69.74 M469 A2 H-Y00630 Plasmninogen activator inhibitor, 45.76 46.OkDa type II (arginine-serpin) M305 El H-Y0071 1 lactate dehydrogenase B 36.85 38.OkDa H2 H-Y00764 ubiquinol/cytocbromne c reductase 10.12 33 ~~hinge H-Y07848 H.sapiens EWS, gar22, rrp22 and 36.3 bam22 genes______ M305 G6 H-ZI 1559 iron-responsive element binding 97.9 98 protein 1 M250 F3 H-ZI 11566 Pr22 protein STATLIMIN 16.5 22.OkDa.

[Homo sapiens], SERVES AS RELAY (VIA PHOSPHORYLATION) FOR DIVERSE SECOND PATHWAYS 169-73 H-Z 1695 H.sapiens 40 kDa protein kinase 50 38.35 to rat ERK2 M475 C8 H-Z 1173 7 Flavin-containing 61.49 _____________monooxygenase 4 WO 99/51766 WO 9951766PCT/US99/07270 Cl H-Z 11898 Octamer binding protein 3 39.71 M266 H4 H-Z12830 SSR, alpha subunit 31.57 42.OkDa A3 H-Z 14000 Ring finger protein 1 41.58 M300 El H-Z 14978 actin-related protein 41.47 49 G 1 H-Z 19002 H.sapiens of PLZF gene encoding 74.14 84 zinc finger protein_____ H I H-Z2 1966 POU homeobox protein 33.22 43 M248 G3 H-Z23139 CLASS II 29.04 34

HISTOCOMPATIBILITY

ANTIGEN, M BETA CHAIN PRECURSOR [Homo sapiens] D3 H-Z26876 ribosomal protein L38 7.81 F2 H-Z28339 H.sapiens mRNA for delta 4-3- 35.97 43 5 beta-reductase M298 B3 H-Z28407 ribosomal protein L8 28.38 39.OkDa M3 13 C3 H-Z29330 ubiquitin-conjugating enzyme 20.24 34 UbcH2, 23 M271 F3 H-Z29677 guanine nucleotide-binding 20.35 28.OkDa protein, ras-related M465 C2 H-Z30425 H-sapiens mRNA for orphan 38.39 34.Okl~a nuclear hormone receptor______ M302 F5 H-Z3 1357 cysteine dioxygenase 22.11 31I.OkDa M340 Cl H-Z3 1695 inositol polyphosphate 5- 40.04 49.OkDa phosphatase, 43 kDa E3 H-Z32564-2 H.sapiens FRGAMMA mRNA 26.84 36 9bp) for folate receptor______ M236 HI H-Z35227 small G protein, TTF, RAS- 21.12 PROTEIN RACI AIO H-Z35491 H.sapiens mRNA for novel 30.25 glucocorticoid receptor-associated protein______ M440 G5 H-Z37986 H.sapiens mRNA for 25.41 28.OkDa phenylallcylaniine binding protein M297 E2 H-Z47087 cyclin Alcyclin-dependent kinase 18.04 2-associated p19 F 1 H-Z4805 1 H.sapiens gene for myelin 27.28 31 oligodendrocyte glycoprotein (MOG) A2 H-Z48475 Glucokinase regulator 68.86 M302 E4 H-Z48570 sperm zona pellucida-binding 16.72 24 protein M266 A2 H-Z68907 Human clone ID 193225 NAD 43.34 45.0kDa (H)-specific isocitrate dehydrogenase gamma subunit mRNA, alternatively spliced, cds G I H-Z83850 Human DNA sequence from PAC 45.76 82J1 1 and cosmid U 134E6 on chromosome Xq22. Contains NIK like and Thyroxin-binding globulin precursor (T4-binding globulin, TBG) genes, ESTs and WO 99/51766 PCT/US99/07270 H4 H-Z97171 Homo sapiens GLC1A (trabecular 55.55 meshwork induced glucocortcoid response) gene, exon I, joined

CDS

M421 D5 H-Z97632 Human DNA sequence from PAC 28.49 38.0kDa 196E23 on chromosome Xq26.1- 27.2. Contains the TAT-SFI (HIV-1 transcriptional elongation factor TAT cofactor TAT-SFI) gene, the BRS3 (Bombesin Receptor subtype-3 (Uterine Bombesin Receptor, BRS-3) gene, an unknown gene coding for two isoforms, a predicted CpG island, ESTs and STSs Example 3 Construction of Expression Plasmids The following example illustrates the construction of the expression vectors used in the Examples above. Similar modifications can be made in other vectors for use in creating libraries of expressible gene sequences.

The vector pcDNA3.1/V5-His was obtained from Invitrogen (cat #V810-20) and modified slightly so that it carried an gene sequence for ZeocinTM resistance and lacked the multiple cloning site. A 100gg aliquot was suspended in 200 pl medical irrigation (MI) water. A 5pl aliquot was saved for gel analysis. The remainder was transferred to a 1.7 ml Eppendorftube. The vector was digested with HindIII (400 U) using Promega Buffer E (final volume 400 pl). The reaction ran 3 hours at 370 C.

An aliquot was checked for completeness of digestion by running on an 0.8% agarose gel in 1X TAE, and visualizing with ethidium bromide.

The digested vector was treated with 200 gl phenol/chloroform according to standard procedures, and the DNA precipitated from the aqueous phase using 1/10 volume 3M NaOAc and 2 volumes 100% EtOH at room temperature, followed by washing with 80% EtOH. The pellet was resuspended in 100 gl MI water.

WO 99/51766 PCT/US99/07270 86 Two oligonucleotides were added to the resuspended DNA (Topo -H (40 pg) (SEQ ID NO:3), Topo-4 (12 g) (P)AGGGCG (SEQ ID plus 17 ull 10X Promega T4 Ligase buffer. The tube was placed on ice and the volume increased to 170 ul with MI water. The oligos were ligated to the vector using 20U Promega T4 DNA ligase, incubated at 120 C overnight.

The vector was treated with 100 l1 phenol/chloroform and the aqueous phase precipitated as described above. The pelleted DNA was resuspended in 150 pl of sterile water the redigested with HindIII (17 pl Promega Buffer E, 200 U HindIII 37° C, 1 hour). The redigested DNA was re-extracted with phenol/chloroform and precipitated with 1/10 volume 3M NaOAc and 7/10 volume isopropanol, then washed with 80% EtOH.

The pelleted DNA was resuspended in 82 pl TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA, pH A 2 gl aliquot was used to check the foregoing procedure using agarose gel electrophoresis as described above. The remaining 80 pl was transferred to a Falcon tube and mixed with 16 ug Topo-5 oligonucleotide (P)CAACACTATCGGAATA (SEQ ID NO:5). To this mixture was added 190 pl NEB Restriction Buffer #1 (room temperature). The total reaction mixture was adjusted to 1.9 mis with MI water. Vaccinia Topoisomerase I enzyme was added ig) and the reaction tube placed in a 370 C water bath for 15 minutes.

After 15 minutes, 200 pl of room temperature Topo- 1OX stop buffer was added (100 mM Tris 7.4, 110 mM EDTA, bromophenol blue). The entire volume was loaded onto an agarose gel (1.2 gr agarose/ 130 mis IX TAE) and run at 70 volts until the bromophenol blue dye had run down about 1/2 in (volume in the loading well was kept constant by the addition of 1X TE). The voltage was reversed for 90 seconds.

The contents of the loading well were transferred to a 15 ml Falcon tube and placed on ice. 2 mis of cold Topo-2X Wash Buffer (60 mM Tris 7.4, 1 mM EDTA, 4 mM dithiothreitol (DTT), 200 gg/ml bovine serum albumin (BSA)) was added and the WO 99/51766 PCT/US99/07270 87 volume then adjusted to 4 mis with cold Topo-1X Enzyme Dilution Buffer glycerol, 50 mM Tris 7.4, 1 mM EDTA, 2 mM DTT, 0.1% Triton X-100, 100 pg/ml BSA) plus 4 mis Topo-Glycerol mix (90% glycerol, 10% 50 mM TE pH 7.4, 0.1% Triton X-100) and stored until needed.

A similar procedure was used to make Topo-adapted pYES2 (Invitrogen cat V825-20).

While the foregoing has been presented with reference to particular embodiments of the invention, it will be appreciated by those skilled in the art that changes in these embodiments may be made without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims.

Claims (36)

1. A method for producing a library of expressible open reading frames, the method comprising: a) amplifying deoxyribonucleic acid (DNA) molecules comprising a plurality of open reading frames (ORFs) using a primer pair, wherein the primer pair comprises a primer, which comprises a nucleotide sequence starting 5'-CACCATG, thereby producing a plurality of amplified ORFs; 1o b) inserting amplified ORFs of the plurality into an expression vector, thereby producing expression vectors comprising the amplified ORFs; and c) verifying the size and orientation of the amplified ORFs in the expression vectors, thereby producing a library of expressible ORFs.

2. The method of claim 1, wherein the primer pair further comprises a 3' primer, which causes the amplification product to end at the third position of a codon immediately preceding a stop codon of an ORF being amplified.

3. The method of claim 2, wherein the 3' primer further causes the amplification product to comprise a 3' terminal adenine residue.

4. The method of claim 2, further comprising transforming cells with the expression vectors comprising the amplified ORFs, thereby obtaining a library of transformed cells containing the expression vectors.

5. The method of claim 2, further comprising purifying the amplified ORFs prior to inserting the amplified ORFs into the expression vector.

6. The method of claim 5, wherein purifying the amplified ORFs is performed 25 using column chromatography or gel electrophoresis.

7. The method of claim 5, wherein purifying the amplified ORFs is performed using agarose gel electrophoresis.

8. The method of claim 7, wherein the agarose is low melt agarose.

9. The method of claim 1, wherein amplified ORFs of the plurality encode full 30 length proteins. The method of claim 1, wherein inserting the amplified ORFs into the expression vector is performed using an enzyme that cleaves and ligates DNA.

11. The method of claim 10, wherein the enzyme is a type I topoisomerase or a O S 7 ite-specific recombinase. [I:\DayLib\LIBUU)52780.doc:MCC 89

12. The method of claim 10, wherein the enzyme is a vaccinia DNA topoisomerase, a lambda integrase, an FLP recombinase, or a P1-Cre protein.

13. The method of claim 10, wherein the enzyme is a vaccinia DNA topoisomerase.

14. The method of claim 1, wherein the expression vector is a eukaryotic expression vector. The method of claim 1, wherein the expression vector is a prokaryotic expression vector.

16. The method of claim 1, wherein the expression vector is suitable for prokaryotic expression and eukaryotic expression.

17. The method of claim 1, wherein verifying the size and orientation of the ORF is performed using a polymerase chain reaction protocol.

18. The method of claim 4, wherein verifying the size and orientation of the ORF is performed using whole cell lysates of transformed cells containing the expression vectors.

19. The method of claim 1, wherein the DNA molecules comprise prokaryotic DNA or eukaryotic DNA. The method of claim 19, wherein the eukaryotic DNA is obtained from yeast cells or mammalian cells.

21. The method of claim 1, wherein the amplified ORFs of the plurality encode members of a family of proteins.

22. The method of claim 21, wherein the members of the family of proteins are human proteins. S. 23. The method of claim 21, wherein the members of the family of proteins 25 comprises members of a family of kinases, phosphatases, transcription factors, oncogenes, or tumor suppressors.

24. The method of claim 1, which is performed in a high throughput format.

25. The method of claim 1, which is performed in a multiwell microtiter plate.

26. The method of claim 4, further comprising verifying the size and orientation 30 of the ORF in the expression vector in the transformed cells.

27. The method of claim 4, wherein the transformed cells are eukaryotic cells or prokaryotic cells.

28. The method of claim 4, wherein the transformed cells are bacteria, yeast, fungi, insect cells, mammalian cells, or plant cells. [I:\DayLib\LIBUU]52780.doc:MCC

29. The method of claim 4, wherein the transformed cells are Chinese hamster ovary cells or Saccharomyces cerevisiae cells. The method of claim 1, wherein the expression vector comprises a nucleotide sequence encoding an affinity purification tag or an epitope tag, and wherein the s expressible ORF and the nucleotide sequence encode a fusion protein comprising a polypeptide encoded by the ORF and the tag.

31. A library of expressible ORFs when obtained according to the method any one of claims 1 to

32. The library of claim 31, wherein the ORFs encode yeast proteins or human to proteins.

33. A method for producing a library of selected expressible open reading frames (ORFs), the method comprising: a) amplifying deoxyribonucleic acid (DNA) molecules comprising a plurality of ORFs using a primer pair, wherein the primer pair comprises a 5' primer, which comprises a nucleotide sequence starting 5'-CACCATG, and a 3' primer, which causes the amplification product to end just prior to a stop codon, thereby producing a plurality of amplified ORFs; b) purifying amplified ORFs of the plurality, thereby obtaining purified amplified ORFs; c) inserting the purified amplified ORFs into expression vectors using a vaccinia DNA topoisomerase, thereby producing expression vectors comprising the amplified ORFs; d) transforming cells with the expression vectors comprising the amplified ORFs; and 25 e) selecting transformed cells containing expression vectors comprising ORFs in an orientation for expression of a polypeptide encoded by the ORF.

34. The method of claim 33, wherein purifying the amplified ORFs comprises separating the amplified ORFs using agarose gel electrophoresis, and isolating the amplified ORFs from the agarose gel. 30 35. The method of claim 34, wherein the agarose is low melt agarose.

36. The method of claim 33, wherein inserting the purified amplified ORFs into the expression vectors is performed using an enzyme that cleaves and ligates DNA.

37. The method of claim 36, wherein the enzyme is a vaccinia DNA topoisomerase, a lambda integrase, an FLP recombinase, or a P1-Cre protein. 91

38. The method of claim 73, wherein the expression vectors are suitable for prokaryotic expression and eukaryotic expression.

39. A method for producing a library of expressible open reading frames, said method substantially as hereinbefore described with reference to any one of the examples.

40. A library of expressible open reading frames produced by the method according any one of claims 1 to 32 or 39.

41. A method for producing a library of selected expressible open reading frames, said method substantially as hereinbefore described with reference to any one of the examples.

42. A library of selected expressible open reading frames produced by the method according to any one of the claims 33 to 38 or 41. Dated 14 August, 2002 INVITROGEN CORPORATION Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON *o *o [l:\DayLib\LIBUU]52780.doc: MCC