CA2340465A1 - Genes that regulate hematopoietic blood forming stem cells and uses thereof - Google Patents

Abstract

The present invention provides an isolated nucleic acid derived from an isolated hematopoietic stem cell. The present invention additionally provide s an isolated hematopoietic stem cell specific protein or a portion thereof encoded by the provided nucleic acid. In addition, the present invention provides a method for generating a stem cell/progenitor cell from a primitiv e hematopoietic cell. Also, the present invention further provides a method fo r identifying the presence in a sample of a compound that modulates hematopoietic stem cell activity. The present invention additionally provide s a molecularly defined primitive hematopoietic stem cell. Finally, the presen t invention provides a method of ex vivo expansion of hematopoietic stem cells .

Description

GENES THAT REGULATE HEMATOPOIETIC BLOOD FORMING STEM
CELLS AND USES THEREOF
This invention was made with the support of National Institutes of Health Grant No. ROl CA45339-09 and American Cancer Society No. DHP-144/01. The United to States government may have certain rights to this invention.
Throughout this application, various publications are referenced by number.
Full citations for these publications may be found listed at the end of the specification and preceding the Claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art. A Sequence Listing is provided.
FIELD OF~'HE INVENTION
The present invention relates to hematopoietic stem cells and the stem cell and support cell genes that support stem cell replication and differentiation.
BACKGROUND OF THE INVENTION
The adult hematopoietic system is organized as a hierarchy of cells with decreasing self renewal and multilineage differentiation potential. This is accompanied by progressively larger numbers of more mature cells and an increasing tendency to be in active cell cycle (Lemischka, LR., 1992; Mornson, S.J., et al. 1995). Collectively, the properties of this hierarchical system result in the balanced, lifelong production of at least eight distinct cell lineages. A population of stem cells establishes the entire hierarchy;
therefore, in order to understand the fundamental mechanisms responsible for normal hematopoiesis it is ultimately necessary to understand the biology of the stem cells.
3o Most information concerning the biology of stem cells has been obtained from the mouse model. In this system, the most critical, characteristic property of the stem cell population has been defined; that is, its ability to reconstitute a normal blood system in a transplanted host. A number of variations on the basic transplantation assay have been described (Harrison, D.E. 1980; Spangrude, G.J., et al. 1995). All of these systems, together with the appropriate donor vs. host or clonotypic markers have rigorously defined the most primitive stem cells and have provided a description of their developmental properties. Perhaps the most striking characteristics of this cell population come from retrovira) "marking" studies (Leminschka, LR. 1992). These studies clearly show that a single stem cell clone is both necessary and sufficient, not only to sustain lifelong, multilineage hematopoiesis in one primary recipient but in numerous secondary animals. This illustrates the remarkable proliferative potential of the stem cell and directly demonstrates stem cell self renewal. A major advance in mouse stem cell to biology was the development of strategies which facilitate the substantial enrichment of stem cell activity (Bauman, J.G., et al. 1988; Spangrude, G.J., et al. 1988;
Jordan C.T. , et al 1990). Purification procedures enabled the first direct approaches to unravel the mechanisms responsible for the unique biological properties of the stem cell population.
A key observation was that the phenotypically defined stem/progenitor cell population is t5 heterogeneous with respect to in vivo functional properties (Fleming, W.G., et al 1993;
Li, C.L. and Johnson G.R. 1992; Spangrude, G.J. and Johnson, G.R. 1990, Jones R., et al. 1990; Uchida, N., et al. 1993). In addition to the in vivo repopulating cells, other primitive progenitor cells are often contained in a purified population (Weilbaecher, K., 1991; Trevisan, M. and Iscove, N.N. 1995; Ogawa, M. 1993). These can be assayed in a 2o variety of in vitro systems. Whether all of these in vivo and in vitro activities represent discrete cell subpopulations or whether there is a continuum of functional potential is still an unanswered question. Recent studies have suggested distinct physical properties for functionally different activities within the primitive population Mornson, S.J., and Weissman, LL. 1994); Mornson, S.J. et al. 1997; Jones, R.M., el al. 1996). A
second set 25 of observations revealed an inverse correlation between a tendency for active cell cycling and primitive, uncommitted developmental potential in BM (Spangrude, G.J., and Johnson, G.R. 1990). In fetal liver a higher proportion of primitive stem cells is actively cycling (Fleming, W.H., et al. 1993). Moreover, it has been shown that fetal stem cells are more potent than adult stem cells in LTRA (Jordan, C.T., et al. 1995;
Pawliuk, R., et 3o al 1996). These are exciting observations because they suggest that rapid stem cell cycling can be compatible with the maintenance of primitive in vivo activity.
Very recent studies suggest that the adult BM stem cell compartment may in fact be cycling at a very slow rate (Bradford, G.B., et al. 1997) . Clearly, stem cell cycle regulation is a critical area for investigation. Stem cell purification has facilitated studies aimed at ex vivo maintenance or expansion of the most primitive, transplantable stem cell. Most culture systems strongly favor a differentiation process (Van der Sluijs, J.P., et al.
1993;
Traycoff, C.M., et al. 1996; Peters, S.O., et al. 1995; Knobel, K.M., et al.
1994).
However, several recent reports have been encouraging. It has been shown that colonies grown in defined cytokines can retain not only myeloid and erythroid but also lymphoid potentials (Ball, T.C., et al. 1995). Moreover, the short-term (2-3 weeks) maintenance of LTRA has been demonstrated in suspension cultures supported by IL6, IL11, together with ckit ligand (KL) or flk2/flt3 ligand (FL) (Yonemura, Y.H., et al. 1997).
A recent to report has shown that colonies initiated in cytokine-supplemented semisolid cultures retain LTRA (Trevisan, M., et al. 1996). The studies described herein have developed a stromal cell line supported system which quantitatively maintains LTRA for an extended (4-7 weeks) time (Moore, F.A., et al. 1997).
In the human system it is clearly not possible to do the same kind of extensive t5 marking and transplantation assays. However, several xenograft model systems have been developed to assess the in vivo behavior of human stem cells (Traycoff, C., et al.
1994; Turner, C., et al. 1996; Cashman, J., et al. 1997) . Some of these experiments can be done quantitatively in limiting dilution (Bhatia, M., et al. 1997). A very recent study has demonstrated a common provira) integration site in granulocyte macrophages and T-2o cells derived from beige/nude/XID mice 7-11 months after engraftment with genetically transduced human stem cells (Nolta, J.A., et al. 1996) . This important study paves the way for precise in vivo clonal analyses. The largest amount of functional information about human stem/progenitor cells has been obtained in vitro using a wide range of stromal cell and cytokine supported culture systems. It is not possible herein to describe 25 and properly accredit all of the important studies, however several advances deserve mention. The long-term culture-initiating cell (LTCIC) assay measures the in vitro production of colony forming cells (CFC) after periods of at least five weeks in culture (Sutherland, H.S., et al. 1989) . The cells producing these CFC derive from a population of cells which, at least to some extent, probably overlaps with the most primitive 3o compartment. The maintenance and expansion of primitive functional abilities in this culture system has recently been documented (Petzer, A.L., et al. 1996) . A
variation on this assay system, the extended LTCIC (ELTCIC) has been suggested to measure an even more primitive cell population in BM and CB (Hao, Q.L., et al. 1996) . A very exciting prospect for the near future will be the integration of the various in vivo xenograft assays with the in vitro LTCIC and ELTCIC systems. Some very recent efforts have suggested that the NODSCID xenograft system and the LTCIC assay may measure distinct stem/progenitor subsets (Larocheile, A..J., et al. 1997). Clearly, much more work needs to be done, however, it may be anticipated that the ELTCIC system will provide the "bridge" in this continuum. Collectively, and including the various strictly cytokine-driven systems, the above studies illustrate the current possibility to accurately and quantitatively reveal the majority (if not all) functional entities in the human stem/progenitor cell hierarchy. The physical characterization and purification of human to stem/progenitor cells has proceeded along lines which are parallel to the mouse system.
Indeed, because of clinical impetus, it can be argued that they are further advanced.
Thus, as measured in the range of assays discussed above, the consensus physical phenotype of the most primitive portion of the human stem/progenitor hierarchy is CD34+Lin-CD38- (Terstappen, L.W.M.M., et al. 1991). The CD34+Lin-CD38+ subset contains less primitive, more committed cellular entities. Other studies have shown that, similar to the mouse, low level expression of Thyl (CD90) is a feature of the primitive human stem cell (CD34+Lin-CD90+)(Baum, C.M., et al. 1992; Craig, W., et al.
1993).
Most CD90+ cells in this compartment are CD38-. Therefore, the consensus phenotype can be described as CD34+Lin-CD90+ (Craig, W., et al. 1993). Two potential 2o differences with the murine system can now be highlighted. First, a very recent and elegant study has shown that the most primitive mouse stem cell may in fact be CD34-/l0 (Osawa, M., et al. 1996) . Whether this is a genuine difference or whether it reflects the ability to perform more accurate long-term engraftment studies in the mouse remains to be determined. Second, it has been suggested that in the mouse, CD38 expression is a positive indicator for primitive stem cell function in a purified population (Randall, T.D., et al. 1996) . As in the mouse, human stem/progenitor cells have been identified and purified from various sources. These include: adult BM (Baum, C.M., et al.
1992), CB
(DiGiusto, D.L., et al 1996) , fetal liver (Craig, W., et al. 1993) and peripheral blood stem cells after various mobilization protocols (Murray, L., et al. 1994).
Similar to the 3o data obtained in the murine system, comparative studies reveal that, in general, the basic and fundamental functional properties of stem/progenitor cells are shared regardless of the tissue source. There are however, significant functional and physical differences.

Numerous insights into hematopoietic molecular control mechanisms have come from gene-targeting studies in mice. Mutations in specific genes, most notably, those encoding transcription or DNA-binding proteins, have profound cell-intrinsic, global or lineage-specific effects on hematopoietic development (Shivdasani, R.A., and Orkin, S.H.
1996; Orkin, S.H. 1996). In the latter cases, it is tempting to speculate that the phenotypes result from defects in the commitment process. However, malfunctions in the commitment decision to "set up" a program of differentiation are difficult to distinguish from malfunctions in the differentiation program itself. Two gene products, (CBF2) and SCL (tal-1) appear to be necessary global regulators of hematopoiesis (Wang, W., et al. 1996; Okuda, T., et al. 1996; Porcher, C., et al. 1996;
Robb, L. , et al.
1996) . Whether these molecules act to specify a hematopoietic stem cell or by other means is an open question. Interestingly, both of these molecules play roles in leukemic transformation. A very important gain of function study documents the apparent ability of HOXB4 to increase primitive cell numbers without significant impairment of ~ 5 differentiation abilities {Sauvageau, G., et al. 1995) . Together with observations that HOXA9 is translocated in myeloid leukemia (Nakamura, T., et al. 1996; Borrow, J., et al.
1996) , these studies suggest an important hematopoietic role for homeobox proteins.
Without question, the above and other studies have identified important regulators of hematopoiesis. However, in almost all cases these regulators were first identified in other systems. The opposite approach is to directly search for stem cell regulators in stem cells, Graf, L., and Torok-Storb, B. 1995; Yang, Y., et al. 1996). The present invention solves these problems.
SUMMARY O~' THE INVENT~nnr The human hematopoietic stem/progenitor cell population has been extensively characterized according to physical and antigenic criteria as well as in a variety of in vitro and in vivo assay systems. Collectively the human studies have revealed similarities to the hierarchical stem/progenitor cell organization defined in the murine system. In spite of significant strides in the identification of cytokines which can act on stem cells, it has 3o not been possible to define a system where undifferentiated expansion of the most primitive stem cell population occurs. Similarly, it has not been possible to direct differentiation along lineage-specific pathways. These limitations, which also apply to the murine system, have hampered the elucidation of regulatory mechanisms which mediate the most fundamental aspect of stem cell biology; that is, the decision to self renew or commit to differentiation. As a consequence, very little is known about the molecular biology of the most primitive hematopoietic stem cell in any organism. It was hypothesized that the regulation of primitive stem cells will be mediated at least in part by the products of genes which are uniquely or predominantly expressed in these cells. One precedent for an important, differentially-expressed molecule is the flk2/flt3 receptor tyrosine kinase. There presumably are other important and differentially expressed gene products. Therefore, it is an object of the present invention to identify these molecules and address their functional roles. Specifically, an aspect of identification of gene to expression patterns specific to primitive human stem cells is the molecular phenotype of the human stem cell. The present invention describes methods to define the profile of genes specifically expressed in undifferentiated human stem/progenitor cell populations.
A primary focus of the present invention is on primitive cells isolated from normal bone marrow (BM) samples. The present invention further comprehends use of other t5 sources of stem cells, such as umbilical cord blood (CB).
The methods of the present invention combine diverse technical approaches and sophisticated bioinformatic analyses.
This invention further provides methods to identify genes whose expression can be modulated by cytokine or stromal-dependent culture and/or by cell-cycle status.
2o Another object of the present invention is to provide methods for the functional characterization of human stem cell-specific gene products. An aspect of this invention is a method to facilitate the functional characterization of specifically expressed gene products as candidate regulators of a variety of stem/progenitor cell processes. In particular, a provided method uses an in vitro system which approximates many 25 characteristic properties of normal stem cells to analyze positive and negative regulation of proliferation, cell-cycle parameters, apoptosis and commitment.
It is a further goal of the present invention to provide a necessary (and usually missing) component for stem cell gene-expression screens; that is, the ability to quickly assess the function of extensive panels of genes.
3o It is also an object of the present invention to provide a method for the functional identification of stem cell regulators. An aspect of this invention is a facile screening method for "categorizing" large populations of specifically-expressed molecules according to their potential roles in a variety of stem/progenitor cell processes. Gain of WO 00/1116$ 7 PCT/US99/19052 function as well as loss of function approaches are contemplate by the present invention.
This method does not rely on any prior knowledge of nucleotide or predicted protein sequence.
Accordingly, the present invention provides an isolated nucleic acid derived from an isolated hematopoietic stem cell, the isolated nucleic acid comprising the following characteristics: (1) specifically expressed in the hematopoietic stem cell; and {2) encoding a hematopoietic stem cell - specific protein.
The present invention additionally provides an isolated hematopoietic stem cell to specific protein or a portion thereof encoded by the provided nucleic acid.
The present invention further still provides a nucleic acid probe capable of specifically hybridizing with the provided nucleic acid under standard hybridization conditions.
Also, the present invention provides an antibody capable of specifically binding to the provided protein without substantially cross-reacting with a non-stem cell specific is protein or homologs thereof under conditions permissive to antibody binding.
Additionally, the present invention provides a cell capable of producing the provided antibody.
In addition, the present invention provides a method for identifying the presence of a primitive hemopoietic stem cell in a sample comprising nucleic acids specifically 2o expressed in hematopoietic stem cells. Further still, the present invention provides a method for generating a stem cell/progenitor cell from a primitive hematopoietic cell in a sample.
The present invention further provides a method for identifying the presence in a sample of a compound that modulates hematopoietic stem cell activity.
25 The present invention even further still provides a method for identifying primitive hematopoietic stem cell-specific nucleic acids.
Also the present invention additionally provides a molecularly defined primitive hematopoietic stem cell.
Yet additionally, the present invention provides a method for treating a condition 3o in a subject comprising administering to the subject a therapeutically effective amount of a provided pharmaceutical composition. The present invention provides a method of introducing an exogenous nucleic acid into a hematopoietic stem cell.

WO 00/11168 g PCT/US99/19052 Finally, the present invention provides a method of ex vivo expansion of hematopoietic stem cells. The expanded cells are available to receive exogenous genes, including by retroviral or other vectors which require a round of replication.
Alternatively, the cells are available for transplantation either autologously or heterologously.
FIGURE 1. Schematic "flow of information" strategy.
F1GURE 2. Mammalian stem cell system (black circle = strong hybridization signal, gray circle - detectable hybridization signal, and open circle - no detectable hybridization signal). The Smc-34 cDNA is a completely novel sequence with a predicted ieucine zipper and several potential membrane spanning domains.
FIGURES 3A-3B. (right panel), the control, non-subtracted RDA cDNA population (38-contains b-actin sequences which are missing in the two subtracted RDA
populations, 38- and 38-)38+. A differentially expressed gene (HDD-2, described below) is enriched in the 38- RDA population and at least retained in the 38-)38+ RDA population (Figure 3A, left panel). Two, bi-directional, RDA cDNA populations (38- and the converse 34 )38) were used to probe (See Figure 3) duplicate arrays of a subtracted 38-library (Figure 3B).
FIGURE 4A-4E. Figure 4A: 34B4 (SEQ.ID.No.: G9) is closely related to a gene encoding TINUR The sequence homologies and restricted expression pattern of 34A5 is shown in Figures 4B and 4C. In Figure 4C and 4D {and also Sa, Sb, and 6b) there are twenty-one samples of capfinder-amplified cDNA from various hematopoietic populations. From left to right these are: four CD34+Lin- populations, three CD34+Lin-CD90+ populations, two CD34- populations, four CD34+Lin-CD38+ (obtained from the 3o same BMs as the CD38- samples in lanes 1-4), two CD34+Lin- samples (obtained from the same BMs as the CD90+ samples in lanes 5 and 6), three CD34+Lin-populations obtained after 1, 2 or 4 days of culture and finally their three CD34+Lin-CD38+
counterparts. The 3862 cDNA (SEQ.ID.No.: 70) is closely homologous to the LTG9/MLLT3 gene located on 9q22 and involved in t(9;11 ) leukemia (Iida, S., et al.
1993) (Figure 4E).
FIGURES SA-5B. GOS3, a fos-related gene (Heximer, S., et al. 1996) (Figure SA) and (2) HLA-DR (Figure SB).
FIGURES 6A-6D. Figure 6A. HDD-2 is about 500 bp. It contains a short open reading frame of 89 amino acids (SEQ.ID.No.: 71 ). The predicted peptide sequence is shown.
Figure 6B. The expression profile of HDD-2 demonstrates that it is stem cell restricted.
o Figure 6C. HDD-2 hybridization to a dot blot with numerous human pA+ mRNA
samples (Clontech). HDD-2 hybridization is only visible in kidney (the other "spots" are background). Figure 6D. Genomic Southern blot confirmed that HDD-2 corresponds to a single-copy human gene.
FIGURE 7. Immunoprecipitation analysis of protein extracts using rat IgG2b isotype control antibody (IgG) or AA4.1 mAb. Protein extracts were prep-ared from D2N
cells;
M2.4 cells; AA4-depleted fetal liver cells (FLAA4-); AA4enriched fetal liver cells (FLAA4+); AA4-depleted bone marrow cells (BMAA4-); AA4-enriched bone marrow cells (BMAA4+). Indicated on the right are positions of molecular weight markers.
zo FIGURES 8A-8C. AA4 expression in retrovirus infected cells. Figure 8A. Flow cytometry analysis of NIH 3T3 and EML Cl cells using PE-conjugated AA4.1 antibody before and after infection with REBNA/AA4. ~jhure . Imrnunoprecipitation of biotin-labeled surface proteins using rat IgG2b isotype control antibody (lanes 1, 3, 5) or AA4.1 mAb (lanes 2, 4, 6). Cellular extracts were prepared from D2N cells (lanes 1 and 2), REBNA/AA4 infected NIH 3T3 fibroblasts (lanes 3 and 4), and REBNA/AA4 infected EML Cl cells (lanes 5 and 6). Figure 8C. Immunoprecipitation of cellular extracts using AA4.1. REBNA/AA4 NIH 3T3 cells (lanes 2 -- 4) and REBNA/AA4 EML
CI cells (lanes 6 -- 8) were labeled with 35S-methionine and chased with nonradioactive media for 10 min. (lanes 3 and 7) or 20 min. (lanes 4 and 8). REBNA/GFP NIH

(lane 1 ) and REBNA/GFP EML CI cells (lane 5) are shown as controls. Indicated on the right are positions of molecular weight markers.

WO 00/11168 t0 PCT/US99/19052 FIGURF~9. Nucleotide sequence (SEQ.ID.No.: 72) and the deduced amino acid sequence of AA4 (SEQ.ID.No.: 73). Amino acid sequences is numbered 1 with initiator methionine. Underlined is the putative signal peptide in AA4. The transmembrane domain is double underlined. Light grey line indicates C-type lectin carbohydrate recognition domain. Dark grey lines show EGF-like repeats, while broken grey lines indicate calcium-binding EGF motifs.
FIGURES l0A-IOB. Comparison of the primary structures of AA4 and human CIqR.
_Fiaeure 10A. Alignment of amino acid sequences of AA4 (SEQ.ID.No.: 73) and CIqR.
to Figure. Comparison of the domain structures of AA4 and CIqR. The proteins contain N-terminal signal peptides (SP), a C-type lectin recognition domain, six EGF-like domains, including three calcium-binding (cb) EGF-like domains, and a transmembrane domain (TM).
F1GURE 11. In normal tissues and transformed cell lines, a 7kb RNA species hybridizes with the cloned cDNA (Figures I 1 A and 1 I B). In addition to the 7kb species, poly(A)-RNA from D2N cells contains a minor band corresponding to a 3.2 kb mRNA
(Figure I 1 A, lane 8).
2o F1GURES 12A-12B Northern blot analysis of AA4 expression in transformed cell lines Figure 12A and normal mouse tissues Figure 12B. Indicated on the left are positions of 9.44 - 0.24 kb RNA molecular weight markers. Hybridization with D2N
poly(A)+
RNA is shown after a 2 hr and overnight exposures. Hybridizations with GAPDH
are shown as controls for equal loading.
FIGURES 13A-13C. RT-PCR of cDNAs prepared from murine fetal liver, (A) bone marrow-derived hematopoietic cells (B), or differentiating ES cells (C). F~ure 1,~A.
Lanes 1 and 2, AA4- cells; lane 3, AA4+ cells; lane 4, AA4+Sca-1+c-Kit+LinlO
cells;
lane 5, AA4+Sca-1-c-Kit+LinIO cells. Figure 13B. Lane 1, RholOSca-1+Thy-1 IOLincells; lane 2, RhohiSca-1+Thy-1 lOLin- cells; lane 3, Sca-I+Thy-l-Lin-cells; lane 4, Lin+ cells. Figure 13C_. ESO, undifferentiated ES cells; BLI and 2, blast cell colonies;
ENT1 and 2, differentiated endothelial cells; HMT1, 2 and 3, differentiated hematopoietic cells. The cDNAs probes used for hybridization are indicated on the right.

FIGURE 14. AFT024 maintains in vivo repopulating stem cells. The ability of 3 different stromal cell lines to support highly purified fetal liver stem cells was studied.
Freshly purified day 14 fetal liver cells were transplanted directly (103 AA4.1+, lin'~°, s Sca-1+, c-kit+ cells plus lOG Ly5.1 competitor BM per mouse; n=6) into Ly5.1 congenic mice (Control). Additional stem cells from the same purification were also used to initiate Dexter-LTC over inradiated AFT024, 2012, and 2018 stromal cell monolayers (104 cells/10 cm dish}. After 4 weeks of culture, 10% aliquots of each culture (103 stem cell equivalents) were transplanted into Ly5.1 recipients (n=8) together with 10~' to competitor Ly5.1 BM cells. Peripheral blood cells from mice were analyzed for the presence of Ly5.2~ cells at S, 12, 24, and 56 weeks after transplant. Error bars represent SEM.
FIGURE 15. Long-term culture on AFT024 maintains greater levels of repopulating 15 stem cell activity than short-term cytokine- or short-term AFT024-supported cultures.
The levels of stem cell activity maintained in short-term cytokine-supported and short-term AFT024-supported cultures were compared to those maintained in long-term AFT024 coculture. Purified fetal liver cells were cultured for 5 days in suspension with cytokines or on an AFT024 monolayer (3000/well-12-well tray). Additional cells from 20 the same purification were seeded onto AFT024 monolayers (3000/6 cm dish) and maintained in Dexter-LTC for 5 weeks. At completion of both culture periods the cells were harvested, mixed with Ly5.1 BM and used to transplant mice. Each mouse received 20% of each culture (600 stem cell equivalents) and 4X105 competitor BM cells (4 mice/culture). Peripheral blood cells from mice were analyzed for the presence of Ly5.2+
2s cells at 15 weeks after transplant. FL 1.0+0.57; FL/SL 0.75+0.25; FL/IL-6 1.8+0.14;
SL/IL-6 3.2+p.46; FL/IL-6/SL 1.7~0.2I; AFT024 5 days 2.8+0.11; AFT024 5 weeks 32.2~7.4 * (p<p.004) Students t-test. See Methods for culture conditions and cytokine concentrations. FL, flk2/flt3-ligand; SL, steel factor. Error bars represent SEM.
3o FIGURE 16. Time course of cobblestone area formation on AFT024. The formation of stromal dependent CA derived from purified fetal liver stem cells was studied in AFT024 cocultures. Characteristic clusters of at least 50 cells were scored as CA
over 28 days of culture. Results are expressed as the mean number of CA/1000 input stem cells from 3 separate fetal liver purifications (300-600 cells/well in 12-well trays).
Error bars represent the SEM. The frequency of CA after 28 days is approximately 1 for every 20 input stem cells.
FIGURE 17. High-proliferative potential multilineage clonogenic progenitors are selectively expanded on AFT024. The clonogenic progenitor content of stem cells maintained in AFT024 supported Dexter-LTC was deternnined. Enriched fetal liver stem cells were seeded onto AFT024 monolayers, at various time points, an individual well was harvested and the cells placed into semi-solid clonogenic progenitor assay (CFU-C).
to Colonies were scored at 8-14 days. Colonies were designated as HPP upon reaching a size __>1 mm after 8 days. CFU numbers at days 0, 4, and 28 are averaged from individual stem cell purifications. Error bars represent the SEM for these experiments.
Other time points are individual determinations. CFU are normalized to 1000 input stem cells in the stromal cocultures for comparison to day 0 progenitors. *CFU-Mix (p=.O1 ) t5 and *CFU-HPP-Mix (p=.001) are significantly expanded at day 28 compared to day 0, (Student's T-test).
FIGURES 18A-18B. Cobblestone area-initiating cells are expanded on AFT024.
Figure 18A. A quantitative estimate of the number of 28 day cultured stem cell 20 equivalents required to form a CA after replating on secondary AFT024 monolayers was determined. Four different AFT024 cocultures from separate fetal liver purifications were studied (A, B, C, and D). In limiting-dilution assay, the frequency of stem cell equivalents required to form a CA in another 7 days was 1 in 4 (3.56+0.64, r2=0.96).
Figure 18 B. CA maintenance in one of the 4 above cultures was followed for an 25 additional 4 weeks. The frequency of stem cell equivalents maintaining CA
was determined. At 37% negative wells the frequencies were: 2 weeks 1 in 3, 3 weeks 1 in 10, 4 weeks 1 in 19, and at 5 weeks 1 in 29.
FIGURES 19A-19B. Dlk expression analysis in stromal cell lines. Figure 19A.
(Top) A
30 1.6 kb dlk transcript is visualized in the parental AFT024 and 2012 cell lines and their subclones, but not in 2018, CFC034 and BFC012. (Bottora) The same filter hybridized with a b-actin probe. Figure 19B. RT-PCR analysis of 14 fetal liver-derived stromal cell lines and other cell lines.

FIGURE 20. Cobblestone area formation by hematopoietic stem cells in the presence of soluble dlk protein. Data are from 4 experiments; 2 each with adult BM cells (Sca-1+, c-kit+, line°~-) and day 14 fetal liver cells (AA4.1+, Sca-1+, c-kit+, line°~-). Results are expressed as the ratio/fold-increase in CSA number for fourteen data points each (bars represent, none vs control; dlk vs none; dlk vs control) for the 4 different experiments.
Error bars represent SEM. *P= 0.01 comparing dlk vs none to none vs control, **P= 0.001 comparing dlk vs control to none vs control (Student's t-test).
to FIGURE 21. Membrane bound dlk expression in transfected BFC012 cells. Full length dlk cDNA was transfected into BFC012 cells. (Left) A flow diagram of dlk expression in transfected BFC012 populations (BFC-dlk) and cells transfected with the selection plasmid alone (BFC-Zeo). (Right) Expression of dlk in a cloned line (BFC-dlk-5) derived from the expressing population and a control clone (BFC-Zeo-1 ).
FIGUR .~ 22A, CSA formation by hematopoietic stem cells in the presence of membrane-bound dlk. Figure 22A. Bars labeled BFC are from 5 groups (nontransfected BFC012 cells, two control pZeo transfected BFC012 populations, and two clones derived from the pZeo transfected populations). Bars labeled BFC-dlk are from three groups shown to express 2o transfected dlk; one dlk-transfected BFC012 population (BFC-dlk) and two individual transfected clones (BFC-dlk-1 and BFC-dlk-5). Error bars represent SEM. ** P<
0.001 days 3, 4, and 5; *P< 0.01 days 6 and 7 (Student's t-test). Figure 22B. A
clone derived from the dlk transfected populations of BFC012 cells (BFC-dlk-5) and a clone derived from pZeo transfected populations (BFC-Zeo-1 ) were used for CSA assay with purified fetal liver stem cells. CSA/1000 input stem cells are expressed as the mean of three individual experiments, error bars represent the SEM. **P< 0.001 at days 4, G, and 8 (Student's t-test).
IfIGURES 23A-2'~R f-lpp multilineage clonogenic progenitors and irr vivo repopulating stem cells are maintained in short-term dlk-expressing cocultures. Figure 23A.
Fetal liver stem cells were purified as described and assayed for their progenitor content immediately after purification and after culture on BFC-dlk-5, and BFC-Zeo-1. At day 4 the cultures were used for clonogenic progenitor (3 experiments) and transplantation assay (2 experiments). Bars represent data from 3 experiments with day 0 cells (Fresh) and day 4 cocultured cells (BFC-dlk-5 and BFC-Zeo-1 ), error bars represent SEM. *P=
0.01 for total CFU-C from fresh stem cells compared to total CFU-C from BFC-dlk-5 cocultures at day 4;
** P= 0.001 for total CFU-C from BFC-dlk-5 compared to total CFU-C from BFC-Zeo-1 (Student's t-test). Figure 23 B. Analysis of in vivo repopulating ability of purified fetal liver stem cells cocultured for 4 days on BFC-dlk-5, BFC-Zeo-1, and AFT024 monolayers.
Results are from nine individual mice in two experiments (4-5 mice in each experiment) at weeks after transplantation. P= 0.05 for BFC-dlk-5 vs BFC-Zeo-I (Student's t-test).
FIGURE 24. Genes and predicted proteins isolated from primitive stem cells by the o techniques of the present invention. (Sequence Identification Numbers are indicated on the figure).
DETAILED DE~RmTmN
The present invention provides an isolated nucleic acid derived from an isolated hematopoietic stem cell, the isolated nucleic acid comprising the following characteristics: (1) specifically expressed in the hematopoietic stem cell;
and (2) encoding a hematopoietic stem cell - specific.protein.
An embodiment of this invention further comprises the following characteristic:
2o capable of hybridizing under standard conditions with a sequence selected from the group consisting of SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: 5, SEQ.ID.No.: G, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.:
14, SEQ.ID.No.: 15, SEQ.ID.No.: IG, SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.:
19, SEQ.ID.No.: 20, SEQ.ID.No.: 21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.:
24, SEQ.ID.No.: 25, SEQ.ID.No.: 2G, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.:
29, SEQ.ID.No.: 30, SEQ.ID.No.: 31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.:
34, SEQ.ID.No.: 35, SEQ.ID.No.: 36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.:
39, SEQ.ID.No.: 40, SEQ.ID.No.: 41, SEQ.ID.No.: 42, and SEQ.ID.No.: 43, SEQ.ID.No.:
45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.: 51, SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: GI, SEQ.ID.No.: G3, SEQ.ID.No.:
65, SEQ.ID.No.: G7, SEQ.ID.No.: 72 or a portion thereof. A portion thereof, in a preferred embodiment of this invention is the 5' end region or the 3' end region of the nucleic acid.

In another preferred embodiment, a portion thereof is at least a 8-18 nucleotide portion of the coding region. In yet another preferred embodiment, a portion therof is at least a 8-18 nucleotide portion of a non-coding regulatory region or a binding region such as a stem-cell specific promoter or enhancer region. According to still another embodiment of the provided isolated nucleic acid further comprises the characteristic of encoding a protein capable of modulating hematopoietic stem cell activity. According to this invention modulating hematopoietic stem cell activity includes up-regulating, down-regulating or otherwise changing the activity of the hematopoietic stem cell. Such activity is contemplated as inducing differentiation or inhibiting differentiation of the cell.
to However, directing differentiation toward one or another daughter cell type is also within the scope of a preferred embodiment of this invention. Other preferred embodiments include but are not limited to modulation of transcription, translation, gene splicing, transport, proteolytic processing, replication, expression of cell surface markers and transplantation. According to still another embodiment of the present invention, the activity is selected from the group consisting of hematopoietic stem cell differentiation and hematopoietic stem cell replication. According to yet another embodiment of this invention, the protein is selected from the group consisting of a growth factor, a transcription factor, a splicing factor, a capping factor, a transport protein, a translation factor, and a replication factor. In one preferred embodiment of this invention, the provided nucleic acid comprises the nucleotide sequence of SEQ.ID.No.: 72, an analog thereof, or a portion thereof. According to another preferred embodiment of this invention, the hematopoietic stem cell is a primitive hematopoietic stem cell.
In one embodiment of this invention, the primitive hematopoietic stem cell is selected from the group consisting of an umbilical cord cell , a bone marrow cell and a fetal liver cell. In a preferred embodiment of this invention, the primitive hematopoietic stem cell is selected from the group consisting of a AFT024 cell, a 2012 cell and a 2018 cell.
The present invention further provides a composition comprising the provided nucleic acid, wherein the nucleic acid comprises one selected from the group consisting of SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: S, 3o SEQ.ID.No.: 6, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.: 14, SEQ.ID.No.:
15, SEQ.ID.No.: 1 G, SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.: 19, SEQ.ID.No.:
20, SEQ.ID.No.: 21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.: 24, SEQ.ID.No.:
25, SEQ.ID.No.: 26, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.: 29, SEQ.ID.No.:
30, SEQ.ID.No.: 31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.: 34, SEQ.ID.No.:
35, SEQ.ID.No.: 36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.: 39, SEQ.ID.No.:
40, SEQ.ID.No.: 41, SEQ.ID.No.: 42, SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.:
47, SEQ.ID.No.: 49, SEQ.ID.No.: 51, SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.:
57, SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.:
67, SEQ.ID.No.: 72 or a portion thereof. According to one embodiment of this invention, the the nucleic acid is selected from the group consisting of DNA, RNA and cDNA.
Another embodiment of this invention is a vector comprising the provided nucleic acid.
to According to yet another embodiment, the vector comprises viral or plasmid DNA. A
further embodiment of this invention is an expression vector comprising the provided nucleic acid and a regulatory element. A still further embodiment of this invention is a host vector system which comprises the expression vector in a suitable host.
In a preferred embodiment of this invention, the suitable host is selected from the group t S consisting of a bacterial cell, a eukaryotic cell, a mammalian cell and an insect cell.
The present invention additionally provides an isolated hematopoietic stem cell specific protein or a portion thereof encoded by the provided nucleic acid.
According to one embodiment of this invention is the provided protein further comprising the characteristic of being capable of modulating hematopoietic stem cell activity. According 20 to this invention modulating hematopoietic stem cell activity includes up-regulating, down-regulating or otherwise changing the activity of the hematopoietic stem cell. Such activity is contemplated as inducing differentiation or inhibiting differentiation of the cell.
However, directing differentiation toward one or another daughter cell type is also within the scope of a preferred embodiment of this invention. Other preferred embodiments 25 include but are not limited to modulation of transcription, translation, gene splicing, transport, proteolytic processing, replication, expression of cell surface markers and transplantation. According to still another embodiment of the present invention, the activity is selected from the group consisting of hematopoietic stem cell differentiation and hematopoietic stem cell replication. According to yet another embodiment of this 3o invention, the protein is selected from the group consisting of a growth factor, a transcription factor, a splicing factor, a capping factor, a transport protein, a translation factor, and a replication factor. According to one embodiment the activity is selected from the group consisting of hematopoietic stem cell differentiation and hematopoietic WO 00/11168 t ~ PCTNS99/19052 stem cell replication. According to another embodiment, the protein is selected from the group consisting of a growth factor, a transcription factor, a splicing factor, a capping factor, a transport protein, a translation factor, and a replication factor.
According to still another embodiment, the protein has substantially the same amino acid sequence as one selected from the group consisting of SEQ.ID.No.: 42, SEQ.ID.No.: 44, SEQ.ID.No.: 46, SEQ.ID.No.: 48, SEQ.ID.No.: 50, SEQ.ID.No.: 52, SEQ.ID.No.: 54, SEQ.ID.No.:
56, SEQ.ID.No.: 58, SEQ.ID.No.: 60, SEQ.ID.No.: 62, SEQ.ID.No.: 64, SEQ.ID.No.:
66, SEQ.ID.No.: 68, SEQ.ID.No.: 70, SEQ.ID.No.: 71, and SEQ.ID.No.: 73.
The present invention further still provides a nucleic acid probe capable of to specifically hybridizing with the provided nucleic acid under standard hybridization conditions.
Also, the present invention provides an antibody capable of specifically binding to the provided protein without substantially cross-reacting with a non-stem cell specific protein or homologs thereof under conditions permissive to antibody binding.
Additionally, the present invention provides a cell capable of producing the provided antibody.
In addition, the present invention provides a method for identifying the presence of a primitive hemopoietic stem cell in a sample comprising nucleic acids specifically expressed in hematopoietic stem cells comprising (a) contacting the sample with the 2o provided antibody under conditions permissive to the formation of an antibody complex;
(b) detecting the presence of the complex formed in step (a), the presence of a complex formed indicating the presence of a primitive hemopoietic stem cell in the sample.
According to one embodiment of this invention, the antibody is labeled with a detectable marker. In a preferred embodiment, the detectabie marker is selected from the group consisting of a radioactive isotope, enzyme, magnetic bead, dye, flourescent marker and biotin.
Further still, the present invention provides a method for generating a stem cell/progenitor cell from a primitive hematopoietic cell in a sample comprising contacting the sample with the provided protein . Another embodiment of this invention, provides a 3o method for generating a stem cell/progenitor cell from a primitive hematopoietic cell in a sample comprising contacting the sample with the provided nucleic acid.
According to an preferred embodiment, the nucleic acid is in an expression vector. According to another preferred embodiment the nucleic acid is introduced into the cell under conditions WO 00/11168 i g PCT/US99/19052 permissive to the expression of the nucleic acid.
The present invention further provides a method for identifying the presence in a sample of a compound that modulates hematopoietic stem cell activity comprising: (a) contacting the hematopoietic stem cell with the sample; (b) determining the hematopoietic stem cell activity; and (c) comparing the hematopoietic stem cell activity determined in step (b) with the activity determined in the absence of the compound an increase or decrease in hematopoietic stem cell activity indicating the presence in the sample of a compound that modulates hematopoietic stem cell activity.
According to one embodiment, the activity is selected from the group consisting of gene expression, o replication, differentiation, transplantation, and self regeneration. The present invention also still further provides a compound identified by the method of this invention, previously unknown.
The present invention even further still provides a method for identifying primitive hematopoietic stem cell-specific nucleic acids , comprising: (a) creating a primitive hematopoietic stem cell cDNA library and a non-primitive stem cell immune cell cDNA library; and (b) subtracting the two libraries, thereby identifying primitive stem cell specific nucleic acids. According to one embodiment is (i) contacting the nucleic acids of the stem cell and non-stem cell libraries with each other under conditions permissive to hybridization, thereby forming hybrid complexes; (ii) separating the hybrid zo complexes formed in step (b) from the nucleic acids which did not form complexes; and (iii) isolating the nucleic acids which did not form complexes, thereby identifying hematopoietic stem cell specific nucleic acids. In still another embodiment, step (ii) further comprising amplification of the nucleic acids. Yet another embodiment is step (iii) further comprising ampliciation of the nucleic acids which did not form complexes.
Even still another embodiment is further comprising displaying the amplified DNA on a chromatography gel. A further embodiment is step (b) comprising differential display of the two libraries, thereby identifying primitive stem cell specific nucleic acids. Also yet another embodiment is step (b) comprising representation difference analysis of the two libraries, thereby identifying primitive stem cell specific nucleic acids. Yet even another 3o embodiment is further comprising cloning the stem cell specific nucleic acid. According to a preferred embodiment, the stem cell is selected from the group consisting of AF024, 2012, and 2018. The present invention further provides a nucleic acid identifted by the provided method.

The present invention additionally provides a composition comprising the provided compound and a carrier. An embodiment of this invention is a pharmaceutical composition comprising the provided compound and a pharmaceutically acceptable carrier.
s Also the present invention additionally provides a primitive hematopoietic stem cell specifically expressing one selected from the group consisting of:
SEQ.ID.No.: I, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: 5, SEQ.ID.No.: 6, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: Il, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.: 14, SEQ.ID.No.: 15, SEQ.ID.No.:
16, to SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.: 19, SEQ.ID.No.: 20, SEQ.ID.No.:
21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.: 24, SEQ.ID.No.: 25, SEQ.ID.No.:
26, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.: 29, SEQ.ID.No.: 30, SEQ.ID.No.:
31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.: 34, SEQ.ID.No.: 35, SEQ.ID.No.:
36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.: 39, SEQ.ID.No.: 40, SEQ.ID.No.:
41, is SEQ.ID.No.: 42, and SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.: 47, SEQ.ID.No.:
49, SEQ.ID.No.: 51, SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a portion thereof. An embodiment of this invention is a primitive hematopoietic stem cell specifically expressing a nucleic acid identified by the provided method.
20 Yet additionally, the present invention provides a method for treating a condition in a subject comprising administering to the subject a therapeutically effective amount of the provided pharmaceutical composition. According to an embodiment of this invention, the condition is an immune system condition. In a further embodiment of this invention, the condition is leukemia.
25 The present invention provides a method of introducing an exogenous nucleic acid into a hematopoietic stem cell comprising contacting the stem cell with the provided composition.
Finally, the present invention provides a method of ex vivo expansion of hematopoietic stem cells comprising contacting the cell with the provided composition.
3o According to an embodiment of this invention, the ex vivo expanded hematopoietic stem cells are available for therapeutic use. The expanded cells are available to receive exogenous genes, including by retroviral or other vectors which require a round of replication. Alternatively, the cells are available for transplantation either autologously or heterologously.
As used herein, the term, a sequence is conserved if there is substantial homology of sequence between multiple gene species.
As used herein, the terms, "hybridization" and "binding" in the context of probes, primers and denatured DNA are used interchangeably. Probes which are hybridized or bound to denatured DNA are aggregated to complementary sequences in the polynucleotide. Whether or not a particular probe remains aggregated with the polynucleotide depends on the degree of complementarity, the length of the probe, and to the stringency of the binding conditions. The higher the stringency, the higher must the degree of complementarity, and/or the longer the probe.
As used herein, the term, probe, refers to an oligonucleotide designed to be sufficiently complementary to a sequence in a denatured nucleic acid to be probed, in relation to its length, to be bound under selected stringency conditions.
Primers may vary in length. Preferably such primers should be sufficiently long to hybridize to the modified RNAs in a specific and stable manner. A semi-random primer as the term is used herein, encompasses a class of primers wherein either a discrete portion of the primer is random, while another discrete portion is conserved as well as primers which have nucleotide preferences at particular positions within a sequence. For example, the 2o discrete portion-type primer may have a predetermined adaptor sequence at its 5' end and a random sequence at its 3' end. Alternatively, several preferred primers have nucleotide preferences at specific positions within the primers while other positions are random. A
degenerate primer as the term is used herein, encompasses a cocktail or mixture of primers wherein one or more of the possible triplet nucleotide sequences encoding an amino acid is incorporated into the primer sequence. For example, Serine may be encoded by six separate triple sequences (AGU, AGC, UCU, UCC, UCA, and UCG).
Thus, a "degenerate" primer may reflect the degeneracy of the nucleotide triplet code.
Alternatively, a randomized primer, as the term is used herein, encompasses a primer wherein, the nucleotide at one or more positions may be randomized in order to yield a 3o triplet sequence encoding an alternative or a random amino acid at the position.
An end region as the term is used herein, consists of the end nucleotide and a portion of the region including as much as that half of the entire sequence.
For example, the "3' end region" or "3' region" of a primer may include the 3' half of the primer.

A preferred method of hybridization is blot hybridization. See Sambrook et al.
1989 Molecular Cloning: A Laboratory Manual 2nd Ed. for additional details regarding blot hybridization. Using this method, separated amplification products are transferred onto a solid matrix, such as a filter. The probe, which is detectable, either directly or indirectly, is hybridized to the solid matrix under appropriate conditions.
The matrix is washed to remove excess probe. Thereafter the probe which specifically hybridized to the solid matrix can be detected.
The probe can be DNA or RNA and can be made detectable by any of the many labeling techniques readily available and known to the skilled artisan. Such methods to include, but are not limited to, radio-labeling, digoxygenin-labeling, and biotin-labeling.
A well-known method of labeling DNA 15 3zP using DNA polymerise, Klenow enzyme or polynucleotide kinase. In addition, there are known non-radioactive techniques for signal amplification including methods for attaching chemical moieties to pyrimidine and purine rings (Dale, R.N.K. et al, 1973 Proc. Natl. Acid. Sci. USA 70:2238-42), methods which allow detection by chemiluminescence (Barton, S.K. et al, 1992 J. Am.
Chem. Soc.
114:8736-40) and methods utilizing biotinylated nucleic acid probes (Johnson, T.K. et al, 1983 Anal. Biochen:. 133:125-131; Erickson, P.F. et al, 1982 J. Immunol.
Methods 51:241-49; Matthaei, F.S. et al, 1986 Anal. Biochem. 157-123-28) and methods which allow detection by fluorescence using commercially available products. Non-radioactive labeling kits are also commercially available.
A basic description of nucleic acid amplification is described in Mullis, U.S.
Patent No. 4,683,202, which is incorporated herein by reference. The amplification reaction uses a template nucleic acid contained in a sample, two primer sequences and inducing agents. The extension product of one primer when hybridized to the second 2s primer becomes a template for the production of a complementary extension product and vice versa, and the process is repeated as often as is necessary to produce a detectable amount of the sequence.
The inducing agent may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
Suitable 3o enzymes for this purpose include, for example, E.coli DNA polymerise I, thelmostable Taq DNA polymerise, Klenow fragment of E.coli DNA polymerise I, T4 DNA
polymerise, other available DNA polymerises, reverse transcriptase and other enzymes which will facilitate combination of the nucleotides in the proper manner to form amplification products. The oligonucleotide primers can be synthesized by automated instruments sold by a variety of manufacturers or can be commercially prepared based upon the nucleic acid sequence of this invention.
The degree of hybridization depends on the degree of complementarity, the length of the nucleic acid molecules being hybridized, and the stringency of the conditions in a reaction mixture. Stringency conditions are affected by a variety of factors including, but not limited to temperature, salt concentration, concentration of the nucleic acids, length of the nucleic acids, sequence of the nucleic acids and viscosity of the reaction mixture.
More stringent conditions require greater complementarity between the nucleic acids in order to achieve effective hybridization.
"Hybridization" and "binding" in the context of probes, primers and denatured DNA are used interchangeably. Probes which are hybridized or bound to denatured DNA
are aggregated to complementary sequences in the polynucleotide. Whether or not a particular probe remains aggregated with the polynucleotide depends on the degree of is complementarity, the length of the probe, and the stringency of the binding conditions.
The higher the stringency, the higher must the degree of complementarity, and/or the longer the probe.
"Probe" refers to an oligonucleotide designed to be sufficiently complementary to a sequence in a denatured nucleic acid to be probed, in relation to its length, to be bound 2o under selected stringency conditions.
Primers may vary in length. Preferably such primers should be sufficiently long to hybridize to the modified RNAs in a specific and stable manner.
A semi-random primer as the term is used herein, encompasses a class of primers wherein either a discrete portion of the primer is random, while another discrete portion is 25 conserved as well as primers which have nucleotide preferences at particular positions within a sequence. For example, the discrete portion-type primer may have a predetermined adaptor sequence at its 5' end and a random sequence at its 3' end.
Alternatively, several preferred primers have nucleotide preferences at specific positions within the primers while other positions are random.
3o A degenerate primer as the term is used herein, encompasses a cocktail or mixture of primers wherein one or more of the possible triplet nucleotide sequences encoding an amino acid is incorporated into the primer sequence. For example, Serine may be encoded by six separate triple sequences (AGU, AGC, UCU, UCC, UCA, and UCG).

Thus, a "degenerate" primer may reflect the degeneracy of the nucleotide triplet code.
Alternatively, a randomized primer, as the term is used herein, encompasses a primer wherein, the nucleotide at one or more positions may be randomized in order to yield a triplet sequence encoding an alternative or a random amino acid at the position.
Solid matrices are available to the skilled artisan. Solid phases useful to serve as a matrix for the present invention include but are not limited to polystyrene, polyethylene, polypropylene, polycarbonate, or any solid plastic material in the shape of test tubes, beads, microparticles, dip-sticks, plates or the like. Additionally matrices include, but are not limited to membranes, 96-well microtiter plates, test tubes and Eppendorf tubes.
o Solid phases also include glass beads, glass test tubes and any other appropriate shape made of glass. A functionalized solid phase such as plastic or glass which has been modified so that the surface carries carboxyl, amino, hydrazide, or aldehyde groups can also be used. In general such matrices comprise any surface wherein a iigand-binding agent can be attached or a surface which itself provides a ligand attachment site.
As used herein, "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. A pharmaceutically acceptable carrier encompasses any of the standard pharmaceutically accepted carriers, such as phosphate buffered saline 2o solution, water emulsions such as an oil/water emulsion or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. The invention 2s also provides for pharmaceutical compositions together with suitable diluents, preservatives, solubilizers, emulsifiers and adjuvants. Other embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including but not limited to intravenous, intramuscular, parenteral, pulmonary, nasal 3o and oral.
As used herein, an "effective amount" is the amount required to achieve a clinically significant effect. For example a significant reduction of infection, or reduction of cell growth or reduction of tumor size is a reduction of preferably of at least 30 percent, more preferably of at least 50 percent, most preferably of at least 90 percent. Accordingly, the effective amount will vary with the subject being treated, as well as the condition to be treated. For the purposes of this invention, the methods of s administration are to include, but are not limited to administration cutaneously, subcutaneously, intravenously, parenterally, orally, topically, or by aerosol.
The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or 1 o more of a polypeptide analog or fragment of the provided peptide or peptide composition, a peptidomimetic composition thereof as described herein as an active ingredient. A cocktail of the provided pharmaceutical composition in various combinations is also contemplated.
The preparation of therapeutic compositions which contain polypeptides, analogs is or active fragments as active ingredients is well understood in the art.
Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and 2o compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
25 A polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, 3o tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
The therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit s dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
to The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and ~ 5 are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at 20 one or more hour intervals by a subsequent injection or other administration.
Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
As used herein, the term "synthetic amino acid" means an amino acid which is chemically synthesized and is not one of the 20 amino acids naturally occurring in nature.
25 As used herein, the term "biosynthetic amino acid" means an amino acid found in nature other than the 20 amino acids commonly described and understood in the art as "natural amino acids."
As used herein, amino acid residues are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino 3o acid residue, as long as the desired functional property is retained by the polypeptide.
NHz refers to the free amino group present at the amino terminus of a polypeptide.
COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. Abbreviations for amino acid residues are used in keeping with standard polypeptide nomenclature delineated in J. Biol. Chem., 243:3552-59 (1969).
It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-s terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
Amino acids with nonpolar R groups include: Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan and Methionine. Amino acids with to uncharged polar R groups include: Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine and Glutamine. Amino acids with charged polar R groups (negatively charged at Ph 6.0) include: Aspartic acid and Glutamic acid. Basic amino acids (positively charged at pH 6.0) include: Lysine, Arginine and Histidine (at pH
6.0).
Amino acids with phenyl groups include: Phenylalanine, Tryptophan and Tyrosine.
is Particularly preferred substitutions are: Lys for Arg and vice versa such that a positive charge may be maintained; Glu for Asp and vice versa such that a negative charge may be maintained; Ser for Thr such that a tree -OH can be maintained; and Gln for Asn such that a free NHz can be maintained. Amino acids can be in the "D" or "L"
configuration. Use of peptidomimetics may involve the incorporation of a non-amino 2o acid residue with non-amide linkages at a given position.
Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid 2s in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces -turns in the protein's structure.
As used herein, "pM" means picomolar, "nM" means nanmolar, "uM, means micromolar, "mM" means millimolar, "ul" or "ul" mean microliter, "ml" means milliliter, "1" means liter.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. While the invention is described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art.
EXAMPI ES
EXAMPLE 1: Molecules specifically expressed in murine stem/progenitor 1 o cells Protein tyrosine kinases and phosphatases expressed in murine hematopoietic stem cells have been previously identified (Matthews, W., et al. 1991;
Matthews, W., et al. 1991; Dosil, M., et al. 1996). These molecules play important roles in hematopoiesis and development (Dosil, M., et al. 1996; Mackarehtschian, K., et al. 1995;
Shalaby, F., et al. 1995; Kabrun, N. , et al. 1997). The present invention contemplates an even more global approach in order to identify molecules specifically expressed in the murine stem/progenitor cell hierarchy. For most of these studies purified (AA4.1+Lin-/loSca+ckit+) fetal liver cells were used. This population is approximately 1,000-fold 2o enriched for in vivo repopulating activity (LTRA) measured by competitive repopulation (LyS.I/Ly5.2 congenic system) (Moore, K.A., et al. 1997) . All myeloid and lymphoid lineages in primary and in secondary recipients are repopulated by these cells. This degree of enrichment is comparable to the current "state of the art." Other primitive members of the stem/progenitor cell hierarchy share this same cell surface phenotype.
These include: ( 1 ) LTCIC or cobblestone area forming cells (Ploemacher, R.E., et al.
1989; Ploemacher, R.E., et ai. 1991) , (2) CFU-blast progenitors , (3) HPP-CFC
progenitors (Lowry, P.A., et al. 1995) and (4) stromal-dependent B-lymphoid progenitors (Whitlock, C.A., and Miiller-Sieburg, C.E. 1990) . The AA4.1+Lin-/loSea-ckit+
subset is depleted of LTRA but contains significant in vitro progenitor activity. In contrast, no 3o stem/progenitor cell activity is found in the AA4.1 subset (Jordan, C.T., et al. 1990) .
Short-term (5-7 days) cytokine cultures of stem cells were used to generate committed progenitor populations at the expense of LTRA (Traycoff, C.M., et al. 1996;
Peters, S.O., et al. 1995; Knobel, K.M., et al. 1994; Yonemura, Y., et al. 1996). In summary, several WO 00/1116$ 28 PCTNS99/19052 cell populations were defined which represent the beginning, the middle and the end points of the hematopoietic hierarchy. This sets the stage for a comparative analysis of gene expression patterns. A goal of the present invention is to complement the physical and functional phenotypes of stem/progenitor cells with profiles of uniquely expressed genes. It was hypothesized that some of these gene products contribute to the unique biological properties of primitive stem/progenitor cells and therefore are regulators of self renewal, proliferation, commitment and other processes.
There are a number of ways to compare gene expression profiles which are available to one of skill in the art. It is possible to do this by exhaustive sequencing of to representative cDNA collections obtained from stem cell and mature cell sources followed by "electronic subtraction". This approach has several drawbacks.
Most importantly, the number of sequences which must be obtained is prohibitive.
For a homogeneously pure population this number is on the order of 50,000 (based on approximately 10-20,000 expressed genes in an average cell type and a statistical calculation). In practice, even the most purified stem cell population is heterogeneous.
Stem cell enrichment values are only meaningful in relation to an unenriched standard and cannot be converted into an absolute stem cell number. It has been documented that cell populations with the same cell-surface phenotypes can differ in biological activity .
The unique properties of stem cells also suggest caution when extrapolating from 2o expressed gene numbers in other cells. In short, the extent of sequencing necessary to ensure complete coverage of gene expression in stem cells is not possible to estimate.
Normalization procedures (Uchida, N., et ai. 1995; Patanjali, S.R., et al.
1991; Soares, M.B., et al. 1994) designed to "equalize" the mRNA abundance classes are not advisable because they obliterate potentially important quantitative expression differences.
Additionally, a high-throughput sequencing effort is not applicable to numerous libraries.
Comparisons of gene expression in diverse sources of stem cells will provide valuable information. An elegant technique, Serial Analysis of Gene Expression (SAGE) permits the rapid acquisition of thousands of DNA sequence "tags" (Zhang, L., et al.
1997) . This technology was not considered herein because the size of each sequence "tag"
is very 3o small ( 10 base pair, bp). Therefore, SAGE is only informative in two extremes; exact nucleotide matches or no matches to sequences in the databases. This limits database comparison to the same species from which the "tags" originate. A key component of the strategy presented herein relies on broad bioinformatic database comparisons.
In addition, even with a specific "tag" one still needs to obtain a full-length cDNA clone for functional studies. Analysis of gene expression can be done in single progenitor cells after these form a colony "start" (Brady, G., et al. 1990; Brady, G., and Iscove, N.N.
1993). Replating of the sibling cells in a "start" colony allows the approximation of the lineage potential present in the starting cell. This technique suffers from several drawbacks. First, it is limited to 3', non-coding ends, thus preventing protein database comparisons. Second, the technique relies on cell growth, thus it is not suitable for analysis of quiescent cells. Moreover, approaches to allow colony-formation by true stem cells with a retention of primitive properties are in their infancy (Ball, T.C., et al. 1995;
to Trevisan, M., et al. 1996). Third, the technique does not take into account stochastic models of stem cell behavior (Ogawa, M. 1993). To accurately reveal physical and functional properties of stem cells it is wise to analyze populations where stochastic differences would average out. Single-cell derived cDNA populations could however, provide valuable "adjunct" material for more refined gene expression screens.
There are many techniques to physically identify differentially expressed genes. For these and other reasons, most notably the technical and economic ease with which physical pre-enrichments of cDNA libraries can be achieved, the studies described herein began with cDNA libraries which are highly enriched in differentially expressed sequences. The integration of individual techniques served to overcome the inherent limitations of each 2o technique. Three strategies were successfully employed: ( 1 ) Differential Display (DD) (Liang, P., et al. 1994; Bauer, D., et al. 1993), (2) Representational Difference Analysis (RDA) (Braun, B.S., et al. 1995; Hubank, M., and Schatz, D.G. 1994;
Diatchenko, L., et al. 1996) and (3) standard subtractive hybridization (Li, W.-B., et al. 1994;
Harrison, S.M., et al. 1995) . The latter underlies the present invention. A key feature is that the differentially expressed cDNAs have a high probability of being full-length.
This facilitates a rapid transition to functional studies. The two former techniques were utilized because of "visual" nature (DD) and the ability to generate representative, differentially expressed probe populations (RDA) in a rapid manner.
Murine stem cell eene expression rofiles. As a first step, a series of high quality, 3o representative cDNA libraries were generated. The cDNA populations were directionally cloned into the pSport-1 or pSport-2 plasmids (BRL-Gibco). The most important libraries originate from purified stem cells. In one case, enough AA4.1+Lin /IoSca+ckit+cells were purified to allow construction of a non-based library using standard methodologies. This library contains 4x106 independent recombinants (average cDNA insert size of 1-2 kilobases, kb). A second library was constructed using a new PCR-based technology called cap-finder (Clontech) designed to yield full-length cDNA copies. cDNAs ranging from 1 to 4 kb were commonly attained using this technique. For cap-finder procedures the purified cells were processed into DNAse-I
digested, poly-A+ mRNA according to microscale procedures routinely in use.
The synthesis of cDNA was done with an aliquot of mRNA corresponding to approximately 20,000 cell equivalents. It has not been necessary to use less material, therefore this is not a lower Limit. A Not I restriction site was included in the 3' reverse transcriptase o primer (cap-finder, version 2) to facilitate directional cloning. An aliquot of the cDNA
was amplified for varying PCR cycle numbers, and analyzed by Southern blots (pseudo-Northerns) for the presence of full-length copies of b-actin, GAPDH, CD 18, flk2/flt3, cdk4, CD34 and other mRNAs. Optimal cycle numbers were used to amplify the remaining cDNA. The cDNA was cloned into the pSport-1 plasmid. The AA4.1+Lin-~ 5 /loSca+ckit+ cap-finder library contains ~3 x 106 independent recombinants. Single-pass sequence analysis of random clones from this library indicated that 95% were full-length (based on sequences with an exact match in Genbank). Much more extensive sequencing of numerous clones from subtracted libraries has confirmed this. Other libraries constructed in similar ways include two libraries from AA4.1 cells, and two libraries from 2o AA4.1+Lin-/IoSca-ckit+ cells. Finally, a library was constructed from AA4.1+Lin-/loSca+ckit+ cells cultured for 5-7 days in a differentiation promoting cytokine cocktail (IL3, IL6, KL). Competitive repopulation and in vitro progenitor assays confirmed a complete loss of LTRA with a significant retention of progenitor cells. All of the cDNA
libraries are large (> 2x106 independent recombinants) and of high quality {1-2 kb 25 average insert size). In summary, the most primitive, intermediate and most mature members of the hematopoietic hierarchy were "converted" into representative panels of expressed genes.
Subtractiv-~ Hybri ization. These cDNA libraries were used in subtractive hybridizations to enrich for differentially expressed genes. Target libraries from 3o AA4.1+Lin-/loSca+ckit+ cells were subtracted with an AA4.1 driver cDNA
library. This yields a population of cDNA clones which is enriched in sequences expressed in primitive stem/progenitor cells but not in mature cell types. In practice, a single-stranded target library is hybridized to an excess of in vitro synthesized biotinylated RNA from the driver library as described . The opposite orientations of the cloning sites in pSport-1 and 2 insure target and driver complementarity. The target library was enzymatically converted to a single-stranded form using gene II protein and exonuclease III
(Gene-Trapper protocols, BRL-Gibco). Previously, this was done by infection with M13 helper phage often resulting in a bias for small cDNA inserts. Here, driver/target combinations were subtracted two times in order to facilitate the removal of commonly expressed sequences (verified by elimination of "housekeeping" genes such as b-actin and GAPDH). Concomitant enrichment of known, differentially expressed genes is also verified. Generally flk2/flt3 and CD34 probes were used. Both are expressed in the to AA4.1+Lin-/loSca+ckit+ subpopulation but not in the AA4.1 population.
Following subtraction, the relative number of clones is reduced by up to 200-fold. In some cases (subtractions with AA4.1 material) the number of clones which "survive" the subtraction is on the order of 10-20,000. Because individual sequences may be represented more than one time, this does not necessarily imply that there are 10-20,000 differentially is expressed genes. The exact number of unique sequences (complexity) in the pool of subtracted clones must be determined. A more thorough discussion of complexity is found in a subsequent section. These subtracted libraries should be enriched for sequences expressed in the primitive portion of the stem/progenitor cell hierarchy; that is in stem cells and/or in primitive clonogenic progenitor cells. Two other subtracted 20 libraries, potentially enriched for sequences expressed in the most primitive stem cell but not in clonogenic progenitors were derived by subtracting the AA4.1+Lin-/loSca+ckit+
libraries with material from the closely related AA4.1+Lin-/IoSca-ckit+
subpopulation.
Each subtracted library is arrayed at high density onto nylon membranes. Each clone in the array has a unique address in microtiter plates. A density of 20-30,000 clones 25 on a 22 x 22 cm. membrane is practical. Because of the inherent "noise" in any subtraction scheme, a positive selection criterion may be imposed in order to focus on true differentially expressed sequences. Details are presented in a subsequent section. To analyze the subtracted libraries, a high throughput sequencing effort was employed using three libraries. These are: 1) two AA4.1+Lin-/loSca+ckit+ cell libraries (standard and 3o cap-finder) subtracted extensively with AA4.1 cell material and 2) a standard AA4.1+Lin-/loSca+ckit+ library subtracted with AA4.1+Lin-/IoSca-ckit+
material. An average of 400 by of 5' sequence was obtained from about 1000 clones. To facilitate a rational handling of sequence information and to focus attention on a small number of clones for functional analysis a "flow of information" strategy was devised.
(See, Figure 1).
A major component of this strategy is bioinformatics. This can be global (comparisons with outside databases) and local ("in-house" analyses within an individual library or comparison of several libraries). A relevant example of the latter is comparison of genes from murine stem cells with genes derived from their human counterparts.
Global bioinformatic analysis provides much information. First, it establishes if a given sequence corresponds identically or closely to an already identified gene in the mouse, human or other mammalian species. Such homologies can be detected at the nucleotide io level. This can provide evidence of hematopoietic expression for a previously described gene. Second, where the homologies are statistically significant but not identical, novel members of gene/protein families can be identified. Third, a wider net can be cast over the databases by using conceptual translation of a sequence in the homology comparisons.
The Examples provided herein illustrate the power of this approach in revealing similarities to proteins from invertebrate organisms such as Drosophila, C.
elegans, and even yeast. In many cases these proteins have functions which have been uncovered by the analysis of mutants. Based on protein homologies "virtual links" are drawn between developmental regulation in invertebrates (such as in germ-line development) and in hematopoietic stem cells. The Notch/Notch ligand pathway first defined in invertebrate 2o cell-fate determination and recently implicated in hematopoietic regulation is a good example .
A number of other putative proteins which share homology with key Drosophila proteins have been identified. Bioinformatics facilitates the recognition of peptide motifs such as EGF-like repeats, Ig-like domains or Zn-finger modules. Fourth, because the databases are annotated, predicted protein sequences can be assigned to cellular processes such as signal transduction pathways or apoptosis. It is also possible to categorize clones according to potential involvement in other mammalian stem cell systems such as the intestine. Fifth, it is feasible to perform virtual expression studies and to construct overlapping EST contigs which can yield virtual full-length cDNAs.
3o The following discussion summarizes the general murine findings and highlights a panel of "interesting" subtracted clones. A number of full-length sequences have been determined. Bioinformatic analysis summaries on a collection of 863 clones derived from AA4.1+Lin-/loSca+ckit+ libraries subtracted with AA4.1 RNA are shown in Table 1.
The present invention encompasses numerous bioinformatic search and comparison parameters. This is the first analysis of its kind in the hematopoietic system and several important points emerge. First there is a high proportion (~SO%) of novel sequences. Sixteen percent do not show homologies in any EST database queried.
A
preliminary analysis of a small sequence set from an AA4.1+Lin-/loSca+ckit+
library depleted of sequences in common with the AA4.1+Lin-/loSca-clot+subset has indicated an even higher percentage of novel genes. These are higher percentages than would to emerge from a random analysis of an unsubtracted library. This was directly addressed by performing these analyses on a similar number of sequences selected at random from one of the mouse EST databases. Second, among the ~50% of the clones which are significantly homologous to previously identified genes or proteins, the proportion of homologies to "housekeeping" genes is low. This underscores the effectiveness of the subtraction strategy. Third, an internal analysis of the data base has revealed few redundancies. This illustrates the degree of gene expression diversity between the two endpoints of the mouse hematopoietic hierarchy; thus supporting the starting hypothesis.
A recent report has suggested that many sequences in mature blood cells are expressed in primitive, non-committed cells (Hu, M., et al. 1997). The data indicate that many of these 2o are removed by the subtraction; thus uncovering a large, previously not described set of genes. Fourth the sequences with homologies to known genes or proteins can be subdivided according to protein families and putative function. Interestingly, a large percentage (32%) fall into the signaling protein category. Examples of these are described herein below.
As part of a more sophisticated bioinformatics approach, automation of database searches, information cross-referencing, and annotation was employed. An illustrative example is automated weekly database queries with the sequence set. New, previously-unidentified homologies were automatically noted and reported. In the mouse studies several genes were encountered whose expression would be predicted to differ between 3o the AA4.1+Lin-/loSca+ckit+ and the AA4.1- populations. Both murine CD34 and flk2/flt3 were identified in the sequenced population. This provides a good internal control for the screening strategy. As shown in Table 2, a "short list" of identified WO 00/11168 34 PC'T/US99/19052 molecules was generated based on bioinformatics and in many cases, expression-specificity verification. The features of some of the molecules merit discussion, The SA49P1 clone is homologous to sex comb on midleg; a member of the Drosophila Polycomb group of zinc-finger transcriptional repressors (Bornemann, D., et al. 1996). Polycomb proteins are regulators of homeobox (HOX) genes and maintain the developmental stability of transcriptional states (Simon, J. 1995) . There is currently great interest in HOX gene function in the biology of hematopoietic stem cells (Sauvageau, G., et al. 1995; Lawrence, H.J., et al. 1996) . The C4-80 gene is homologous to the Drosophila cornichon gene (Roth, S., et al. 1995) . Cornichon is required during to oogenesis for the induction of follicle cells, which provide the environment that supports oocyte development. Cornichon is a component of the Drosophila EGFR signaling pathway (Neuman-Silverberg, F.S., and T. Schupback 1996). The LL2-12 gene is similar to the Drosophila neurogenesis gene brainiac. Mutants defective in this extracellular gene product show neural hyperplasia. Brainiac also plays a role in oogenesis (Goode, S., et al.
t5 1992; Goode, S., et al. 1996) . Mosaic experiments show that brainiac is required in the developing oocyte. Brainiac involvement in the Drosophila EGFR pathway has been suggested. The LL2-35 gene is similar to the Drosophila germ cell-less gene (Jongens, T.A., et al. 1992) . The product of this gene is required for the specification of the Drosophila germ line. The ectopic expression of germ cell-less causes somatic cells to zo adopt the characteristics of pole cells (destined for the germ line). The homologies of the above clones to Drosophila genes were identified at the predicted amino acid level. This underscores the utility of the bioinformatic approach. In all four cases the cDNAs represent novel unpublished murine genes. Clearly, the involvement of three of the Drosophila genes in the ultimate stem cell system, the germline, coupled with the 25 identification of homologous genes expressed in mouse hematopoietic stem /progenitor cells gives food for thought. Four other cDNAs identified are homologous to the Drosophila genes dishevelled and smoothened (mouse homologs are already described) as well as to ketch and discs large (not previously described in the mouse).
DD116 was originally isolated in a DD comparison of AA4.1+ vs. AA4.1 cells. A cDNA clone 3o identified with the DD116 probe was sequenced; the predicted protein is homologous but not identical to the murine beige gene product. The beige mutation in the mouse causes bleeding, immune system disorders and a coat-color phenotype (Perou, C.M., et al. 1996;
Fukai, K., et al. 1996; Barbosa, M.D., et al. 1996). Murine beige is thought to be the homologue of a gene responsible for the Chediak-Higashi syndrome in human.
This novel gene is likely to be the second member in the beige family; it is designated herein as "taupe". Two of the cDNAs in Table 2 were identified in a DD comparison of AA4.1+Lin-/IoSca+ckit+ cells and cytokine differentiated, cultured cells. One focus was molecules whose expression disappeared with the loss of stem cell activity in culture.
Cyt28 and Cytl9 fulfilled this criterion and were used to isolate full length clones which have been completely sequenced. Cyt28 encodes a novel, seven-transmembrane domain receptor in the same family as mouse F4/80 and human CD97 (secretin receptor superfamily) (Hamann, J., et al. 1995; Baud, W., et al. 1995) . Cyt 19 is a novel putative to methyltransferase. In both of these cases protein family assignations were made after extensive bioinformatic analyses. At least in the case of Cyt28, an antibody will be a useful reagent because it may provide a means to further subdivide the stem/progenitor cell population. Such antibodies are provided by the present invention. The cDNA SA61 is similar to a newly discovered molecule called p62dok. p62dok is a tyrosine ~ 5 phosphorylated protein which binds to rasGAP and is likely to be a common target for numerous tyrosine kinases including Abl and ckit (Carpino, N., et al. 1997;
Yamanashi, Y., 1997) . Interestingly, p62dok is also constitutively phosphorylated in hematopoietic progenitor cells from chronic phase CML patients (Carpino, N,, et al. 1997) .
The predicted protein encoded by SA61 appears to be a second member of this protein class.
20 Other representative molecules are listed in Table 2. Two cDNAs were identified as homologous to putative apoptosis regulators (SBSA56 and LLS-68, two cDNAs homologous to genes translocated in leukemias (LLS-03 and B2-67), several homologies to putative chromatin proteins (C4-23, C3-25 and LL2-89) and a LIM-domain encoding cDNA (LL5-96). Most interestingly, three eDNAs (B3-77, C2-48, and LL2-76) are 25 homologous to genes expressed in intestinal crypts or during intestinal development.
Isolated cDNAs were hybridized to slot blots representing globally amplified cDNAs from pools of individual progenitor and mature cells. Because the developmental potentials of these cells have been measured (by replating of siblings) it is possible to graphically represent the expression of a gene in various stages of the hierarchy. Three 3o examples are shown in Figures 2A-2C. The Smc-34 cDNA is a completely novel sequence with a predicted leucine zipper and several potential membrane spanning domains. These examples underscore the value of "interfacing" the above-described approach with a single cell approach. Thus, it was demonstrated that a given gene is WO 00!11168 36 PCTNS99/19052 expressed in at least multipotential progenitor cells and other members of the progenitor cell hierarchy.
~fuman stem cell gene expression profiles. It is also a goal of the present invention to identify genes expressed specifically in human stem cells. It is useful to use a mufti-species (mouse and human) approach to define stem/progenitor cell-specific gene profiles. One goal is to integrate mouse and human information. However, rather than proceeding directly to human homologs of the mouse genes already identified, an independent human effort was initiated, The rationale for this is mufti-faceted. First, it is possible that some aspects of human and mouse stem cell biology are regulated in to different ways. Although it is likely that most regulatory pathways will be conserved, it must be kept in mind that many properties ascribed to the most primitive stem cell population have been rigorously proven only in the mouse. Clearly a human lymphoid-myeloid stem/progenitor cell exists. However, the exact degree of proliferative capacity (the ability to give rise to oligoclonal hematopoiesis) as well as the exact spectrum of differentiation potentials of human stem cells have not yet been accurately measured.
Several potential differences between mouse and human have already been mentioned.
One additional indicator of differences may be the difficulty of gene-transfer into stem cells from large animals (Larochelle, A., et al. 1997). This is not likely to result solely from the quiescent status of the most primitive cells. Second, while it is usually possible 2o to find human homologs for individual mouse genes by manipulating hybridization stringency, such conditions will vary for different genes. Therefore, to find human homologs for a large pool of mouse genes (i.e. 100) may be more labor intensive and costly than to independently determine a sequence profile of differentially expressed genes from human stem/progenitor cells. Relationships to the murine panel can then be determined electronically where it is easy to manipulate comparative parameters.
Clearly, for some individual mouse genes it will be of great importance to physically identify human homologs. In some cases it may be possible to use existing human EST
databases to quickly obtain the sequence of a human homolog. Third, the availability of a large panel of human sequences specifically expressed in stem/progenitor cells lends itself to the application of various chip and array technologies. Such technologies will be instrumental in identifying which subsets of human stem cell specific genes are up or down-regulated in the highly clonogenic stem cells from diseases such as Acute Myelogenous Leukemia (AML) and other leukemias.- Highly purified human stem cells for cDNA libraries were obtained from normal donor BM. CD34+ cells were obtained from the mononuclear fraction using an immuno-magnetic affinity device (Miltenyi MACS column) and stained with a mixture of monoclonal antibodies (mAbs) designed to identify both primitive and mature cell surface markers. To identify antigens on mature cells a lineage (Lin) cocktail of FITC conjugated mAbs was used .
These mAbs were directed against: CD3, CDllb, CD15, CD19, CD20, glycophorin A, and CD71 (CD3, CD15, CD19 and CD20 from Becton Dickinson, CDllb and glycophorin A
from Coulter, and CD71 from Ortho Diagnostics). To identify primitive cells mAbs were used which were directed against CD34 (biotin conjugated and detected via a 1o streptavidin-allophycocyanin reagent; Coulter) and either CD38 (phycoerythrin conjugated, Becton Dickinson) or CD90 (Thyl) (phycoerythrin conjugated;
Pharmingen).
The stained cells were analyzed using a dual laser Becton Dickinson FACStar Plus flow cytometer. Cells of the desired phenotype were sorted into siliconized tubes.
To obtain material from cultured cells, CD34+ enriched populations (isolated as described above), t5 were first cultured in serum-free medium (IMDM, 5 mg/ml low density lipoprotein, 2 mM glutamine, human serum albumin, insulin, and transfernn, ) supplemented with IL-3 (Sng/ml), KL (25ng/ml), and FL (25ng/ml). After 1-4 days of culture, the cells were purified as described above. Human libraries were constructed using the cap-finder technology, version 2. Amplified cDNAs originating from several independent BM
2o donors were pooled. Representative cDNA libraries have been constructed from the following sources: ( 1 ) BM CD34+Lin- cells (7.1 X/ 05 independent recombinants), (2) BM CD34+Lin-CD38+ cells (1.9X106 independent recombinants), (3) BM CD34- cells (1.6X106 independent recombinants), (4) CB CD34+ cells (4.3X105 independent recombinants}, and (5) CB CD34- cells (2.9X105 independent recombinants). The 25 average cDNA insert size in all libraries is 1-2 kb. According to the comparative biological properties of the source material used for the mouse and human cDNA
libraries, the following parallels can be drawn: (1) mouse AA4.1+Lin-/loSca+ckit+
human CD34+Lin-, (2) mouse AA4.1+Lin-/loSca-ckit+ = human CD34+Lin-CD38+ and (3) mouse AA4.1 - human CD34-. Thus, in both species, collections of clones 3o representing the beginning, the middle and the end-points of the hematopoietic hierarchy have been generated. Using procedures described above, the human libraries were subtracted and arrayed 3,000 clones from two libraries: (1) BM CD34+Lin-subtracted with BM CD34+Lin-CD38+ material (refereed to as: 38-)38+) and (2) BM CD34+Lin-subtracted with BM CD34- material (referred to as: 38-). Before initiating a sequencing effort pilot studies were performed to improve the resolution of the screen.
Specifically, in order to develop techniques which would eliminate as much non-specific "noise" as possible from the libraries. RDA (PCR-Select, Clontech) were used . There are several salient features of RDA. First, it is rapid (2-3 days) and applicable to small cell numbers (ie. several thousand). It does not require the generation of cloned cDNA
libraries therefore, numerous comparisons can be made simultaneously. Second, it yields short cDNA fragments (generated by 4-cutter restriction enzymes) which are representative of to differentially expressed mRNAs. Third, it is based on the suppression PCR
technique which prevents overamplification of abundant messages (Diatchenko, L., et al.
1996) . In addition, sequences expressed in both populations in an RDA comparison do not amplify exponentially. Differentially expressed populations of cDNA fragments are not obtained by physical enrichment but rather by selective PCR amplification. Fourth and most importantly, it simultaneously yields two populations each representing differentially expressed genes in one of the two starting samples. This bi-directionality is valuable because as discussed below it can simultaneously reveal up and down regulated genes.
RDA comparisons were made between CD34+Lin- and CD34+Lin-CD38+ and between CD34+Lin- and CD34- cDNAs. The latter comparison was verified by hybridization. As 2o shown in Figure 3A (right panel), the control, non-subtracted RDA cDNA
population (38 contains b-actin sequences which are missing in the two subtracted RDA
populations, 38- and 38-)38+. A differentially expressed gene (HDD-2, described below) is enriched in the 38- RDA population and at least retained in the 38-)38+ RDA population (Figure 3A, left panel). Two, bi-directional, RDA cDNA populations (38- and the converse 34 )38) were used to probe (See Figure 3) duplicate arrays of a subtracted 38-library (Figure 3B). The correct RDA probe hybridizes to considerably more clones than the incorrect probe (compare the greater numbers of spots in the left as compared to the right panel).
Hybridization signals for each clone are doublets due to the arraying technique.
Hybridization signals with the incorrect probe (right panel} suggest that further 3o improvements to this strategy are worth pursuing. Because the subtractions are based on different technologies, it was reasoned that clones which "survived" the library subtraction and hybridized preferentially to the "correct" RDA probe would be more likely to represent true differentially expressed genes. Preliminary data from the mouse studies had already suggested that this was indeed the case. The arrayed clones were also hybridized to probe populations from the entire starting non-subtracted libraries in order to eliminate residual cDNA clones corresponding to abundant mRNA species.
In one analysis, a total of 77 clones (49 from the 38- subtraction and 28 from the 38-)38+ subtraction) which "obeyed" the RDA criteria were sequenced from the 5' end.
In order to verify expression specificity, a series of pseudo-Northern blot analyses were performed. cDNA populations from independent purified sources of material was amplified. This is of particular importance with an outbred species such as humans. In these experiments additional material from CD34+Lin-CD90+ (Thyl+) (three separate l0 purifications) and their CD90- (Thyl-) counterpart subpopulations was also included.
Cytokine-cultured samples were also included. These were CD34+Lin- cells and their CD38+ counterparts purified after 1, 2 and 4 days of culture. The collective panel of cDNAs also includes four independently purified CD34+Lin- populations and their CD34+Lin-CD38+ counterparts. All of these ampiified cDNAs have been arranged on numerous pseudo-Northern blots. In short, this allowed us to evaluate the expression of a given "sequence tag" in stem cell populations isolated by two different criteria, from a number of independent donors and after cytokine culture. Several interesting genes emerged from the sequence data set. Individual clones obtained from the -38-)38+
subtraction are designated 38 letterlnumber (i.e., 38A1) while those from the 38-)34+
subtraction are designated 34 letter/number (i.e., 34A1). One clone (38B5) was identified and determined to be human flk2/flt3. A second interesting clone (34B4) is closely related to a gene encoding TINUIt (Figure 4A). Clone 34B4 may be a novel variant of TINUR due to a 25 amino acid, in-frame deletion. TINUR was identified as an orphan member of the steroid receptor superfamily (NGFI-B/nur77 subfamily)(Okabe, T., et al.
1995). TINUR has also been implicated in apoptosis. An additional clone (34F4 ) is highly homologous to DAP-kinase. This protein is a serine-threonine kinase which has been implicated in cytokine (IFN- -induced apoptosis (Deiss, L., et al. 1995) . Clone 34F4 (DAP-Kinase) and 34B4 (TINUR) both exhibit a stem-cell restricted expression pattern. Clearly, the identification of two genes whose products are implicated in 3o apoptosis and whose expression is largely restricted to human stem cells is of interest. In addition, a cDNA (34A5) was identified which is closely related to the MLF1 gene which is a translocation partner in t(3;5)(q25.1;q34)(Yoneda-Kato, N., et al. 1996) . This translocation is associated with Myelodysplastic Syndrome (a stem cell disease) and AML. The sequence homologies and restricted expression pattern of 34A5 is shown in Figures 4B and 4C. In Figures 4C and 4D (and also SA, SB, and 6B) there are twenty-one samples of capfinder-amplified cDNA from various hematopoietic populations.
From left to right these are: four CD34+Lin- populations, three CD34+Lin-CD90+
populations, two CD34- populations, four CD34+Lin-CD38+ (obtained from the same BMs as the CD38- samples in lanes 1-4), two CD34+Lin- samples (obtained from the same BMs as the CD90+ samples in lanes 5 and 6), three CD34+Lin- populations obtained after 1, 2 or 4 days of culture and finally their three CD34+Lin-CD38+
counterparts. A recent study shows that, at least with some types of AML, the disease to can be transferred into NODSCID mice only by the leukemic CD34+CD38-subpopulation (Bonnet, D., and Dick, J.E. 1997) . These demonstrate MLFI
expression in normal stem cells. As shown in Figure 4D, nucleophosmin (NPM), the partner in this translocation was also identified. This is an example of non-specifically expressed "noise" in the screens. The 3862 cDNA is closely homologous to the LTG9/MLLT3 t5 gene located on 9q22 and involved in t(9;11) leukemia (Iida, S., et al.
1993) (Figure 4E).
A degree of stem cell expression specificity has also been observed. All of the above cDNA clones are likely to be identical to the homologous, previously identified genes.
However, the suggested involvement of DAP-kinase and TINLTR in apoptosis necessitates their inclusion in any comprehensive consideration of stem cell apoptotic pathways.
2o Similarly, the expression of two genes associated with myeloid leukemias bears on any speculation regarding the primary transformation target cell as well as the origins of the ultimate clinical phenotype in these and other leukemic disorders.
Other known genes to were found that exhibit identity or very close homology including: (I) GOS3, a fos-related gene (Heximer, S., et al. 1996) (Figure 5A) and (2) 25 HLA-DR (Figure 5B). GOS3 shows a specific expression pattern, while HLA-DR
expression appears to be more variable. The expression status of Class II MHC
on the most primitive human BM stem cell population is not entirely clear. It has been suggested to be present in a primitive, multipotent progenitor population but not in the most primitive stem cells (Sutherland, H.S., et al. 1989; Verfaillie, C., et al. 1990) . If 3o true, this may suggest an additional negative selection parameter for future experiments designed to subdivide the stemlprogenitor cell hierarchy. Of the first 75 sequences, 22 have no homologies in the databases or homologies only to ESTs. Expression analyses on these clones are in progress. One gene of particular interest is called HDD-2 as well as 34B5, 34E1 and 38A11. The three latter designations reflect its isolation from both the 38- as well as the 38-)38+ subtracted libraries. The designation HDD-2 reflects its independent isolation in a limited DD "first look" at molecular differences in the purified cell populations. The likely full-length sequence of HDD-2 is 500 bp. It contains a short open reading frame of 89 amino acids (SEQ.ID.No.: 7i). The predicted peptide sequence is shown in Figure 6A. The 3' cDNA sequence contains a poly-A tail preceded by the canonical AATAAA poly-adenylation signal. Neither the nucleotide nor the predicted protein sequences of HDD-2 show homologies in any known gene or EST
database. The expression profile of HDD-2 shown in Figure 6B, demonstrates that it is to stem cell restricted. Also shown below (Figure 6C) is HDD-2 hybridization to a dot blot with numerous human pA+ mRNA samples (Clontech). HDD-2 hybridization is only visible in kidney (the other "spots" are background). It was confirmed that corresponds to a single-copy human gene by genomic Southern blot (Figure bD).
In summary, the results of this very low throughput human sequencing effort substantiate the overall approach; that judicious pre-enrichments and selections will result in rapid identification of biologically interesting and often novel genes. Most importantly, these studies firmly establish the existence of genes whose expression correlates with the most primitive stem cell phenotype.--Other data were generated using (1) a murine stromal cell line to support enriched 2o human stem/progenitor cells, and (2) tetracycline regulatable retroviral expression vectors. The stromal cell line AFT024 is efficient in long-term, in vitro maintenance of LTRA in purified murine fetal liver and adult bone marrow populations (Terstappen, L.W.M.M., et al. 1991) . Additional data demonstrated highly-efficient retroviral-mediated gene-transfer into murine LTRA as well as into primitive in vitro progenitors during the AFT024 cocultures. Further, it has been shown that AFT024 is very effective in supporting ELTCIC. Specifically, the CD34+CD38- immunophenotype as well as the functional capacity of these cells is maintained. The latter was measured in limiting-dilution replating experiments. Moreover, it has been shown that limiting numbers of human CD34+Lin- cells can give rise to both B cells and NK-cells when cultured on 3o AFT024. However, the supportive activities of AFT024 on mouse and human stem/progenitor cells have been indistinguishable. In addition, more than 500 sequences have been analyzed from an AFT024-specific subtracted cDNA library. A number of candidate stem cell regulators have been identified. Three of these are dlk (Moore, K.A., et al. 1997), a novel BMP/TGF-b superfamily member and a novel selectin-related molecule. The present invention contemplates identifying human stem/progenitor cell receptors and/or ligands for the AFT024 specific proteins.
In order to facilitate functional studies of stem cell gene products several retroviral s gene-transfer vector systems were constructed and characterized. All of these employ the 293T cell retroviral packaging system (Kinsella, T., and Nolan, G. 1996). High titers of virus can be produced transiently without the time and labor consuming effort required for stable producer cell lines. Large cDNA populations can also be converted into virus populations (Kitamura. T., et al. 1995). It is preferable to have inducible (or repressible) vectors which are also selectable. Also, the single-transcription unit tetracycline (tet) repressible vector was modified (Hofmann, A., et al. 1996). This vector includes an enhancer/promoter deletion in the 3' LTR. The tet system is currently one of the best inducible expression systems available; regulation over a several hundred-fold range of expression is observed (Shokett, P.E., and Schatz, D.G. 1996) . Cloning sites were 15 introduced in order to insert cDNAs in a sense or antisense orientation. A
green fluorescent protein (GFP) marker was also included in these vectors driven by a thymidine-kinase promoter (TK-GFP) (Yang, T., et al. 1996; Cheng, L., and Kain, S.
1996). The cDNA fragment of interest is under tet regulation while GFP is constitutively expressed in transduced cells. In order to confirm this, NIH3T3 cells are infected with a 20 LacZ virus and GFP~ cells sorted. The GFP+ cells express LacZ in the absence of tet, while LacZ expression is undetectable in most cells after the addition of tet.
The titer of this model construct is approximately l04/ml. which is suitable for tissue culture studies.
'table 1. Known genes (or extensive as homology):
436 (50..~%) Grouped by function by nucleotide: 407 Signaling/receptors: 133 by amino acid only: 29 Translational/post-: 62 Structural: 49 to mouse: 230 Transcriptional/post-: . 45 to human/other: 206 Cell fate: 35 Other. 87 Unknown function: 25 Novel genes: 427 (49.5%) Homologous only to expressed sequence tags: 288 (33.3%) No homology to any known nt or as sequences: I 39 ( I6.2%) Table 2.
~ueative familr Clone hlethod Cells compareddotes 7-transctumbnne Cyt-28 difL SC' fresh vs. secretin R supetfamiiy receptor display 7d-cultured MethyltransferaseCyt-19 dill. SC fresh vs.
display Td-cultured C3-54 subtraction5C vs. AAd' Aspartyl proteaseSAT subtractionSC vs. AAt' r Signal tnnsductionSA6l subtractionSC vs. AA4' dok fancily member molecules LL2-02 subtractionSC vs. AA4' BTK associated beige-related DDt dill. AAd' vs. AA4' contains WD repeat proteins 16 display Transcriptional SA.i9PlsubtractionSC vs. AAd' polyeomb homol.
regulators LLS-96 subtractionSC vs. AA4' includes LIM domain G-protein signalingLL.t-39subtractionSC vs. AA4' cell cycle transition B l-66 subtractionSC vs. AA.t' similu to BL34 Apoptosis-relatedSBSA56 subtractionSC vs. AA4' SVtT3A-related genes LL3-68 subtractionSC vs. AA4' sim. to Requiem transcript. factor Chromatin proteinsC.a-23 subtractionSC vs. AA4' yeast HST3like C3-25 subtractionSC vs. AA4' NHP2like LL2-89 subtractionSC vs. AAd' yeast SIS2-like Other stem cells B3-T7 subtractionSC vs. AAi' homologous to A4 (intestinal gene crypts) C2-48 subtractionSC vs. AA.t- homologous to C101 LL2-76 subtractionSC vs. AAd' homologous to EDPF

Genes involved LLS-03 subtractionSC vs. AAd' t(1:11)(q21:q23) in leukemogettic translocations B2-67 subtractionSC vs. AA4' t(X:14)(q28:q11) Homologues of LL235 subtractionSC vs. AAt' germ cell-less Drosophila developmental LL2-l2 subtraction5C vs. AA4' brainiac gents C.t-$0 subtractionSC vs. AA.t' cornichon B.i-1s subtractionSC vs. AA4' discs-large ~The dcsignanon "SC" refers to sorted day Lt fetal liver cells having the phenotype AAi. I' Lin''Sca cktt'.

FI~A_MPL li; 2: Molecular cloning and characterization of AA4, an early marker of hematopoietic development In this example, the expression cloning and molecular characterization of AA4, a surface marker expressed on hematopoietic stem and progenitor cells is described. The results demonstrate that AA4 is a 130kDa type I glycosylated membrane protein whose structural organization suggests a role in cell adhesion. Expression analysis showed that high levels of AA4 are found in lung, heart, and bone marrow. It is not found in undifferentiated ES cells, but its expression is upregulated as these cells differentiate into colonies of hematopoietic precursors and endothelial cells. In the hematopoietic system, t0 expression of AA4 correlates with the expression of stem cell markers CD34 and CD43.
Functional studies revealed that AA4 coimmunoprecipitates with CD34 and CD43, suggesting that these proteins form a macromolecular complex which functions in the regulation of cell adhesion, proliferation and/or differentiation of hematopoietic cells.
Introduction Monoclonal antibody AA4.1 was first described more than a decade ago (McKearn et al., 1984) and since then it has become a useful marker for the isolation and analysis of hematopoietic cells (McKearn et al., 1985; Jordan et al., 1990;
Fujimoto et al., 1996). A number of works have shown that the antigen recognized by AA4.1 is present on a subset of primitive hematopoietic progenitors found at various stages of development 2o in sites of active hematopoiesis in yolk sac (Cumano et al., 1993; Auerbach et al., 1996;
Yoder et al., 1997), AGM region (Godin et al., 1995; Marcos et al., 1997), fetal liver (McKearn et al., 1985; Jordan et al., 1990; Cumano and Paige, 1993), and bone marrow (Li et al., 1996; Szivassy and Cory, 1993). In yolk sac, AA4-positive cells are first detected at day 8-10 of gestation (Cumano et al., 1993; Sanchez et al., 1996).
At day 9-10 of gestation AA4+c-Kit+ progenitors are found in the P-Sp/AGM region (Sanchez et al., 1996; Marcos et al., 1997), and by day 14 of development, AA4 defines 0.5-1.0%
of the fetal liver tissue that contains the entire hierarchy of primitive hematopoietic cells (Jordan et al., 1990). Proliferation within each successive compartment results in increased total number of progenitor cells. Antigen density per cell also increases with developmental 3o progress, which is especially marked for c-Kit and AA4 (Marcos et al., 1997). In bone marrow, HSC are found in both AA4+ and AA4- subpopulations, although in adult marrow AA4 is largely regarded to be a marker of early B lymphoid lineage. In addition, recent studies demonstrate that the expression of AA4 parallels the onset of hematopoietic development in differentiating ES cells (Kabrun et al., 1997; Lin and Neben, 1997;
Potocnik et al., 1997). Taken together, these results indicate that AA4 plays an important role in hematopoiesis and has to be studied in more detail.
In order to achieve this goal, AA4 was molecularly cloned and characterized.
The results demonstrate that AA4 is a 130kDa type I transmembrane glycoprotein whose structure suggests a role in cell adhesion. Expression analysis showed that high levels of AA4 are found in lung, heart, and bone marrow. In the hematopoietic system, expression of AA4 correlates with the expression of stem cell markers CD34 and CD43.
Functional 1o studies indicate that AA4 coimmunoprecipitates with CD34 and CD43, suggesting that these proteins form a macromolecular complex which may function in the regulation of cell adhesion, proliferation and/or differentiation of hematopoietic cells.
Results Cloning n, f AA4. In order to identify AA4, AA4.1 monoclonal antibody was used to screen various cell lines and primary hematopoetic cells. The murine B
lymphoid cell line D2N was found to expresses relatively high levels of AA4 antigen (see Table 3).
Immunoprecipitation of protein extracts prepared from D2N cells showed that AA4.1 recognizes a protein with apparent molecular weight (Mr) 130kDa (Figure 7).
This protein was also present in extracts prepared from B lymphoid cell line M2.4, and AA4 2o positive hematopoietic cells derived from bone marrow and fetal liver.
To isolate AA4, a cDNA library was prepared from D2N cells and cloned into the polylinker region of a retroviral expression vector REBNA (see Materials and Methods).
Following production of retroviruses, NIH 3T3 cells were infected with the recombinant cDNA library and selected for AA4 expression by flow cytometry using PE-conjugated 2s AA4.1. After two rounds of sorting, genomic DNA extracted from AA4-positive cells was analyzed by pcr amplification using viral vector primers. This resulted in the amplification of a 3.1 kbp cDNA which was gel-purified and subcloned for further analysis. Infection of NIH 3T3 fibroblast or EML C1 hematopoietic cells with REBNA/AA4, a recombinant retrovirus expressing the cloned cDNA, has lead to the 3o acquisition by cells of high affinity to AA4.1 mAb (Figure SA). In infected cells, AA4.1 detects a 130kDa surface protein which comigrates with the endogenous AA4 from cells (Figure 8B), thus indicating that the cloned cDNA encodes AA4.

Seauence analvsis ofAA4. Sequence analysis of the cloned cDNA (SEQ. ID
No.: 72) showed that it has a single open reading frame encoding a protein of 644 amino acids (see Figure 9)(SEQ. ID No.: 73. The deduced amino acid sequence includes a putative leader peptide and the mature protein which starts at position 20.
The protein contains a long N-terminal extracellular region, a single putative hydrophobic transmembrane region, and 47 amino acids of the C-terminal cytoplasmic domain.
The extracellular part of AA4 is composed of two major structural moieties. The N-terminal region contains a C-type lectin domain (CTL) which has 32% sequence homology to endothelial cell receptor thrombomodulin. This region is followed by a cysteine-rich t0 domain composed of six epidermal growth factor (EGF)-like repeats, three of which are consistent with the calcium-binding EGF motifs. Similar repeats are found in the extracellular domains of a large number of membrane-bound proteins and in proteins known to be recreated (Bork et al., 1996).
Database searches revealed that AA4 exhibits high homology to C 1 qRP, the is human receptor for complement component Clq expressed on surfaces of myeloid cell lineage and endothelial cells (Nepomuceno et al., 1997). Sequence alignment showed that AA4 and C 1 qR have approximately 68% identical amino acid positions and similar domain structures. Highest homologies were found within the N-terminal parts of the proteins and their C-terminal cytoplasmic domains (see Figure l0A and lOB), suggesting 20 that AA4 and C 1 qR may share functional properties.
The amino acid sequence of AA4 contains numerous potential O-linked and N-glycosylation sites. Although the predicted Mr of AA4 is 67.4 kDa, two protein bands exhibiting Mr lOSkDa and 130kDa respectively, are immunoprecipitated by AA4.1 mAb in cells infected with REBNA/AA4 (Figure 8C). When 35S-labeled cells are chased with 25 nonradioactive media, the intensity of the lOSkDa band rapidly decreases, while the intensity of the 130kDa band increases, thus indicating that p130 is the mature form of the protein, whereas p 1 OS is its precursor. In agreement with this conclusion, immunoprecipitation of biotinyated cells using AA4.1 reproducibly results in the detection of a 130kDa surface protein (see Figure 8B).
30 ~xinression inatterns ofAA4. Northern blot analysis showed that in adult mouse tissues, AA4 is expressed at high levels in lung, heart, and bone marrow. No detectable expression was found in brain, testis, spleen, and thymus (Figure 10B). In normal tissues and transformed cell lines, a 7kb RNA species hybridizes with the cloned cDNA
(Figure 11A and 11B). In addition to the 7kb species, poly(A)-RNA from D2N cells contains a minor band corresponding to a 3.2 kb mRNA (see Figure 11A, lane 8). Similarity search against expressed sequence tags (ESTs) showed that databases contain at least seven different sequences corresponding to the 3'-untranslated region of the cloned aa4cDNA
which were isolated from the following mouse tissues: colon (Genbank accession No AA929174), heart (AA435107), lymph node (AA185911 and AA267407), lung (AA220480), mammary gland (AI021507), and spleen (AA145088). Sequence identity with the ESTs abrogates upstream of nucleotide G at position 2481 in the 3'-untranslated 1o region of aa4cDNA, thus indicating that the cloned cDNA corresponds to an alternatively spliced 3.2 kb aa4mRNA.
~;xnression ofAA4 in hematonoietic ~ro~enitor cells AA4 is produced in murine hematopoietic progenitor cells and immature B lymphocytes found at various stages of development in yolk sac (Godin et al., 1995; Marcos et al., 1997), fetal liver (MeKearn et t 5 al., I 985; Jordan et al., 1990), and bone marrow (Cumano et al., 1992; Li et al., 1996). To confirm that the cloned cDNA encodes AA4, RT-PCR was performed on hematopoietic cells fractionationated using several different techniques. The cells analyzed included AA4+ and AA4- fetal liver (FL) cells; AA4+ FL cells fractionated into Linloc-Kit+Sca-1+ and Linloc-Kit+Sca-1- populations; and hematopoietic progenitors isolated from bone 2o marrow (BM) by cell sorting using combinations of different surface markers.
Figure 12A shows that aa4 was amplified from AA4+ FL cells, whereas in AA4-cells it was only present at low levels. AA4 expression was high in Linloc-Kit+Sca-1+
cells enriched for HSC activity. In adult marrow cells, aa4 was amplified from Lin+ cells and Lin-c-Kit+Sca-1+CD34+ multipotential progenitors. At lower levels aa4 was present 25 in Lin-c-Kit+Sca-1+CD34- long term reconstituting HSCs (Figure 12B). In all tested cells aa4 expression correlated with the expression of stem cell markers CD34 and CD43.
Embryonic stem (ES) cells which have been shown to generate progenitors for most hematopoietic lineages during differentiation in vitro (Keller, 1995) were also tested in this experiment. Figure 12C shows that aa4 was not found in undifferentiated ES cells 3o but its expression was upregulated as these cells differentiated and formed blast cell colonies (BL) and colonies of more differentiated hematopoietic cells (HMT).
These results are in line with previous studies which showed that AA4 is expressed in ES-derived hematopoietic precursors (Kabrun et al., 1997; Ling and Neben, 1997;
Scott et al., 1997) and show that this expression is not abrogated upon differentiation of blast cell colonies into endothelial cells (ENT in Figure 12C).
Fcto~ic xnressiorr ~,f~~has no mitoQerric effect EML C1 and NIH 3T3 cells expressing exogenous AA4 did not exhibit morphological changes indicative of alterations in their growth properties. To examine the effect of AA4 on growth phenotypes in more detail, NIH 3T3 and primary mouse embryo fibroblasts were infected with a retrovirus expressing AA4 and maintained in high and low serum conditions.
FACS analysis of the transduced cells confirmed that in each case the efficiency of to infection was close to 100%. However, examination of growth rates showed that overexpressed AA4 had no apparent effect on proliferation of both cell types as compared to control uninfected cells or the corresponding cells infected with a retrovirus expressing GFP.
~A4 coimmrmonrecinitates mith CD34 and CD43 To investigate interactions ~ 5 with other proteins, hematopoietic and fibroblast cells expressing AA4 were imrnunoprecipitated with AA4.1 mAb and then examined by immunoblot analysis using a panel of antibodies directed against membrane-associated proteins. This analysis revealed that AA4 coimmunoprecipitates with CD34, a membrane glycoprotein selectively expressed within the hematopoietic system on stem and progenitor cells, and CD43 which 2o is a major O-glycosylated sialomucin found on the surfaces of most leukocytes. This result is in line with previous studies which showed that CD43 coimmunoprecipitates with the human C 1 qRP (Guan et al., I 991; 1994). Figure 13B shows that in the murine D2N and EML C1 cells, a 52kDa protein is the major isoform of CD43 that associates with AA4. This 52kDa protein was found to be reactive with both the N-terminal (S 19) 25 and C-terminal (M19) anti-CD43 antibodies, indicating that it is not a breakdown product.
In NIH 3T3 fibroblasts coexpressing CD43 and AA4, a 54kDa and a 170kDa CD43 isoforms coimmunoprecipitated with AA4 pointing to the glycosylation differences brtween CD43 expressed in different cell types. A 115kDa CD43 isoform which previously have been shown to be sialylated and thus overly negatively charged (Guan et 3o al., 1994) did not form macromolecular complexes with AA4 in NIH 3T3 cells.
Similarly, AA4 did not coimmunoprecipitate with a 115kDa CD43 isoform found in D2N cells and a 120kDa isoform found in EML C1 (Figure 13B).

To examine associations of AA4 with CD34, both proteins were expressed in Rat-1 cells following infection with the corresponding retroviruses. See Figure 13C.
In Rat-1 cells coexpressing AA4 and CD34, AA4.1 the results also demonstrate that AA4 coimmunoprecipitates with CD34. Studies have suggested a role for both CD34 and CD43 in the regulation of adhesion, growth and differentiation of hematopoietic precursors (Ardman et al., 1992; Bazil et al., 1997; Chen et al., 1996; Suzuki et al., 1996;
Wood et al., 1997; Stockton et al., 1998). Coexpression of AA-Fc fusion protein in NIH
3T3 cells stably producing AA4 showed that AA4-Fc forms heterodimeric complexes with AA4. Figure 11A shows that equimolar amounts of AA4 and AA4-Fc were to coprecipitated from these cells by protein A, indicating that AA4 is prone to homo- or heterodimerization.
Discussion This Example describes the expression cloning and sequence analysis of AA4, a molecular marker expressed on hematopoietic stem and progenitor cells. The CDNA
encoding AA4 was isolated from a retroviral cDNA library prepared from the murine D2N lymphoid cell line. Sequence analysis of the cloned eDNA revealed that AA4 is a type I transmembrane protein composed of 625 amino acids. The extracellular part of the molecule contains two major structural moieties, a C-type lectin carbohydrate recognition domain and six EGF-like domains. Similar repeats have been found in a large number of 2o membrane-bound proteins or in proteins known to be secreted. The cytoplasmic domain of AA4, in contrast, bears no structural similarity with known protein families. Instead, AA4 revealed strong homology to CIqR, the human receptor for complement component Clq which is predominantly expressed in phagocytic cells such as monocytes, neutrophils, and endothelial cells (Nepomuceno et al., 1997). Sequence alighnment shows that AA4 and CIqR have approximately 68% identical amino acid positions and share similar domain structure. Highest homologies were found within the Nterminal parts of the two proteins and their C-terminal cytoplasmic domains, suggesting that AA4 and CIqR may have similar functional properties. Expression of AA4 correlates with the expression of CD34 and CD43, two other surface markers normally present on murine hematopoietic 3o stem and progenitor cells. Previous studies have shown that AA4-positive cells are first detected in yolk sac at day 8-10 of gestation (Cumano et al., 1993; Sanchez et al., 1996).
At day 9-10 pc AA4+c-Kit+Mac-I+ progenitors are found in the P-Sp/AGM
region (Sanchez et al., 1996; Marcos et al., 1997), and by day 14 of gestation, AA4 defines 0.5-1.0% of the fetal liver tissue that contains the entire hierarchy of primitive hematopoietic cells (Jordan et al., 1990). As proliferation within each successive compartment results in increased total number progenitor cells, antigen density per cell also increases with developmental progress, which is especially marked for c-Kit and s AA4 (Marcos et al., 1997). A significant proportion of hematopoietic progenitors from yolk sac also express CD34, CD38, CD43, CD44, and Mac-1 however show little or no expression of CD4, CDB, CD45R and Sca-. These same markers are present on FLderived HSC which also express Sca-1. Adult marrow HSCs also express cKit, CD38 and Sca-1, but do not normally express Mac-I or AA4. In BM, HSC are found in both AA4+
and to AA4-subpopulations, although in adult marrow AA4 is largely regarded to be a marker of early B lymphoid lineage 0. These results indicate that the expression of cell surface antigens changes on HSC during ontogeny and that differential display of theses cell surface markers may reflect relationship between HSCs that contribute to multilineage hematopoiesis and distinct anatomical sites during development.
15 Methods ~.11s and tissue culture. NIH 3T3 fibroblasts were grown in DME medium supplemented with 10% fetal calf serum (FCS). D2N cells were grown in RPMI

medium containing 10% FCS. EML C1 cells were grown in IMDM supplemented with 20% horse serum and 8% BHK/MKL conditioned medium (Tsai et al., 1994). To 2o maintain the multipotentiality of EML Cl, the cells were kept at low density (0.5 - 5x10-5/ml) and subcultured every two days. Cell lines constitutively expressing AA4 and GFP
were derived from NIH 3T3 or EML Cl cells by infecting with the corresponding recombinant retroviruses.
Retroviral-mediated gene ~ n_~. Retrovirus expression vector REBNA was 25 constructed by substituting the LacZ gene contained within the EcoRI-Notl fragment of plasmid LZRSPBMN-Z (Kinsella and Nolan, 1996) with a synthetic polylinker composed of EcoRl, Xhol, Sfil, and Notl sites. Retroviral vector REBNA/IRESGFP contains a poliovirus IRES element and the CDNA encoding color-enhanced GFP (S.
Zolotukhin, Gainesville, FL) inserted into REBNA.
30 For DNA transfections, cells were plated at a density 2x106 cells per 60mm dish and transfected with Sug of plasmid DNA using 20u1 of lipofectamine reagent (GibcoBRL). REBNA-transfected cells were selected in puromycin (2ug/ml) and grown to confluence prior to collecting virus supernatant. For infections" the culture medium was supplemented with polybrene (Sigma) at Sug/ml. The appropriate virus was added and incubated overnight. Multiple infections were carried sequentially, with the appropriate selection between.
Isolation o.t RNA and CDNA cloning. Poly(A) RNA prepared from D2N cells s was converted into CDNA using Superscript II Reverse Transcriptase (GibcoBRL) and an oligo(dt) primer containing NotI site, S'-TGGTGTCGACGCAGAGTAGCGGCCGCT18 (SEQ.ID.No.:74). The second strand was synthesized using DNA polymerase I in combination with E. coli RNAse H and E. coli DNA lipase as described (Gubler and Hoffman, 1983). An adaptor composed of complementary oligonucleotides, S'-to GGCCCGGGCCGGCC (SEQ.ID.No.:75) and S'-TCGAGGCCGCCCGGGCC
(SEQ.ID.No.:76), was ligated to the CDNA and cut with Notl to produce CDNA
molecules with NotI and Xhol termini for directional cloning. After size fractionation in agarose gel, eDNAs larger than 2.5 kbp were Iigated into Notl and XhoI cut plasmid REBNA and electroporated into electrocompetent DH12S cells (GibcoBRL). Plasmid 15 DNAs were transfected into 293-derived packaging cell line for retrovirus production.
Virus-containing supernatants were collected and stored at -80C.
NIH 3T3 cells infected with the recombinant retroviruses representative of D2N
CDNA library were selected for the production of AA4 by flow cytometry using phycoerythrin-conjugated AA4.1 mAb. After two rounds of sorting, genomic DNA
2o isolated from AA4-positive cells was subjected to pcr amplification using retroviral vector primers, S'CAGCCCTCACTCCTTCTC (SEQ.ID.No.: 77) and S'-GGTGGGGTCTTTCATTCC (SEQ.ID.No.: 78) (Kitamura et al., 1995). Amplified CDNA was gel purified and subcloned into pbluescript SK and REBNA plasmid vectors.
Nucleotide sequences were analyzed using NCBI Blast database search programs and 25 ExPASy molecular biology server from the Swiss Institute of Bioinformatics.
Northern blot, hybridization. RNAs prepared using acid guanidinium thiocyanate-phenol extraction procedure (Chomczynski and Sacchi, 1987) were separated.on formaldehydeagarose gels and blotted onto the Hybond-N nylon membranes (Amersham). Hybridization probes were derived from cloned cDNAs using Ready To 3o Go DNA labeling beads {Pharmacia Biotech). Hybridizations were performed as described previously (Petrenko et al., 1997).
Cell labeling and Immunonrec_ipitations. The ECL protein biotinylation system (Amersham) for the detection of cell surface proteins was used as recommended by the manufacturer. For radioactive labeling, 2x106 cells were incubated with 200uCi Translabel (ICN) in 2m1 of cysteine and methionine-deficcient medium for 2hr at 370C.
Cells were washed in PBS and lyzed l Omin. on ice in 500u1 of NP40 buffer containing 2OmM TrisHCI pH 7.6; ISOmM NaCI; 0.5% NP40; ImM PMSF; SmM benzamidine;
ImM sodium vanadate; LOug/ml aprotinin; IOug/ml leupeptin. Lysates were cleared by centrifugation and tug of soluble antibody were added to the supernatants followed by 25u1 of Protein G-Sepharose suspension (Sigma). After 2 to 4hr incubation at 40C with rotation, protein Gantibody complexes were pelleted and washed in the successive changes of wash buffer I (1M NaCI; IOmM TrisHCI pH 8; 0.1 % NP40); wash buffer (O.IM NaCI; IOmM TrisHCI pH 8; 0.1 % NP40); wash buffer 3 (IOmM TrisHCI pH 8;
0.1 % NP40). Samples were eluted by boiling 2min. in 2Xge1 loading buffer, separated by SDS/PAAG, dried and exposed to X-ray film.
Western blot analysis. Protein extracts for Western blot analysis were prepared as described (Mornson et al., 1991 ). The antibodies used included goat anti-mouse CD43 ~5 polyclonal IgG (M19 and S-19, Santa Cruz) in combination with HRPconjugated secondary antibodies and ECL detection system (Amersham).
Flow c_~~~e~ and RT-PCR. Timed-pregnant mice and 5- to 7-week-old female mice (C57BI/6j) were purchased from Jackson Laboratory (Bar Harbor, ME). AA4-positive cells were isolated from day 14 fetal liver by immunopanning on Petri dishes 2o coated with AA4.1 antibody (l0ug/ml). Hematopoietic stem cells were purified from AA4-positive fraction by staining with lineage-specific antibodies as described previously (Moore et al., 1997). Three-color fluorescence-activated cell sorting for lineage negative to low, Sca-I(+), c-Kit(+) cells was performed on a multilaser FACS Vantage with CeIIQuest software (Beckton Dickinson). ES cells differentiated into blasts cell colonies, 25 hematopoietic progenitors, and endothelial cells prepared as described previously.
For RT-pcr, poly(A)-RNA isolated from sorted cells was converted into CDNA
using Superscript II Reverse Transcriptase (GibcoBRL) and CapFinder CDNA
amplification kit (Clontech). Gene-specific primers for pcr amplification included: 5'-TTCAGCAAGCCCTGACTC (SEQ.ID.No.:79) and 5'GCCACCTTCGAAGCAATC
30 (SEQ.ID.No.:80) (AA4); 5'-GAGCGGTACAGGAGAATG (SEQ.ID.No.:81) and 5'GCCCACCCAACCAAATCA (SEQ.ID.No.:82) (CD34); 5'-ACCGCGTTCTTCTGTAAC (SEQ.ID.No.:83) and S'CAGCTAACAGCAGGATCC

WO 00!11168 53 PCT/US99/19052 (SEQ.ID.No.:84) (CD43); G3PDH Control Amplimer Set (Clontech) for the amplification of GAPDH.

EXAMPLE 3: IN VITRD MAINTENANCE OF HIGHLY PURIFIED, TRANSPLANTABLE HEMATOPOIETIC STEM CELLS
The cellular and molecular mechanisms which regulate even the most primitive hematopoietic stem cell are not well understood. This example details a systematic dissection of the complex hematopoietic microenvironment in order to define some of these mechanisms. An extensive panel of immortalized stromal cell lines from murine fetal liver was established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacities. This example describes a t0 long-term in vitro culture system, utilizing a single, stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations. Disclosed is multi-lineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells which were cultured for 4-7 weeks on AFT024. The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population. The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in short-term cytokine-supported cultures. In addition, the maintenance of input levels of transplantable stem cell activity is accompanied by the expansion of other classes of stem/progenitor cells.
This suggests that the stem/progenitor cell population is actively proliferating in culture and 2o that the AFT024 cell line provides a milieu which stimulates progenitor cell proliferation while maintaining in vivo repopulating activity.
Introduction Mammalian blood formation originates in a small population of hematopoietic stem cells. The hallmark features of these cells are: (1) a hierarchical multilineage differentiation potential with the ability to clonally give rise to at least 8 distinct cell lineages, (2) self renewal capacity which is reflected in the life long continuous activity of few, in some cases single, stem cells and (3) a dramatic proliferative potential which is ultimately responsible for the production of large numbers of mature blood cells. (Leminschka, LR.
1992;
Morrison, S.J. et al. 1995; Harrison, D.E. 1980). During the past decade much progress has 3o been made in providing a physical phenotype for this rare population of stem cells.(Spangrude, G.J., et al. 1995; Lemischka, LR. 1992; Bauman, J.G., et al.
1998).
However, currently, the only reliable functional assay system for the most primitive stem cell compartment is long-term in vivo transplantation. No in vitro system has been developed which adequately recapitulates stem cell behaviors. Therefore, the cellular and molecular mechanisms that regulate the biology of stem cells have remained obscure.
A major challenge in stem cell research is the establishment of culture systems which facilitate in vitro maintenance of long-term transplantable stem cell activity. This is a necessary first step towards a cellular and molecular understanding of the regulatory mechanisms which mediate commitment versus self renewal decisions. Moreover, the establishment of such culture systems is a prerequisite for the potential expansion of undifferentiated stem cell populations as well as for the generation of stemlprogenitor cells committed to selected lineages.
to Efforts to develop culture systems for the maintenance of transplantable stem cells can be subdivided into two broad categories: ( 1 ) those utilizing defined cytokine combinations as the only culture supplements and (2) those relying on a pre-established stromal monolayer as an additional supportive component (with or without exogenously added cytokines). Both of these strategies have met with only limited success.
In the first case, it has been repeatedly demonstrated that combinations of cytokines can exert potent stimulatory effects on stem/progenitor populations. (Spangrude, G.J., et al.
1988; Jordan, C.R. et a(. 1990; Fleming, W.H., et al. 1993). In some studies, highly purified stem cells (Li, C.L., and Johnson, G.R. 1990; Spangrude, G.J. and Johnson, G.R. 1990). have been used and the direct effects of cytokines have been demonstrated at the single cell level. (Jones, R.
2o et al. 1990; Uchida, N. et al. 1993). While informative, the vast majority of these studies are limited by their strictly in vitro nature. Thus, it is feasible to expand, replatable in vitro progenitor populations (Li, C.L., and Johnson G.R. 1992; Uchida, N. et al.
1993) and to stimulate colony-formation by cells with both myeloid-erythroid and lymphoid potentials, Jones, R., et al. 1990; Uchida, N. et al. 1993; Trevisan, M., and Iscove, N.N.
1995; Ogawa, M. 1993) however, the equivalence of these progenitor cells with the in vivo transplantable stem cell population remains speculative. Several studies have clearly demonstrated a dramatic loss of in vivo repopulating potential as a result of cytokine driven in vitro proliferation.(Knobel, et al., 1994; Peters, et al., 1995; Traycoff, et al., 1996). A small number of studies have shown that defined cytokine combinations promote the maintenance of transplantable activity.(Rebel., et al., 1994). However, most of these are limited both by the use of very short culture periods, the exact nature of the in vivo assay, and the use of non-enriched stem cell sources.(Muench, et al., 1993; Holyoake, et al., 1996;
Soma, et al., WO 00/11168 56 PCC/iJS99/19052 1996). This precludes interpretations suggesting a direct action of the given cytokine(s) in maintaining transplantable activity.
A further complication with defined cytokine studies is the inability to ascribe in vivo physiological relevance to the observed effects. It has long been accepted that in the intact animal, stem cells are found in close association with discrete cellular microenvironments.(Lord, et al., 1975; Trentin, et al., 1970; Weiss, et al., 1991; Wolf, 1979).
These observations suggest both the existence of stem cell niches and the notion that in vivo stem cell regulatory mechanisms are likely to require cell-cell contact or short range interactions.(Dorschking, 1990). Efforts to understand the features of the hematopoietic to microenvironment began with the establishment of the Dexter long-term culture (LTC) system. (Dexter, et al., 1977). In this culture system hematopoiesis is maintained for weeks or months by a heterogeneous adherent cell monolayer derived from bone marrow (BM).
While some degree of transplantable stem cell maintenance and self renewal (Eraser, et al., 1990) has been demonstrated, a general feature of the Dexter-LTC is a dramatic net decrease t5 of stem cell activity over time. (Harrison, et al., 1987; Van der Sluijs, et al., 1993). Although much progress has been made, especially in studies of human stem/progenitor cells, (Sutherland, et al., 1989; Verfaillie, et al., 1995; Hao, et al., 1996) a further drawback of this system is the heterogeneity of the stromal cell types present in the supportive monolayer.
This hampers the identification of regulatory mechanisms. Studies have been reported zo where the heterogeneous stromal monolayer is replaced with cloned stromal cell lines.
(Roberts, et al., 1987; Kodama, et al., 1984; Issad, et al., 1993; Wineman, et al., 1993). Many of these cell lines can support in vitro myelopoiesis, (Suzuki, et al., 1992;
Neben, et al., 1993; Kodama, et al., 1992) B-lymphopoiesis (Collins, et al., 1987; Whitlock, et al., 1982) or in some cases both.Wineman, et al., 1993; Wineman, et al., 1996) However, very few 25 studies have focused on the in vitro maintenance of the most primitive transplantable stem cell compartment. Moreover, with one exception (Szilvassy, et al., 1996) the studies which have focused on the in vitro maintenance of this stem cell population begin with heterogeneous unpurified sources of hematopoietic activity. Wineman, et al., 1992, 1996;
Deryugina, et al., 1994). Such populations contain numerous non-hematopoietic stromal cell 3o types. Therefore, it has not yet been possible to assign a direct stem cell supporting phenotype to a given stromal cell line.
It was hypothesized that the rare frequency of primitive, stem cells may suggest an equally rare frequency of stem cell supporting microenvironmental niches.
Accordingly, we established and characterized a large panel of conditionally immortalized, cloned stromal cell lines from mid-gestation fetal liver. This organ was chosen because, during development, it is here that the stem cell compartment is undergoing self renewal expansion in addition to differentiation. (Moore, et al., 1970). The cell lines were generated as previously described, (Wineman, et al., 1996) by immortalization with a temperature sensitive SV40TAg. (Frederiksen, et al., 1988) The clonal nature of the AFT024, 2018, and 2012 cell lines was verified by Southern blot analysis which detected a single, unique proviral integration locus in their genomic DNA. In order to identify potentially interesting cell lines, we used a "cobblestone area" (CA) assay (Ploemacher, et al., 1989) was initiated 1o with BM taken from mice injected two days previously with 5-fluorouracil (5-FU). It has been suggested that CA colonies which appear after a prolonged culture period are derived from more primitive stem cells, possibly identical to some in vivo transplantable entities.
(Ploemacher, et al., 1991 ). Therefore, a goal was in identifying cell lines which support such late arising CAs. Of 225 lines, 77 (34%) were capable of supporting limited in vitro hematopoiesis, while, consistent with the initial hypothesis, only 2% were able to maintain long-term (>6 weeks) hematopoietic CA activity. Subsequent studies with a selected subset of these lines, showed that the ability to effectively support in vivo reconstituting BM stem cells is infrequently observed. (Wineman, et al., 1996). Two out of sixteen cell lines maintained significant levels of long-term reconstituting stem cell activity for an in vitro 2o culture period of three weeks. Several other cell lines supported low levels of such activity or transiently repopulating stem cells. The cell inoculum, in these studies, was whole BM
which was not enriched for stem cell activity. Therefore, it was not possible to suggest that the effective stromal cell lines were directly supporting stem cell activity.
This example demonstrates that a single clonal cell line, designated AFT024, can maintain quantitative levels of transplantable stem cell activity present in highly purified stem cell populations.
These data were generated using a competitive repopulation assay system, which employs uncornpromised competitor BM cells. The in vitro-maintained stem cells satisfy all criteria which currently define the most primitive stem cell population including the ability to reconstitute secondary recipients. This example also shows that the in vitro maintenance of 3o primitive transplantable stem cells is compatible with the concurrent generation of large numbers of committed progenitors.

Methods Mice. Timed-pregnant mice and 5-7 weeks old females (C57BI/6J, Ly5.2) were purchased from the Jackson Laboratory (Bar Harbor, ME). Congenic C57B1/6, Ly5.1 female mice were purchased from the National Cancer Institute (Frederick, MD).
All mice were housed in the Princeton University Barrier Animal Facility, in autoclaved micro-isolator cages on ventilated cage racks. The animals received sterile, irradiated food, and acidified, autoclaved water ad libitum.
Stromal cell lines and culture conditions. The fetal liver stromal cell lines used in this study were derived as previously described. (Wineman, et al., 1996).
Stromal cell lines to were routinely cultured in Dulbecco's modified Eagles's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), SX10-5 mol/L b-mercaptoethanol (2-ME), at 32°C, 5%
CO2, 100% humidity. Sera were obtained from Hyclone, Logan, UT. Other biochemical reagents were obtained from Sigma, St. Louis, MO. Two of the lines used in this study (2012 and 2018) were previously characterized for their ability to support long-term repopulating activity present in whole unfractionated BM. (Wineman, et al., 1996). The AFT024 cell line was identified as an additional long-term (>4 weeks) CA
supporter.
Subclones of 2012 and AFT024 were isolated and used in these studies. The AFT024 cell line has remained stable and demonstrated consistent stem cell supporting abilities for over 4 years.
2o Hematopoietic sterrL~~~urification. Stem cells were purified from day 14 fetal livers essentially as described, (Jordan, et al., 1990) with the inclusion of c-kit expression as an additional parameter. Briefly, AA4.1+ cells were isolated by immunopanning on petri dishes coated with AA4.1 antibody (10 ug/mL). The AA4.1+ fraction has been shown to contain all repopulating stem cell activity present in day 14 fetal liver. (Jordan, et al., 1990) AA4.1+ cells were collected and stained with saturating concentrations of fluorescein isothiocyanate (FITC) labeled rat monoclonal antibodies to lineage markers (CD3, CD4, CDS, CDB, B220, Gr-1, Mac-1, and TER-119). The cells were simultaneously stained with phycoerythrin (PE) labeled Ly6A/E (Sca-1) antibody and biotinylated antibody to c-kit. The latter was developed with streptavidin 3o allophycocyanin (APC). The AA4.1 hybridoma was a kind gift from Dr. J.
McKearn, Monsanto, St. Louis, MO. AA4.1 antibody was purified by ImClone Systems Inc.
New York, NY. The TER-119 antibody was initially obtained from Dr. T. Kina, Kyoto University, Japan and subsequently purchased from PharMingen, San Diego, CA.
All other antibodies were purchased from PharMingen. Streptavidin APC was purchased from Molecular Probes Inc., Eugene, OR. Three color fluorescence activated cell sorting for lineage negative to low (lin'"°), Sca-1+, c-kit+ cells was initially done on a dual laser Epics 753 cell sorter (Coulter Electronics, Hialeah, FA) interfaced with Cicero software (Cytomation Inc., Fort Collins, CO) and subsequently on a mufti-laser FACS
Vantage with Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).
Enriched fractions of BM stem cells were obtained from CS7B1/6 Ly5.1 mice as described. (Okada, et al., 1992). Briefly, BM mononuclear cells were isolated by density centrifugation over Ficoll Hypaque (<1.077, Pharmacia, Piscataway, NJ).
Lineage 1o negative or low staining cells (fin"°) were obtained by magnetic bead depletion (anti-rat immunoglobulin coated beads, Dynal, Oslo, Norway) of mononuclear cells using the same lineage cocktail described herein above. The cells were further stained with antibodies to Sca-1 and c-kit as described herein above. Sorting for fin ~~°, Sca-1+, and c-kit cells was accomplished with the Epics 753 as described above.
SfPm cell/stromal cell cocultivation and cytokine-suRp)emented susQension a r . Stromal cell lines were seeded on tissue culture dishes that had been coated with 1 % gelatin (Specialty Media, Lavallette, NJ) and were grown at 32°C, S% COz~ 100%
humidity. Confluent monolayers were irradiated (20 Gy, ' ~~Cesium source, Gammacell 40, Nordion International Inc. Ontario, Canada) and cultured in modified Dexter (Dexter, 2o et al., 1984) media (DMEM, 10% FBS, 10% horse serum, 5X10'5 moUL 2-ME, 1X10-' mol/L hydrocortisone). For Dexter-LTC, enriched hematopoietic stem cells were added and the cultures were maintained at 37° C, S% COz, 100% humidity with weekly media changes. The specific numbers of purified stem cells added to stromal cell cocultures are given in the appropriate figure or table legends. In some experiments, week 4 AFT024/stem cell cultures were harvested and replated in limiting-dilution onto fresh, irradiated (20 Gy) AFT024 monolayers in 96-well trays (Dexter-LTC conditions).
CAs were scored weekly (as described above), for an additional 5 weeks. Irradiated (20 Gy) 2018 monolayers in 96-well trays were used in limiting-dilution Whitlock-Witte assays (LD-WW) (Whitlock, et al., 1982) to assess stromal-dependent B-lymphopoiesis content of both freshly purified and AFT024 cultured fetal liver stem cells. These cultures were established in RPMI media with 5% FBS, 2 mmol/L glutamine, 1 mmol/L Na pyruvate, and SX10-5 mol/L 2-ME at 37° C, S% COz, 100% humidity. 2018 has been identified as a potent B-lymphopoiesis supporting line in W-W conditions. (Deryugina, et al., 1994).

Short-term cytokine-supported suspension cultures and short-term AFT024/stem cell cocultures were established in Iscove's Modified Dulbecco's Media (IMDM), 10%
FBS, 1% BSA, 5X10-5 mol/L 2-ME at 37°C, S% COZ, 100% humidity. Cytokine concentrations: rmflk2/flt3-ligand (FL) 30 ng/mL, rmSteel factor (SL) 20 ng/mL, rhIL-6 10 ng/mL. FL was obtained from ImClone Systems Inc.; SL was purchased from Genzyme Corporation, Cambridge, MA. IL-6 was purchased from Upstate Biotechnology Inc., Lake Placid, NY.
~ransmantation assavs for hemato~oietic stem cell activity. Competitive repopulation was used to measure stem cell activity present in both freshly isolated and cultured stem 1o cell populations. (Harrison, et al., 1993). This assay was performed using the congenic Ly5.1/5.2 mouse system (Morse, et al., 1987). Enriched stem cells were seeded onto irradiated stromal monolayers and maintained in Dexter-LTC conditions. At the end of 4-7 weeks, the cultures were harvested by vigorous trituration. Single cell suspensions were prepared by passage through 22-gauge needles, mixed with fresh congenic BM and transplanted into lethally irradiated congenic mice (10 Gy, split dose 3 hours apart, 1 Gy/min, Gammacell 40). All purified fetal liver stem cells were from Ly5.2 mice. The competitor BM and recipients were LyS.l. Purified BM cells were from Ly5.1 mice; in this experiment, Ly5.2 BM was used as competitor and Ly5.2 mice were used as recipients. In order to assess reconstitution, mice were periodically bled by capillary puncture of the orbital venous plexus. Blood (0.1 mL) was collected into heparin-containing ( 10 U/mL) DMEM and the red blood cells were lysed with NH,~CI
(Mishell, et al., 1980). For the experiments described in Figure 15 and Table 4., the nucleated cells were divided into two fractions and stained with the appropriate biotinylated Ly5 antibody and developed with streptavidin-peridinin chlorophyll protein (PerCP) and; (1) CD4-FITC, CD8-PE and (2) B220-FITC, Mac-1-PE, Gr-1-PE. Cells from each fraction were analyzed on an Epics Profile II, Coulter Electronics. For the experiments described in Figure 16 and Tables 2. and 3., nucleated cells were stained with directly conjugated lineage antibodies (CD4-PE, CD8-PE, Mac-1-FITC, Gr-1-FITC, and B220-APC) and biotinylated Ly5.2 antibody which was developed with streptavidin Texas Red (T.R.).
3o Four color analysis of stained cells was performed on either the Coulter Epics 753 or Becton Dickinson FACS Vantage with the appropriate software interfaces described above. Anti-Ly5.1 was a kind gift of Dr. H. Nakauchi, University of Tsukuba, Japan.
Purified and biotinylated Ly5.2 antibody was originally obtained from a hybridoma (AL14A2) kindly provided by Dr. G. Spangrude, University of Utah Medical Center, Sait Lake City, UT. In later experiments, the Ly5.2 antibody was purchased from PharMingen; CD4-PE, CD8-PE, and B220-APC were also purchased from PharMingen.
Streptavidin-PerCP was purchased from Becton Dickinson Immunocytometry Systems.
Streptavidin T.R. was purchased from Molecular Probes. Competitive repopulating units (CRU) per 105 were calculated according to the formula: (Harnson, et al., 1990).
CRU/105 = %Ly5 positive cells X cell number competitor BM
100-%Ly5 number of test cells to Reconstitution values of less than 2% of the test Ly5 donor allele were not considered sufficiently above background for calculation of CRU.
Retransplantation potential of stem cells was assessed by secondary transplantation. Mice from the experiment presented in Figure 14. were sacrificed 60 weeks after transplantation, BM was harvested and stained with antibody to Ly5.2 followed by streptavidin T.R. Ly5.2+ cells were collected by cell sorting (Coulter Epics 753) and used to transplant lethally irradiated secondary Ly5.1 recipients.
Marrow from mice in the Control and AFT024 groups was used to transplant mice in both radioprotection and competitive repopulation assays. Ly5.2+ BM from the primary 2012 2o transplants was used only in radioprotection assays. Primary 2018 mice did not contain sufficient Ly5.2+ cells for secondary transplantation. Transplanted mice were bled and analyzed by 4-color flow cytometry for the presence of Ly5.2~ cells and multilineage reconstitution as described above.
In vitro hemato oip etic progenitor cell assays. The progenitor content of freshly purified hematopoietic stem cell populations and AFT024/stem cell cocultures was assessed using a variety of in vitro assays. All of the following assays were accomplished with fetal liver stem cells enriched as described herein above. To determine the time course of CA development, enriched stem cells were seeded onto irradiated monolayers (300-600 cells/well in 12-well trays). CA development was followed over 3o time and characteristic clusters were quantitated as described above. At different time points of the stem cell/AFT024 cultures, individual wells were harvested and replated into cytokine-supplemented semisolid clonogenic progenitor assays (CFU-C). The cytokine-enriched (rmIL-3 10 ng/mL, rhIL-6 10 ng/mL, rmSL 50 ng/mL, Epo 3 U/mL) methyl-cellulose mixture was purchased from Stem Cell Technologies Inc., Vancouver BC, Canada. Colonies were scored after 8-14 days of culture at 37°, S% COZ
, 100% humidity according to established criteria. (Testa, et al., 1993). Colonies that reached >lmm in size after 8 days and which contained erythroid bursts and multiple myeloid cell lineages including megakaryocytes were scored as high-proliferative potential-mixed lineage colonies (CFU-HPP-Mix). (Lowry, et al., 1995). Lineage content of typical colonies was determined by Wright's/Giemsa staining of cytospin slide preparation from individual colonies. Colony assays were also done with 103 freshly purified cells. The CFU
progenitor contents of the AFT024 cocultures were normalized to an initial input of 103 t0 stem cells. To assess the ability of the AFT024 cell line to maintain primitive lymphoid progenitors, 4 week cocultures were plated into LD-WW assay on 2018 cells as described above. Resulting pro-B cell colonies were scored after 7 days. The cell number in individual wells (96-well trays, 8 wells/cell number) was normalized from the original number of purified stem cells that initiated the coculture, i.e. stem cell equivalents/well.
As calculated from the line of best fit, the cell number at 37% negative wells is the frequency of pro-B cell colony initiating cells in the starting population.
(Taswell, 1981 ).
In a similar manner, the frequency of CA initiating cells in week 4 stem cell/AFT024 cocultures was also determined by replating them in limiting-dilution onto fresh, irradiated AFT024 monolayers in 96-well trays. CA were scored as described above, at 1, 2, 3, 4, and 5 weeks after replating in Dexter-LTC. The resulting frequencies were calculated as described herein above for the LD-WW assays and are also expressed in relationship to the number of stem cells that seeded the initial cultures (stem cell equivalents).
Results Ice, vivo and in vitro assa,Ys f'or stem cell activity maintained by stromal cell lines.
Highly enriched stem cell populations were used to initiate cultures supported by single stromal cell lines. The focus was on one cell line, AFT024, which exhibited particularly potent stem/progenitor cell support. Two other stromal cell lines, 2012 and 2018, (Wineman, et al., 1996) were included in some experiments. In order to more rigorously 3o establish the clonality of these lines, they were subcloned by limiting-dilution. All subclones obtained from a given cell line contained the same proviral integrant position as the parental cell line. The AFT024 cell line was evaluated both for its ability to maintain in vivo competitive repopulating stem cells, as well as a broad spectrum of stem/progenitor cells defined by a variety of in vitro assays. The in vivo assays focused on the ability of stem cells, cultured for extended time periods (4-7 weeks), to permanently reconstitute mufti-lineage hematopoiesis in transplanted hosts.
The in vitro assays included the enumeration of CA appearing over time in the initial cultures as well as the quantitation of stemlprogenitor cells which can form colonies in cytokine-supplemented replating assays. Cells from four-week AFT024/stem cell cocultures were also assayed by limiting-dilution for the content of progenitors capable of initiating secondary CAs on AFT024 or B-lymphopoiesis in Whitlock-Witte cultures supported by 2018.
to AFT024 maintains auantitative levels of long-term in vivo repo~u'ating stem cell 'vi . One line of investigation inquired if and at what levels in vivo transplantable stem cell activity was present in four to seven week-old cultures initiated with highly enriched stem cells and supported by AFT024, 2012 or 2018. Purified day 14 fetal liver cells (AA4.1+, liri ~~°, Sca-1+, c-kit+) and adult BM cells (lin'~~°, Sca-I+, c-kit+) were used as t5 sources of stem cell activity. Both of these populations are about 1000 to 1500-fold enriched for stem cell activity, as measured by competitive repopulation.
(Harrison, et al., 1993). The LyS.I/Ly5.2 congenic system was utilized for all competitive repopulation studies. (Morse, et al., 1987). The data presented in Figure 14 demonstrate that the cultures supported by AFT024 contain stem cell activity at levels quantitatively identical 2o to those present in the uncultured purified populations. In this experiment, individual Ly5.1 mice received 103 freshly purified Ly5.2 cells or the cultured equivalent of 103 purified Ly5.2 cells. Each mouse also received 10~' LyS.I competitor BM cells.
The percentage of Ly5.2 positive peripheral blood cells was approximately equal in both groups of recipient animals. Moreover, the cultured stem cell activity is as effective as 25 freshly purified activity for in vivo periods of greater than one year. The data in Figure 14 also show that the 2018 cell line is completely ineffective in maintaining highly purified stem cell activity while the 2012 cell line supports intermediate levels of repopulating activity. The data presented in Table 4A. provide quantitative competitive repopulating unit (CRU) value calculations as well as the results of multiparameter lineage analyses.
3o The extremely low levels of reconstitution by 2018-cultured stem cells precluded lineage analysis. The CRU values of the AFT024-cultured and freshly purified populations are nearly identical. Moreover, both fresh and AFT024-cultured stem cells reconstitute myeloid and lymphoid cell populations to a similar degree. In order to further access the supporting activities of AFT024 and 2012 we utilized a 10-fold lower number of fetal liver stem cells from two separate purifications to initiate the cocultures.
The cultures were continued for 4-7 weeks, harvested, and used in competitive repopulation studies.
Each recipient received the cultured equivalent of 100 purified Ly5.2 stem cells plus 4X105 Ly5.1 competitor BM cells. A total of twelve mice were transplanted with AFT024 cocultures ( four each aver four, five and seven weeks of LTC). The parental AFT024 line was used in the 4 week group and two different subclones were used to support the five and seven week cultures. The AFT024-cultured Ly5.2 stem cells contributed to 20-30% of peripheral blood cells in these recipients while cells cultured on l0 2012 demonstrated more limited in vivo function (Table 4B). The 2012 cultures were done with two subclones of the parental line and were maintained for four weeks prior to harvest and transplant (four mice/subclone). The data utilizing different cultures time or sublcones did not vary significantly from each other and are presented together in Table 4B. An additional experiment was undertaken using enriched BM which was cultured on AFT024 and 2018 for six weeks. In this experiment, BM was purified from Ly5.1 congenic mice. Each Ly5.2 recipient mouse in this study received 100 freshly purified cells, or the cultured equivalent of 100 purified cells. Both groups received 105 Ly5.1 competitor BM cells per mouse. Data analysis, for the presence of LyS.I+ cells at 4 months after transplant, is presented in Table 4C. For 6 weeks of culture, AFT024 cells maintained quantitative levels of reconstituting activity present in 100 purified BM stem cells. The 2018 cell line failed to maintain stem cell activity.
These studies were extended to include secondary transplantation as an additional assay for primitive stem cells. BM cells were harvested from the primary recipients of fresh and cultured fetal liver stem cells (see Figure 14. and Table 4A.) and the Ly5.2 positive, fetal liver-derived fraction was collected by cell-sorting.
Secondary radioprotection and competitive repopulation transplants were performed. The data are presented in Table S. The secondary recipient repopulating activities are nearly identical for the AFT024 cultured stem cells and the non-cultured controls. Lineage analysis of the Ly5.2 cells in the secondary recipients revealed similar numbers of myeloid and lymphoid cells derived from both AFT024-cultured and non-cultured stem cells.
Some level of secondary reconstituting cell activity was observed.
Additional experiments were also performed to determine where the levels of stem cell activity present in long-term AFT024 cocultures were compared to those present in short-term cytokine stimulated cultures or in short-term AFT024-supported cultures (Figure 15). Purified fetal liver cells were seeded onto an AFT024 monolayer and maintained in Dexter-LTC conditions for 5 weeks. Simultaneously, the same numbers of purified cells were cultured for 5 days with; ( 1 ) different cytokine combinations or (2) on AFT024. The transplantable activity in the cultured cells was then assayed by competitive repopulation. Each mouse received the cultured equivalent of 600 stem cells together with 4X 105 LyS.I competitor BM cells. It is evident from the data that the levels of in vivo repopulating activity present in the long-term AFT024-supported cultures are much greater than those remaining after a short-term cytokine-supported culture period.
to Of interest also is that short-term AFT024 stem cell cocultures do not maintain significant in vivo reconstituting activity. In fact, these levels of stem cell activity are identical to the levels seen in the cytokine-supported cultures.
In vitro stemlprogenitor populations are expanded by AFT024. AFT024/stem cell cocultures have vigorous hematopoiesis throughout the entire in vitro culture period.
is This is reflected in the large numbers of relatively mature hematopoietic cells which are produced throughout the culture period. In addition, CA colonies are observed throughout the culture period. Figure 16 shows a time course of CA appearance with purified fetal liver stem cells (3 separate experiments). After 28 days in culture, approximately one in every twenty input stem cells is capable of proliferating into a CA.
2o In addition, CA appearance over time follows a biphasic distribution, with many CA
observed early in the culture period. In order to enumerate the various classes of stem/progenitor cells present in AFT024 cocultures, we performed a series of in vitro replating experiments. These included the quantitation of: (l.) progenitor cells capable of colony-formation in cytokine-supplemented semisolid cultures (CFU assay), (2.) zs progenitor cells capable of initiating secondary CA in limiting-dilution AFT024 cultures and (3.) progenitor cells which can initiate B-lymphopoiesis in LD-WW
cultures. In all of these experiments the primary cultures were initiated with purified fetal liver cells. All data presented below are normalized to an initial input of 103 purified cells (CFU assay) or the actual number of initial input stem cells (limiting-dilution assays).
3o Shown in Figure 17 are the numbers and types of cytokine responsive CFU
progenitors present at various times in the AFT024-supported cocultures.
Production of CFU is evident at all time points. However, the content is especially high after four weeks, representing a 5-7 fold increase/expansion when compared to the content in the freshly purified populations. The content of more primitive progenitors (CFU-HPP-Mix) is increased by 12-fold. These HPP-Mix colonies often reach a size of 2 mm in 8 days and contain large numbers of erythroid bursts and megakaryocytes.
Interestingly, there does not appear to be a correlation between CA number and CFU content at different culture times. This is most apparent at day 6, when CA numbers are at their peak but the progenitor content is similar to that observed in non-cultured stem cells.
Furthermore, there is no correlation between CFU content and the absolute numbers of maturing hematopoietic cells present in a given culture.
Next, the content of primitive B-lymphoid progenitors present in the 1o AFT024/stem cell cultures was determined. This was accomplished by plating cells from the four-week AFT024 cocuitures into LD-WW assays over the 2018 stromal cell line.
Two experiments with freshly purified stem cells and AFT024-cultured stem cells showed that the frequency of pro-B cell progenitors is expanded 10-fold in cultures compared to the frequency observed in the freshly purified input population (day 0 frequency 1 in I 1.0, rZ= 0.98; day 28 AFT024-cultured frequency 1 in 1.1, rz= 0.97).
In order to measure the content or frequency of progenitor cells capable of initiating secondary CA, 4 separate, four-week AFT024/stem cell cocultures were replated in limiting-dilution onto fresh AFT024 monolayers. CAs were scored after one week. The data are presented in Figure 18A. Large numbers of secondary CAs were observed. When normalized to the stem cell numbers used to initiate the primary cultures (stem cell equivalents, see Methods), the frequency of these progenitors is 1 in 3 to 4.
Figure 18B. shows data from one of the 4 experiments presented in Figure 18A., where the quantitation of secondary CAs was extended for 4 more weeks. The frequency of CA
decreases slowly over time (1 in 19 after an additional 4 weeks), approximating the frequency seen in the primary cultures at four weeks. In summary, our in vitro replating assays collectively demonstrate a significant expansion of primitive progenitor populations in 4 week AFT024 cultures. In these same cultures there is no decrease in the levels of transplantable stem cell activity present in the total hematopoietic cell population.
Discussion In this example, it was demonstrated, that the AFT024 stromal cell line can maintain quantitative levels of in vivo repopulating stem cells for at least 7 weeks of in vitro culture. Highly enriched stem cell populations in low numbers ( 100 cells) were used and cell activity was measured in a stringent, competitive repopulation assay system. The cultured stem cell activity satisfies all in vivo criteria normally ascribed to the most primitive stem cell compartment; (1) long-term engraftment ability, (2) multilineage potential and (3) the ability to repopulate secondary recipients.
In addition, the studies with the low culture initiating stem cell numbers for both BM and fetal liver, imply that AFT024 stromal cells exert their supportive effects in a direct manner. These studies represent a clear example of an in vitro system capable of directly supporting the most primitive stem cell compartment.
Other data revealed that stromal cell lines isolated from a single tissue source are to heterogeneous with respect to their abilities in maintaining long-term repopulating stem cells. (Wineman, et al., 1996). It was speculated that the rare cell lines which were effective in supporting in vivo reconstituting stem cells may represent immortalized components of in vivo stem cell niches. However, the data argue for the necessity of using purified stem cell populations in order to support such a hypothesis.
Specifically, the 2018 cell line maintained transiently reconstituting activity present in unfractionated BM. However, 2018 fails to maintain measurable repopulating activity when cultured for 4-6 weeks with highly purified BM or fetal liver stem cells. Similarly, in experiments using purified cells, there was a failure to show robust levels of reconstituting stem cell activity in cultures supported by CFC034, the most effective cell line in the whole BM
2o studies (Wineman, et al., 1996). The 2012 cell line which was reported (Wineman, et al., 1996) to be effective in maintaining long-term repopulating whole BM derived stem cells is only partially effective in the present studies. Moreover, only some subclones of 2012 display such activity (in spite of identical proviral integration positions in all subclones).
Recently, studies have shown that the S 17 cell line which consistently supports the stem cell activity present in whole BM, (Wineman, et al.,. 1993) is not similarly effective in the maintenance of purified BM stem cells. (Szilvassy, et al., 1996). Taken together with these current data, the previously observed stem cell supporting stromal cell activities may reflect the actions) of indirect mechanisms and therefore do not permit the identification of cellular stem cell niche components. One previous study has shown that 3o the Sysl stromal cell line can maintain high levels of transplantable activity present in purified BM. (Szilvassy, et al., 1996). The competitor cells in that study were compromised by prior serial transplantation. Moreover, the culture period was extended for only two weeks and effective maintenance required the addition of exogenous leukemia inhibitory factor. In contrast, AFT024 is a cell line that provides a direct-acting long-term stem cell supporting environment without the addition of exogenous factors.
In addition to recovering net input levels of transplantable activity from supported cultures, significantly expanded populations of primitive progenitor cells were also obtained. CFU-HPP-Mix progenitors are expanded by 12-fold after 4 weeks of culture and the numbers of stromal-dependent pro-B lymphoid progenitors are similarly amplified. This suggests that the AFT024-mediated process of stem cell maintenance is in reality a dynamic phenomenon. Specifically, during the first portion of the culture period, the majority of transplantable stem cell activity may be lost, through l0 differentiation or cell death. The remaining primitive stem cells may expand to yield input levels of transplantable activity as well as increases in the numbers of more committed progenitors. One hypothesis is that short-term AFT024 supported cultures should contain reduced levels of transplantable stem cell activity. Figure 17 support this hypothesis. The standard Dexter-type media used in parallel long-term AFT024 cocultures was not used in these short-term cultures. However, in another short-term experiment, utilizing Dexter-LTC media, a similarly dramatic reduction in stem cell activity was observed after 4 days of culture on AFT024.63 These observations are intriguing because they suggest that the AFT024 cell line is able to facilitate some degree of ex vivo transplantable stem cell proliferation and expansion. Indeed, in other studies it 2o was shown that AFT024 can support colony formation initiated by single purified stem cells with B and T-lymphoid, myeloid and erythroid potentials. Moreover, the data suggest that is possible to efficiently introduce retroviral markers into transplantable stem cells at various times during AFT024 cocultures. Extension of such marking experiments and an analysis of proviral integration patterns will be necessary to rigorously ascertain if self renewal replication is occurring during these coculture periods.
The ability of AFT024 to maintain the most primitive stem cell compartment while generating and expanding at least some less primitive members of the stem/progenitor cell hierarchy raises interesting issues regarding the nature of stem cell niches. The present invention suggests that microenvironmental niche models which 3o postulate distinct cellular entities responsible for stem cell self renewal and other cellular entities which support the generation of committed progenitor cells may be overly simplified. (Uchida, et al., 1993). Quite clearly a single microenvironmental cell type represented by AFT024 is sufficient for keeping stem cells in an undifferentiated state as well as allowing commitment and progenitor expansion to take place. A hallmark feature of a stem cell niche is the ability to facilitate the generation of the entire stem/progenitor cell hierarchy from very primitive cells. Therefore, the main functional role of such niches may be to provide an environment which permits the production of the correct numerical balance of more and less primitive stem/progenitor cell entities.
This model contains several testable hypotheses. The most important is that in vitro stem cell maintenance should not be interpreted, literally, as the maintenance of quiescent cells but rather as a phenomenon which results from a balance of self renewal and commitment decisions which occur during stem cell division.
1o A cytokine cocktail of IL-6, SL, and FL is not effective in maintaining fetal liver stem cell activity. It was shown that RNA transcripts for these and 10 other cytokines are present in AFT024, but they are also detected at similar levels in non-supporting lines such as 2018. (Wineman, et al., 1996). These observations suggest the existence of novel AFT024-derived molecules which may act on stem cells. Indeed, using a subtractive hybridization molecular cloning strategy, a number of candidate molecules have been identified. Two of these molecules contain EGF-like repeat motifs that are most closely related to those found in the Notch/Notch-ligand family. Interestingly, one of these molecules appears to have activity on primitive stem cell populations. (Moore, et al., 1997). (See, Examples presented herein above.
2o Table 4. Multilineage stem cell activity, high CRU levels maintained in AFT024 cultures A. With 1000 purified fetal liver cells Lineage Contribution, % Ly 5.2 Cells Total Cells Week CD4 CDS B220 Myeloid % Ly 5.2 CRU/105 Control 5 3.3+1.5 4.4~1.7 48+5.1 32+6.0 28~1.3 3$6~24 12 27~10 24~8.7 63~8.9 43+9.2 44~3.2 800~102 24 42+6.9 34+4.1 71+4.4 62+1.4 48+4.8 984+171 AFT024 5 7.7~6.2 6.0~3.5 58~6.6 36+4.7 39~2.1 641~54 12 44~7.1 38+6.4 70~5.6 53~3.6 48+1.4 942+51 24 56+3.8 49+3.1 722.4 643.7 561.9 1270+96 2012 5 0.8+0.5 1.40.3 6.8+3.6 1411 7.4+2.3 528.5 12 9.1 $.4 106.9 149.3 23+20 14+4.5 19468 24 173.8 182.0 235.9 326.4 21+4.4 19671 WO 00111168 70 PC'T/US99/19052 B. With 100 purified fetal liver cells Lineage Contribution, y5.2 CellsTotal % L

Cells Week GranulocytesB-cells T-Cells % Ly5.2 CRU/105 AFT024 15 334.6 416.0 255.3 284.6 1888315 26 17+4.4 18+4.8 185.9 245.9 1825+571 46 22+5.7 19+6.6 15+6.7 20+6.5 1595628 2012 15 32+7.8 28+7.2 205.2 24+6.6 1390+382 26 102.8 9.7+3.2 9.73.2 143.9 699176 46 11+2.3 6.7+1.6 8.93.2 9.02.0 409+

C. With 100 purified bone marrow cells No. of Lineage Contribution, % Ly 5.1 Cells Total Cells mice CD4 CD8 B220 Myeloid % Ly 5.1 CRU/105 Control 8 38~7.3 30~5.8 43+6.7 31~4.8 32+6.5 583+172 AFT024 7 20~9.8 18+8.8 20~8.7 24~11 22~10 490~296 A. AA4.1 day 14 fetal liver cells (Ly 5.2) were further purified for a lin' ', Sca-1 , ckit stem cell surface phenotype. Fresh purified control cells, 103, were transplanted with 10~' Ly5.1 competitor marrow (n=6 mice). From the same purification, 10 cells were cocultured with stroma! cell lines for 4 weeks.
Subsequently, 10% of each culture was transplanted per mouse (n=8 mice/stroma) together with 10'' competitor BM cells. The contribution to each lineage in peripheral blood is expressed as the percent of the total specific lineage population that was Ly5.2'. CRU/10', relative enrichment of competitive repopulating units. Data are presented +SEM.
B. The lineage and CRU content of low numbers of enriched fetal liver stem cells maintained on AFT024 and 2012 were determined. 500 stem cells were maintained in Dexter-LTC for 4-7 weeks over irradiated monolayers. 20% of each culture was used to transplant groups of 4 mice (i.e.
each mouse received the equivalent of 100 stem cells that initially seeded the cultures) combined with 4X105 competitor BM cells.
Data are presented as ~SEM.
C. Ly 5.1 BM cells with a lin'~°, Sca-1+, and c-kit' cell surface phenotype were purified. 100 fresh cells per mouse (Control) were transplanted with 105 Ly 5.2 competitor BM cells. One thousand of the same purified cells were cocultured with stromal cell lines for 6 weeks. The cultures were then harvested and 10% of each culture was transplanted per mouse with competitor. Data are from peripheral blood samples taken 4 months after transplant and are presented +SEM.

Table 5. LTRSC maintained on AFT024 are able to repopulate secondary recipients at levels comparable to non-cultured stem cells.
Ly 5.2T peripheral blood cells Group Weeks Radioprotection Comp.

Repopulation Control 6 21 (1) 2.60.6 (4) 22 54 ( 1 ) 5.61.6 (4) AFT024 6 130.2 (4) 1.3+0.9 (8) 22 4415.I (3) 4.30.9 (8) 2012 6 6.72.9 (4) ND

22 142.2 (4) ND

The retransplantation potential of LTRSC in primary recipients of stromal cell cultured stem cells was studied in secondary recipients. 60 weeks after transplant, primary mice (see Figure 14, Table 4A.) were sacrificed, BM harvested and stained with antibody to Ly5.2. Ly5.2+ cells were collected by cell sorting and used to transplant secondary recipients (congenic LyS.I mice). Control and AFT024 groups were transplanted with 1.5X106 Ly5.2 cells/mouse for radioprotection (4 mice/group were transplanted) and to 7.5X105 Ly5.2 cells + 7.5X105 Ly5.1 cells for competitive repopulation (4 mice for the Control group and 8 mice for the AFT024 group). 2012 mice were transplanted with 3X105 Ly5.2 cells/mouse (4 mice). Weeks are the times after transplant that the mice were analyzed. (n), number of mice surviving/group; ND, not done. Data are presented +SEM.
EXAMPLE 4: Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs Primitive hematopoietic stem cells are closely associated with discrete in vivo microenvironments. These "niches" are thought to provide the molecular signals that 2o mediate stern cell differentiation and self renewal. The fetal liver microenvironment was dissected into distinct cellular components by establishing an extensive panel of stromal cell lines. One particular cell line maintains repopulating stem cells for prolonged in vitro culture periods. A subtraction cloning strategy has yielded a cDNA which encodes a cell surface glycoprotein with a restricted pattern of expression among stromal cell lines. This molecule, previously identified as delta-like/pre-adipocyte factor-1, contains epidermal growth factor-like repeats which are related to those in the notch/delta/serrate family of proteins. The potential role of this molecule in hematopoietic stem/progenitor cell regulation was investigated. The delta-like protein displays activity on purified stem cells by promoting the formation of "cobblestone areas" of proliferation. These cobblestone areas contain both primitive high-proliferative potential progenitors and in vivo repopulating stem cells.
Introduction The positive and negative regulatory mechanisms that govern the proliferation, self renewal and differentiation of primitive hematopoietic stem cells are complex and poorly understood ( Ogawa, M. (1993) Blood 81, 2844-2853). Numerous cytokines have been identified that, when used iu vitro appear to act directly on purified stem cells by promoting proliferation and differentiation. However, attempts to demonstrate the in vitro maintenance and/or expansion of transplantable pluripotent stem cells using defined cytokine combinations have been largely unsuccessful (Knobel, K. M., et al., 1994;
Peters, S. O., et al., 1995). Moreover, it is unclear to what extent any currently identified cytokines reflect mechanisms that are responsible for regulating normal, in vivo, stem cell behaviors. It is widely accepted that in vivo, stem cells are intimately associated with discrete microenvironmental "niches" (Wolf, N. S. 1979). Such niches are likely sources for the molecular signals which collectively mediate the differentiation and self renewal of stem 2o cells. Indeed, it has long been possible to demonstrate that preestablished stromal cell monolayers derived from hematopoietic tissues can support long-term hematopoiesis in vitro ( Dexter, T. M., Allen, T. D. & Lajtha, L. G. 1977). The long-term nature of these cultures, together with the continuous production of committed progenitor cells suggest that both self renewal and commitment decisions can occur in vitro. At the cellular level.
the hematopoietic microenvironment consists of numerous distinct cell types.
Previous studies have shown that this cellular heterogeneity reflects a similarly broad heterogeneity in terms of hematopoietic supportive abilities (Deryugina, E. L, et al., 1994). Some cloned stromal cell lines can support stem cell activity in vitro, while others are ineffective. Similarly, distinct stromal cell types appear to influence the outcomes of stem cell differentiation 3o processes (Friedrich, C., et al., 1996). Recent studies have shown that stromal cell lines that efficiently maintain long-term transplantable stem cells in vitro for prolonged intervals represent a small fraction of the total stromal cell population ( Wineman, J., et al., 1996). A
fetal liver stromal cell line, AFT024 was identified which maintains high levels of transplantable multilineage stem cell activity for extended in vitro culture periods (Moore, K. A., Ema, H. & Lemischka, I. R. (1997) (See, Example 3). The stem cells used to initiate these cultures are highly purified. It is, therefore, likely that in this system the mechanisms that mediate stem cell maintenance do so by acting directly on primitive stem cells. A
s different fetal liver-derived cell line, 2018, fails to maintain long-term repopulating stem cell activity. A PCR-based RNA expression analysis of 13 cytokines reveals qualitatively identical expression patterns in AFT024 and 2018. It wastherefore, hypothesized that the hematopoietic supportive ability of AFT024 is, at least in part, mediated by novel gene products not expressed in 2018.
to Using a subtractive hybridization approach a number of cDNA clones were identified which were specifically expressed in AFT024 but not in 2018. The sequence of one cDNA
was identical to a molecule that encodes a transmembrane protein that contains six epidermal growth factor (EGF) repeat motifs. The EGF-like repeat sequences of this molecule, variously known as delta-like (dlk) ( Laborda, J., et al., 1993), preadipocyte factor-1 (Smas, is C. M. & Sul, H. S, 1993), and stromal cell protein-1 (Genbank, D16847), are most closely related to those present in the notch/delta/serrate family of signaling proteins. In Drosophila and Cuenorhabclitis, these molecules are required for correct cell-fate specification decisions in a variety of tissues (Rebay, L, et al., 1991 ). Vertebrate homologs of the notch/deltalserrate family have been identified (Ellisen, L. W., et al., 1991;
Bettenhausen, B., 2o et al., 1995; Lindsell, C. E., et a1.,1995). While the exact functional relationship of dlk to the activities of this family of molecules is unclear, in one in vitro study, it has been shown to block adipocyte differentiation ( Smas, C. M. & Sul, H. S, 1993). No studies have been reported that demonstrate a hematopoietic function for dlk. Expression analyses and these observations show a limited temporal pattern of dlk expression during murine fetal 2s development which coincides with the time period of hematopoietic stem cell expansion (Smas, C. M. & Sul, H. S, 1993).
Functional studies were undertaken to determine if dlk can act as a hematopoietic regulator. This molecule affects highly enriched stem cell populations by promoting "cobblestone area" (CSA) colony formation in dexter-type stromal cocultures.
These CSA
3o colonies contain an expanded population of primitive, high proliferative potential myeloid-erythroid progenitors. These cultures also contain stem cells capable of in vivo engraftment at levels equivalent to those present in parallel AFT024 supported cultures.
It is proposed that dlk represents one molecular component responsible for the hematopoietic supportive ability of AFT024. As such, dlk may define a novel molecular pathway of stem cell regulation by the microenvironment.
Materials and Methods Cell Lines and Culture. The fetal liver stromal cell lines used in this study were derived as previously described (Wineman, J., et al., 1996). Cells were routinely cultured in DMEM containing 10% fetal bovine serum (FBS) and 50 uM b-mercaptoethanol (2-ME), and maintained at 31°-33°C, S% CO2. For long-term cocultures with hematopoietic stem cells, confluent monolayers were irradiated (20 Gy), placed in modified Dexter media (DMEM, 10% FBS, 10% horse serum, 50 uM 2-ME, 0.1 uM hydrocortisone) and l0 maintained at 37° C, S% COZ with weekly media changes. NIH3T3 cells were obtained from ATCC.
d(k Expression Ana~rsis. Total RNAs from stromal cell lines were poly A+
selected, Northern blotted, and hybridized to 'ZP-labeled probes according to standard protocols (Sambrook, J., Fritsch, E. F. & Maniatis, T, 1989). A 600 by dlk-cDNA clone from the t 5 AFT024 subtracted library was used as a probe. cDNA templates for RT-PCR
were prepared according to manufacturers' protocols (GIBCO/BRL). Oligonucleotide primers were: sense 5'- GACCCAGGCTGCCCC-3' (SEQ.ID.No.:85) and antisense 5'-GGTACTCTTGTTGAG-3' (SEQ.ID.No.:86). For analysis of dlk expression at the protein level, antisera specific for dlk was generated by immunizing rabbits with a Flag-dlk fusion 2o protein (described below). Resultant antibodies were purified by affinity chromatography.
Ceil surface expression of dlk in stromal cell lines was accomplished by flow cytometry.
Cells were incubated with dlk antibody and a similarly prepared irrelevant control antibody.
Specific labeling was developed by donkey anti-rabbit-fluorescein isothiocyanate (Jackson ImmunoResearch). Stained cells were analyzed on a Becton Dickinson FACScan using Cell 25 Quest software.
dlk Fusion Protein Preparation. The expression plasmid pCD4-Ig contains cDNA
for the extracellular domain of human CD4 fused to genomic sequences of the human immunoglobin heavy chain (Zettlmeissl, G., et al., (1990). cDNA for CH2-CH3 of human IgG~ (Goodwin, R. G., et al., 1990) was cloned into EcoRI and NotI sites of pcDNA3 30 (Invitrogen) to give the plasmid KB52.3.2. cDNA encoding the extracellular domain of dlk was obtained by RT-PCR with primers BP 151 and BP 152 using total RNA from NIH

cells as template. The resulting PCR fragment was cloned into KB52.3.2 via HindIII and EcoRI sites to obtain the soluble dlk-Ig expression plasmid. pdlk-Ig or pCD4-Ig were transfected into NIH3T3 cells together with pSVNeo and stable clones were isolated.
Soluble CD4-Ig and dlk-Ig fusion proteins were harvested and then purified by affinity chromatography on HiTrap Protein G-sepharose (Pharmacia). Primers: sense BP 1 S 1, 5'GAGGGTACCAAGCTTCGTGGTCCGCAACCAGAAG-3' (SEQ.ID.No.: 87);
anti-sense BP 152, S'-CTCAGATCTGAATTCGGCCTGTCCCTCGGTGAGGAG-3' (SEQ.ID.No.: 88).
Flag-dlk fusion protein was used to immunize rabbits for the production of dlk antiserum. The protein expression plasmid pcDNA3-Flag is a modif cation of the plasmid 1o pcDNA3 (Invitrogen) and contains the coding region for the Flag peptide (DYKDDDDKI) {Hopp, T. P., et al., 1988) as well as a BgIII restriction site. A cDNA
fragment encoding the extracellular domain of dlk was obtained by RT-PCR using RNA from NIH3T3 cells.
Primers: sense BP 155, 5'-GACAAGATCTCAGCTGAATAGCGACCCACCCTGTG-3' (SEQ.ID.No.: 89);
antisense BP 154, S'-GCATCTAGAGCGGCCGCTCAGGCCTGTCCCTCGGTGAGGAG-3' (SEQ.ID.No.:
90). The PCR fragment was ligated into pcDNA3-Flag to yield pFlag-dlk. pFlag-dlk was transfected into cos cells. Purification of the Flag-dlk protein from cos-conditioned media was performed according to manufacturer's directions using the Flag monoclonal antibody, M 1, immobilized on agarose (International Biotechnologies).
gJ'asmid Constructs and Table Transfection. Full-length murine dlk cDNA was obtained by RT-PCR with primers BP 1 S 1 (see above) and antisense BP 200:
5'GCATCTAGAGCGGCCGCGAACGCTGCTTAGATCTCCT-3' (SEQ.ID.No.:91), using total RNA from NIH3T3 cells as template. The product was subcloned into the vector pCRII (Invitrogen) and then cloned into a retroviral expression vector (Kitamura, T., et al., 1995), (G. Nolan, Stanford University), via the primer-encoded HindIII and NotI sites.
Supercoiled plasmid was transfected into BFC012 stromal cells together with the pZeo (Invitrogen) selectable marker and selected in 50 ug/ml Zeocin (Invitrogen).
BFC012 cells also were transfected with pZeo alone and selected as above. Clones from both selected 3o populations were isolated and all remaining colonies ( 100-200 per dish) were pooled and expanded as populations.
Hematonoietic Stem Cells and In yitro Hematopoiet~'r Away. Hematopoietic stem cell populations were derived from wild type, Ly5.2-C57B1/6J (Jackson Laboratories), day 14 fetal liver, enriched for the AA4.1~, Sca-1+, c-kit+, and line°~-phenotype, by immunopanning and fluorescence-activated cell sorting as described (Jordan, C.
T., et al., 1995). Adult hone marrow (BM) was used directly after density centrifugation and immunomagnetic bead depletion or was further enriched for Sca-1+, c-kit+, line°~~ cells by flow cytometry as described (Okada, S., et al., 1992). Cell sorting and data analysis was accomplished with a Becton Dickinson FACS Vantage using Celt Quest software.
Stromal cell/stem cell cocultures were initiated in 12-well trays with 300-1,000 enriched stem cells per well. Cobblestone areas were quantitated by inverted-phase microscopy as described (Ploemacher, R. E., et al. , 1991 ). Clonogenic progenitor assays were performed with either freshly purified stem cells or cells harvested from the stromal cocultures.
These were cultured in cytokine-containing semisolid media according to the manufacturer's recommendations (Stem Cell Technologies, Vancouver, BC). Soluble dik and control fusion proteins were added to semisolid progenitor assays at concentrations of 0.1, 0.5 and 1.0 ug/ml and also to BFC012 stromal cocuItures at concentrations of 0.1 ug/ml.
Fusion protein was replenished weekly in the stromal cocultures.
Compgljtive repopulating tra~,~t~lantation assay. Cultured cells were harvested, combined with fresh unfractionated BM obtained from congenic C5'7B1/G Ly5.1 mice (National Cancer Institute) and transplanted into lethally irradiated ( 10 Gy, split dose 3 h apart from a ~3'Cs source, 1 Gy/min) Ly5.1 recipient mice. Each mouse received 2o competitor BM cells and a fraction of the cocultured stem cells. Mice were bled by capillary puncture of the orbital venous plexus and 100 ul was collected; red blood cells were removed by NHaCI lysis. The nucleated cells were stained for the Ly5.2 (CD45.2) allelic marker using either fluorescein isothiocyanate-labeled directly conjugated Ly5.2 monoclonal antibody or a biotinylated form developed with streptavidin conjugated to Texas red. Cells also were stained with directly conjugated antibodies to lineage markers. All antibodies and chromogens were obtained from Pharmingen. Flow cytometric analysis was done on a Becton Dickinson FACS Vantage using Cell Quest software.
Results Genes expressed in AFT024 but not in 2018 were identified by a subtractive cloning approach. Sequence analysis identified one of these AFT024-specific clones as dlk.
Expression studies, (Figure 19A) show high levels of dlk in AFT024 and subclones isolated from this line, but undetectable levels in 2018 and BFC012. The latter two stromal cell lines do not support repopulating stem cells. The PA6 stromal cell line and NIH
Swiss 3T3 cells both show expression of dlk and were the cell sources for identification of SCP-1 and dlk, respectively. PA6 cells have been shown to support in vitro hematopoiesis and long-term, in vivo repopulating stem cells (Kodama, H., et al., 1992). Swiss 3T3 cells are also capable of supporting multipotent hematopoietic stem cells in vitro, promoting CSAs and maintaining in vivo spleen colony-forming units (CFU-S) (Roberts, R. A., et al., 1987).
Interestingly, an additional fetal liver stromal cell line, 2012, which has some degree of stem cell supporting activity (Wineman, J., et al., 1996), and its subclones also express dlk.
Furthermore, an RT-PCR analysis (40 cycles) of an additional 10 fetal liver-derived stromal cell lines and several t0 other lines, shows detectable levels of d1k in only two additional lines (Figure 19B). These two cell lines (CFC032 and CFC008) can maintain some level of long-term transplantable stem cell activity present in whole BM (Wineman, J., et al., 1996). A
correlation between a stromal cell line's ability to support stem cells and the expression of dlk was suggested.
Therefore, functional studies were undertaken in order to delineate if dlk can act on or t 5 modulate hematopoietic stem cells.
Soluble dlk protein was added to progenitor cultures in semi-solid media. The soluble protein consisted of the dlk extracellular domain fused to the Fc portion of human IgG, . The stem cell sources in these assays were highly enriched fetal liver cells (AA4.1+, lin'°~-, Sca-1+, c-kit +). The influence of soluble dlk on hematopoietic progenitor colony-formation was zo assessed. As shown in Table 6, no differences were noted either in the number, sizes, or lineage compositions of colonies. Identical results were obtained at dlk concentrations ranging from 0.1 to 1.0 ug/ml. In addition, no differences were noted in similar studies using enriched BM cells (Sca-1+, c-kilt, lin"°).
Evidence for a positive effect of the dlk protein on stem/progenitor cells was 25 observed when the soluble form was added to dexter-type cocultures. For these studies a stromal cell line (BFC012) was used that neither expresses endogenous dlk (see Figure 19) nor maintains significant in vitro hematopoiesis. In four experiments, two each using highly enriched adult BM and fetal liver stem cells, we monitored the appearance of CSAs over time. These colonies provide a convenient, quantitative estimate of hematopoietic activity 30 initiated by primitive stromal dependent stem/progenitor cells. As shown in Figure 20, the addition of soluble dlk (0.1 ug/mL) results in an approximately 2-fold increase in the number of CSAs initiated by purified fetal liver or BM stem cells over a 2 week time period (P=
0.001 for dlk vs control and P= 0.01 for dlk vs no additive, Student's t-test). There was no difference in the numbers of CSA observed in BFC012/stem cell cultures with or without control fusion protein (mean of no additive/control = 0.96+0.11) In order to assess the activity of the normal transmembrane form of dlk, a full-length dlk cDNA was transfected into BFC012 cells. Expression of the introduced dlk was demonstrated at the RNA (Northern blot) and protein levels using both Western blot and flow cytometric analyses with rabbit anti-dlk antibodies. The flow cytometry data are presented in Figure 21.
Initially, dlk-expressing transfected populations (BFC-dlk) were compared to a negative control "mock" transfected population of BFC012 cells. A 4- to 6-fold increase in to the number of CSAs was observed in two separate experiments. The maintenance of CSAs was transient, lasting less than 2 weeks. No further hematopoietic activity was observed during an additional 2 weeks of culture. Once dlk-expressing clones had been identified from the transfected populations, they were studied for their ability to support CSAs in experiments designed to more precisely identify the time course of hematopoietic activity.
Ficoll-separated, lineage depleted BM was used in these experiments. Five different negative control, non-dlk expressing BFC012 cell groups (parental BFC012 cells, two "mock" transfected populations, and two "mock" transfected clones) and 3 dlk-expressing BFC012 cell groups (one transfected population and two clones) were studied.
The data are presented in Figure 22A. Neither the negative control BFC populations nor the "mock"
2o transfected BFC clones supported high numbers of CSAs. In contrast, the BFC-dlk populations and the two individual dlk-expressing clones supported significantly greater numbers of CSAs at all time points studied (P< 0.001 days 3,4, and 5; P< 0.01 days 6 and 7, Student's t-test). As observed previously, all the CSAs were transient. This experiment also indicated that the dlk-promoted hematopoietic activity peaks early, at 4 days, in this culture system. Three additional experiments using purified (AA4.1+, line°~-, Sca-1+, c-kit+) fetal liver stem cells were performed using two individual clones, BFC-dlk-5 and a "mock"
transfected negative control BFC-Zeo-1. The results are presented in Figure 22B. There was a dramatic and significant difference in the number of CSAs observed in the BFC-dlk-S
cultures compared to the control line (P< 0.001, days 4, 6, and 8, Student's t-test). As 3o before, the effect was transient and the CSA declined in number over 2 weeks. AFT024 was included as a positive control and, in each of the three experiments, verified the quality of the input purified stem cells. In the first week of culture the numbers of CSAs observed on AFT024 were similar to the numbers in the BFC-dlk5 cultures.

In order to address the "primitiveness" of the cells that give rise to the CSA
observed in the BFC-dlk cocultures, a series of in vitro replating experiments were performed.
Individual wells were harvested at various time points of coculture and the cells were plated into semisolid cytokine-containing media. The numbers and lineage compositions of the s colonies were scored after 8-12 days. As shown in Figure 23A, the CSAs obtained from day 4 BFC-dlk-S cocultures contained numerous progenitors capable of extensive proliferation and multilineage differentiation. The total number of progenitors from the dlk-expressing cultures at day 4 was significantly expanded compared to the content in the freshly purified uncultured stem cell population (P= 0.01, Student's t-test). The number and lineage composition of colonies derived from parallel day 4 AFT024 cultures was nearly identical to BFC-dlk-5 derived colonies. The content of CSAs replated at day 6 from the BFC-dlk-5 cocultures was devoid of multilineage colonies, although CFU-granulocyte-macrophages were maintained at high levels; the progenitor content in the BFC-dlk-5 cocultures continued to decrease when next sampled at 10 days. In contrast, few progenitors could be demonstrated in the BFC-Zeo-1 cultures (P= 0.001, BFC-dlk-5 vs. BFC-Zeo-1, Student's t-test) (Figure 23A). Taken together, the data strongly suggest that dlk acts to promote stromal-dependent colony-formation by primitive cells capable of yielding large numbers of committed progenitors, including those endowed with a high proliferative capacity and multilineage differentiation potential. The lack of CSAs and significant progenitor 2o maintenance in the BFC-Zeo-1 cultures argues that expression of dlk in the transfected BFC012 cells is responsible for both their ability to support CSAs and to generate/maintain primitive progenitors.
In order to determine if the CSA-containing cultures supported by BFC-dlk-5 contained stem cells capable of in vivo engraftment, portions of the same day 4 cocultures that were plated into progenitor assays also were used to transplant mice in competitive repopulation assays. Shown in Figure 23B are the results from two independent experiments analyzed at 10 weeks after transplant. The same BFC-dlk-5 cultures that contain CSAs and primitive CFU-high proliferativ potential (HPP)-Mix progenitors also contain repopulating stem cells at levels equal to those maintained in parallel AFT024 cocultures.
In addition, a 3o significant difference exists in the levels of repopulating stem cells derived from dlk-expressing cocultures compared to non-dlk expressing BFC012 cells (P= 0.05, Student's t-test). Mufti-color flow cytometric analyses also demonstrated that both myeloid and lymphoid Ly5.2 cells are present in these animals. A subsequent analysis of these animals at WO 00/11168 go PCT/US99/19052 22 weeks demonstrated lower levels of repopulation with Ly5.2 cells derived from the AFT024 and BFC-dlk-5 supported cultures (data not shown). Most significantly, no repopulation was observed at any time point in mice that received cells cocultured on the non-dlk-expressing monolayers (Figure 23B).
Discussion As part of an ongoing effort to understand the biology of the hematopoietic microenvironment, a panel of stromal cell lines from midgestation fetal liver was established and characterized. Among these cell lines, the AFT024 line has the ability to maintain nearly quantitative levels of transplantable stem cell activity for extended in vitro time to periods (Moore, K. A., Ema, H. & Lemischka, I. R.,1997). Because these cultures are initiated with highly purified stem cell populations it is likely that the AFT024-derived molecular mechanisms responsible for this ability act directly on the stem cell population.
Other stromal lines that fail to maintain stem cell activity were also identified. These observations facilitated a subtractive hybridization approach aimed at identifying potential t5 candidate molecules whose collective actions may be responsible for the AFT024 stem cell maintenance activity. This effort has identified dlk, a transmembrane molecule containing six EGF-like repeat motifs. Although lacking the DSL motif indicative of the notch ligands delta and serrate (Tax, F. E., Yeargers, J. J. & Thomas, J. H., 1994), dlk is most closely homologous to delta/notch/serrate when compared to other EGF-like repeat containing 2o molecules (Laborda, J., et al., 1993; Smas, C. M. & Sul, H. S., 1993). The predominant role of these types of molecules in cell growth and differentiation led us to investigate the potential role of dlk in hematopoiesis. Constitutive expression of translocated human notch (Tan-1) is found in a T-cell leukemia (Ellisen, L. W., 1991). Moreover the expression of Tan-1 in primitive human stem cells has been demonstrated (Milner, L. A., et al., 1994).
25 Nevertheless, a functional role in hematopoiesis for the notch ligands Jagged (Lindsell, C.
E., 1995) and Delta-like-1 ( Bettenhausen, B., et al., 1995) has not been described. dlk expression is highly restricted in a panel of stromal cell lines. Two lines, AFT024 and 2012, which maintain repopulating stem cell activity in vitro, express dlk, whereas two non-supportive cell lines, 2018 and BFC012, do not. Interestingly, the S 17 stromal cell line 3o which is considered to be a potent stem cell supporter (Wineman, J. P., et al., 1993) does not express detectable levels of dlk. The S 17 cell line was derived from adult BM
(Collins, L. S.
& Dorshkind, K., 1987)). The other lines described are all derived from fetal sources (AFT024, 2012, and NIH 3T3 cells) or from newborn calvaria (PA6 cells). It is therefore possible that dlk acts in a developmentally regulated fashion. An extensive analysis of dlk expression in adult BM stroma is currently underway. Taken together, the data suggest that, at least in fetal stromal cell types, there exists a correlation between hematopoietic supporting ability and the expression of dlk.
The potential activity of both soluble and transmembrane dlk protein, on highly purified stem cell populations were directly measured using in vitro and in vivo assays. Initial experiments designed to ask if dlk can enhance colony formation in cytokine-rich semisolid assay systems were negative. These results may indicate that:
(i) progenitor cells capable of colony formation in semisolid assays do not respond to dlk, (ii) the o collection of cytokines present in the semisolid cultures may "mask" an effects) of added dlk or (iii) that the soluble form of dlk requires a stromal monolayer to mediate its effects.
The first possibility can be addressed more extensively in delta-type assays, where stem/progenitor cells are first cultured in suspension in serum-free media containing various cytokine combinations, with and without dlk, and then replated into colony assays (Muench, J. O., Firpo, M. T. & Moore, M. A., 1993). The second possibility can be addressed by more extensive studies using subsets of the cytokines present in our initial studies. These experiments are underway. As a first step to address the third possibility, we added soluble dlk to preestablished BFC012 monolayers. Using both purified BM and fetal liver stem cell populations, a significant increase in CSA colony formation was observed in the dlk supplemented cultures (Figure 20). This was a surprising result, given that dlk is a transmembrane protein; however, before its cDNA cloning, a soluble form of dlk was identified as FA1 or fetal antigen 1 (Jensen, C. H., et al., 1994). A role in hematopoiesis was not indicated in these studies, but expression was detected in stroma of placental villi, in yolk sac blood islands and in fetal liver (Jensen, C. H., et al., 1994). It is of interest to determine if a soluble form is produced by the stromal cell lines that express dlk. An additional explanation, for the effects observed with the soluble form added to stromal/stem cell cocultures, is that they may be facilitated by the Fc portion of the fusion protein. It is possible that Fc receptors expressed by some of the hematopoietic cells in the cultures are able to sequester and present the dlk-Fc fusion protein more effectively. This possibility can 3o be addressed by using a different type of soluble dlk protein. These studies have been initiated. Alternatively, the soluble dlk may be sequestered and thus presented by the stromal cell extracellular matrix. In order to further address the third possibility, an intact transmembrane form of dlk was introduced into the BFC012 stromal cell line.
Initially, dlk transfected BFC012 cell populations were compared to BFC012 cells transfected with the selectable marker alone. In these studies, the dlk expressing monolayers were more effective at promoting CSA colonies. As with the soluble dlk experiments, the CSAs appeared early and were transient. When cells were replated from the BFC-dlk supported cocultures onto an AFT024 monolayer a reiteration of the burst of CSAs was seen that was maintained for 3 weeks. In addition, replating of these cocultures revealed a high progenitor content (~l in 10) that included a high percentage (43%) of multipotential colonies including HPP-Mix. In the BFC-Zeo control populations neither replatable CSA nor CFU progenitors were maintained. Further experiments with individual clones from the transfected populations to confirmed and extended the results obtained with the populations, demonstrating highly significant differences in the numbers of developing CSAs (Figure 22).
However, in two experiments we observed that one dlk-transfected BFC012 clone, which expresses a very high level of dlk, supported fewer CSAs than non-dlk-expressing control cells.
These cultures also suggested differentiation phenomena, as indicated by the number of rapidly accumulating nonadherent cells. Experiments utilizing this cell line were not included in our analyses. It is possible that there may be a threshold level of dlk expression necessary in these cultures and when it is surpassed the cells differentiate and die rapidly in the culture mileau provided by BFC012 cells. In addition, it is possible that an aberrant form of the dlk protein is made by this line. Further studies are necessary to clarify this issue. Nevertheless, in Figure 22A, dlk-transfected BFC012 cells (one population and two clones) show a significant enhancement of CSA formation compared to controls.
The observed low level maintenance of competitive repopulating stem cells in short-term dlk-expressing cocultures is of interest even though the activity diminished over time.
These studies show that the ectopic expression of a single molecule (dlk) in a previously nonsupporting stromal cell line restores or enables hematopoietic support.
This is demonstrated by maintenance of three different stem/progenitor cell compartments: (i) CFU-HPP-Mix, (ii) CSA, and (iii) short-term in vivo repopulating stem cells.
It is also of interest that both qualitatively and quantitatively similar stem/progenitor cell compartments are maintained in short-term AFT024 supported cocultures ( Moore, K. A., Ema, H. &
Lemischka, I. R., 1997).
Two mechanisms underlying the effects of dlk are considered. First, it may be that some level of dlk expression is sufficient to retard potent differentiation signals provided by the BFC012 cell line. Second, dlk may provide a proliferative stimulus not normally produced by BFC012. A more direct and perhaps relevant assay will be to eliminate the expression of dlk in AFT024 cocultures, thus maintaining other components that make up the culture "milieu." Studies to evaluate potential neutralizing antibodies and various dlk antisense strategies are underway.
The failure of BFC-dlk-5 supported CSAs to persist for periods longer than 1 to 2 weeks also may suggest the existence of other molecules in AFT024 that facilitate hematopoiesis. In this regard, it is interesting that our subtraction screen has yielded several other clones with expression patterns very similar to dlk. Eventually, with the addition of dlk and other AFT024 specific molecules it may be possible to reconstruct a 1o supportive phenotype. This should lead towards a further understanding of the in vivo hematopoietic microenvironment. In summary, it is proposed that dlk represents one molecular component responsible for the hematopoietic supportive activities of the AFT024 cell line. As such, dlk may define a novel molecular pathway of stem cell regulation by the hematopoietic microenvironment.
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1o This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

SEQUENCE LISTING
<110> Lemischka, Ihor Moore, Kateri <120> GENES THAT REGULATE HEMATOPIETIC BLOOD FORMING STEM
CELLS AND USES THEREOF
<130> 2275-1-005PCT
<190> UNASSIGNED
<191> 1999-08-20 <150> US 09/138,132 <151> 1998-08-21 <160> 96 <170> PatentIn Ver. 2.0 <210> 1 <211> 400 <212> DNA
<213> Mus musculus <400> 1 cccacgcgtc cggagagcgg cagccatctc catgccaagg ttgaacaaaa tgccaggaat 60 gttcttttct gctagcccaa aggattcgaa agaacacagc cattctcttc tagacnacaa 120 aaagcagaaa aaaaggccaa agacttttgg aatggacgtg aaaacatacc tgagatcgat 180 gatcccacat ctggaatctg ggatgaaatc ggccaagtcc aaagacatac tttctgctga 290 agaagttatg cagtggtctc agtctctgga aaaatccttg ccaaccagac aggtcnaaat 300 gtctttggaa gattctaaag tctgattcag tgaggaaaat attgaattct ggttggcttg 360 tgaggactat nanaaaacan aaactgatct tttgcatanc 900 <2i0> 2 <211> 198 <212> DNA
<213> Mus musculus <400> 2 cggctgctag aagacgacag aaggggactc actcgttttg agaagaccat ganagcggca 60 gccntctcca tgccaaggtt gaacaaaatg ccaggaatgt tcttttctgc tancccaaag 120 gattcgaaan aacacagcca ttctcttc 198 <210> 3 <211> 900 <212> DNA
<213> Mus musculus <400> 3 tacggctgcg agaagacnac agaaggggaa ncagctgcgg tggccgcggg agtctgacaa 60 tgcaaagtgc catgttcctg gctgtccngc acgactgcgt acccatggac aagagtgcan 120 gcaacggccc caaggtcnan gagaagcggg agaaaatgaa tcggacactc ttnaangatt 180 ggaanacccg tttgagctac ttctngcaga gnncctctgc tcccgggaag cccnnaactg 240 gcaataaaac naacagcata cttttatcna nccttctcct nangaagcgc anctctnggc 300 ataaactttt natnanctgc tggccngtat atatgggctg gctgcattca nggcgttttt 360 taaagtccca gttctntgaa gaaaacattg aaattctggt 900 <210> 4 <211> 900 <212> DNA
<213> Mus musculus <400> 9 Page l of 56 gcctgtggtg gctccgga ccctcccttc ctgctcatcc tggtgtgtac gS ccggag 60 cacctgaacc agagaaacgc cattcgggca tcttggggtg ccatccgcga agcccggggt 120 ttcagagtgc agacgctctt cctcctggga aaacctagaa gacagcagct tgctgacctg 180 tcctcagagt cagcagcaca cagggatatc ttgcaggcct ccttccagga ttcctaccgc 290 aacctcaccc tcaagaccct cagtggactg aactgggtga acaaatactg tcctatggcc 300 cgctacatcc tcaagacgga tgatgacgtg tatgtcaacg tcccananct ggtgtcagag 360 ctgatacaaa gaagggggcc ttcggacaat ggcagaaggg 400 <210> 5 <211> 1081 <212> DNA
<213> Mus musculus <900> 5 cccacgcgtc cgatctcctc cagggccacc aagcacctct gaagagccat gttccaagct 60 gccggagccg cccaggccac cccctctcat gaagccaaag gcagcagtgg cagcagcacg 120 gtacagcggt ctaagtcctt tagcttgcgg gctcaggtga aggagacctg tgcagcctgc 180 cagaagactg tgtacccgat ggagcggctg gtggcagaca agctcatttt ccacaactct 290 tgtttctgtt gcaaacactg ccacaccaaa ctcagcctgg gcagttatgc tgcaatgcac 300 ggtgaatttt actgcagacc tcactttcag cagctgttta agagtaaagg caactacgat 360 gaagggtttg gtcgtaaaca gcacaaggag ctctgggccc acaaggaggt ggactcaggc 420 accaagacgg cctgagaccc ctttaacacc cattccctcc cagcacatgg cctcccgctg 980 ggcagtggaa aggagattaa cccgggggcg cggggtggga gaggatgagg ctccctcaca 540 caggtttcag gcataaggct ctgctccagg attccttact tttcccatgg gaggttggcg 600 ttgggaacca gaattggaat tttcaccata ctgtgtcctt tagtccacct catctcaccc 660 cacggctccc tgggaggccc acaagcccag cttccatact taggtgcttt tctccagcaa 720 ggagtcagca tgccctcctc agggtcccaa gctccctcac tgccacctgg gccttgtgta 780 cccccttgtc tccccatcta cctctgcccc ttagcctggt aatgagccac agagactgga B40 agagggagag tgccatntac tgggcctcat agatgccacc tcgctgaggg gggagggctg 900 gggaagaggc aagacagcct gcagccttca gggtctgggg gtcccttgca ccacaaagct 960 aaagctcttg ctagagcctc agctgacagg gtcggcagta gctatgctcc tccatctgtt 1020 gtgctgttct gttgtgatca accctctttt aaaaacattt aaacagcaaa aaaaaaaaaa 1080 a 1081 <210> 6 <211> 793 <212> DNA
<213> Mus musculus <400> 6 attggtacgc ctgcacgnac cggtccggaa ttcccgggtc gaccacgcgt ccgcccacgc 60 gtccgatctc ctccaggggg caccaagcac ctctgaagaa catgttccaa gctgccggan 120 gcgcccangc caccccctct cntgaagccc aangcagcan tggcagcagc acggtncagc 180 ggtctaagtc ctttaacttg cgggctcagg tgaanganac ctgtgcagcc tgccngaaaa 290 ctgtgtnccc natggancgg ctggtggcag acaanctcnt tttccacaac tcttgtttct 300 gttgcaaaca ctgccacacc aaactcancc tgggcagtta tgctgcnatg caccgtgaat 360 tttactgcag acctcacttt cagcagctgt ttnagaatna aggcnactac natgaaaggt 420 ttggtcgtta acagcacaan ganctctggg cccncnagga agtngactca ggcccnanan 980 aggctganaa ccctttaaca cccattccct cccnncacat tgnctcccnc tnggcagttg 540 gaaaagaaaa taancccngg gcnccgggtt ggganaagaa aaaggtcccc cccnccggtt 600 ttccggcnta agggtctccc ccnnaatccc ttcttttncc cctgggaagt ttgggtttgg 660 gaacccaaat tggaatttcc ccctnccgtn ttcctttntt ccccnccnct cccccccnng 720 gtccctggga aggccccaan ccn 743 <210> 7 <211> 743 <212> DNA
<213> Mus musculus <900> 7 gggggagtct tgagtcgcat cgcccgttnc gtaagcttgg atcctctana acgggcgccc 60 tttttttttt gttttgctgt ttaaatgttt ttaaaaagaa ggttgatcac cacagaacng 120 cacnacngat ggaggagcat anctnctgcc gaccctgtca gctgangctc tagcaanaac 180 =ttagctttg tg~tgcaagg gaccccnnac cctgaaagct gcaggctgtc ttgcctcttc 290 Page 2 of 56 WO 00/1116$ PCT/US99/19052 cccanccctc c~~cctc~ gaagtggcat ctatgaagcc cagtanatgg ca :.tccct 300 cntccnttct c~?tggctca ttaccangct aangggcnga ngttnatngg ganacaaagg 360 ggtncacnat gcccangtgg cantnaagga cttgggacct gaagaaggca tgctgactcc 420 ttgctggana aaaagcacct aanttggaac tgggcttgtg ggcctcccnc ggaaccntgg 480 ggtgagatna a:,tggactat aggaccntat ggtgaaaatt ccaattctgg ttcccaaccc 540 nacctcccat gggaaaaant taggaatcct gcaaccaaac ccttatgcct gaaaacctgt 600 tttnaaggga ncctcatcct cttcccancc ccgngccccc nggggtttaa agggaaaaag 660 ggtttttttn aaagggggtt ccccnnggcc ccccnntggt tnccctnaat ttcccccccc 720 cctnntttgg gccccnaaaa ccn 743 <210> 8 <211> 179 <212> DNA
<213> Mus musculus <400> 8 ggattccggt ta~gaatgaa attagaagct gtagatctca tggagccacg gttaatatgt 60 gtagccacag tta~tcgaat tattcaccat ctcttgagga tacattttga tggttgggaa 120 gaagagtatg a~~agtgggt agactgtgag tcccctgacc tctatcctgt aggg 179 <210> 9 <211> 711 <212> DNA
<213> Mus musculus <900> 9 ggggctcagg gaaggccgat cttccgggtg gagggggaag cggcgtgact ggagtggaaa 60 tttttcccaa cacaacttct cggaggcaac atattggaag ggactcgggg aggccggagt 120 ccaaatggaa gtggctgaaa gaaacttctc qccctgctga ttctgagccc cgcgtcgtgc 180 cgcgcgccct caattacctc atcgacctgt ggtcttgaca gaacattctt cacaatccaa 240 aagaaaaagc agactggttc gggatgtttg acggctatga tagctgcagt gaggacacaa 300 gtagcagctc cagctctgag gagagtgaag aagaagttgc tcctttacct tccaatctcc 360 caatcatcaa gaataatgga caagtctaca catacccaga tggtaaatct ggcatggcta 420 cctgtgagat gtgtgggatg gtcggtgtgc gagatgcttt ttactctaaa acgaaacgtt 480 tctgcagtgt tt=ctgttca agaagttact cgtcaaactc taagaaggca agcattctgg 540 ccngacttcn nc~tacgggt tngcctccca cnaagaaagc caaantcctt ccnaaacnac 600 cnttagttgg taaattnnct gcctatgccc ntntccanct accttgcnna atccnnccag 660 acnaaancng gc~.attctgc catctctgtg gaaagggtcc ctgggggtta c 711 <210> 10 <211> 356 <212> DNA
<213> Mus musculus <900> 10 tacggccgcg anaagacgac agaagggtnc gggctgcgan aagacgacag aaagggggcc 60 tttctccgct gc~cccggcg cgcccggcag ctcctccccg gccatggcgt tcactttcgc 120 ggccttctgc tatatgctgg cgctgctgct. caccgccgcg ctcatcttct tcgccatctg 180 gcacatcata nc.-..~ttgatg agctgaagac cgactacaag aaaccctata gaccagtgca 240 ataccctgaa cc=cttgtcc ttccagaagt actcatccac gcgttcttct gtgtcatgtt 300 tctctgtgcg ca~aattggc tgaccctggg cctcaatatg ccccttttgg catacc 356 <210> 11 <211> 400 <212> DNA
<213> Mus musculus <900> 11 acggctgcg aa~agacgac agaaggggag tgctcgccgg tcccagcgtt gccataccat 60 cgagatggca tctcgtagcc ggagacccga acacagcgga ccgccggagc tgttttatga 120 ccagaatgaa gc~cggaaat acgttcgcaa ctcacggatg attgacatcc agaccaanat 180 gactgagcga gc~~tggagc tcctctgttt accaganggt cagccttctt acctgttaga 290 ~attggctgc gc=~ctgggc tgagtggaaa ttatatctca gaagaaggac actactgggt 300 gggcattgac atcagccctg ccatgttgga tgccgccttg gacgagatac anaaggggac 360 Page 3 of 56 ~gctgctang cgactgg~ aggctc~~tt caaaccggcc 400 <210> 12 <211> 400 <212> DNA
<213> Mus musculus <400> 12 acggctgcga gaagacgaca gaagggtacg gctgcgagaa gacgacagaa gggtacggct 60 gcganaagac gacagaaggg tacggctgcg agaagacgac agaaggggga ggaaactgta 120 aaagctatgc tc~ggangca gcgcgganct tgattcacct tcacctgctc tggccacccg 180 ctgacccggg g~~=ccggcc gganagcagt canatatgaa cggacgggtg gattatttag 290 tcacggagga agagatcaac ctgacgagag gaccttcggg gctgggcttc aacatcgtcn 300 gtgggacaga tcaacagtat gtctccaacg acagtggcat ctacntcagc cgtatcaaan 360 aagatggggc t?cngcccan gatgggcggc tccaggaagg 400 <210> 13 <211> 1360 <212> DNA
<213> Mus musculus <900> 13 agctctagtc cccagagatg tcgccactac tgctgctgct gctgtgcctg ctgctgggga 60 atttggagcc tgaggaggcc aaactgatcc gtgtccctct tcaacgaatc caccttggac 120 acagaatctt aaacccactg aatggatggg aacagctggc agagctttct aggacctcca 180 cctctggtgg caacccctcc tttgtgcctc tctccaagtt catgaacacc cagtattttg 290 gaactattgg tttgggaacg cctcctcaga atttcaccgt tgtctttgac acgggttctt 300 ccaacttgtg gg=tccgtcc acgagatgtc atttcttcag tttggcatgc tggtttcacc 360 atcgctttaa tcccaaggcc tccagctcct tcaggcccaa tgggaccaag tttgccattc 920 agtatgggac cgggcggctg agcggaatcc tgagccagga caatctgact atcgggggga 980 tccacgatgc tt~tgtgaca tttggagagg ctctgtggga gcccagcctg atctttgctt 590 tagcccactt tgatgggatc ctgggcctcg gcttccccac tctggctgtg ggcggagttc 600 agcctccgct ggatgcgatg gtggagcaag ggctgctgga gaaacccgtc ttctcctttt 660 acctcaacag ggattctgaa gggtctgatg ggggagagct ggtcctaggg ggctcagacc 720 ccgctcacta cg~acctccc ctcaccttca taccagtcac catccctgcc tactggcagg 780 tccacatgga ga~tgtgaag gtcggcacag ggctgagcct ctgtgcccag ggctgcagtg 890 ccatcctaga cacaggcaca tccctcatca caggacctag tgaggagatc cgggccttga 900 ataaagccat tg~gggatat cccttcctga atgggcagta cttcattcag tgttccaaga 960 cgccaacgct tccccctgtc tccttccacc ttggtggagt ctggtttaac ctcacaggcc 1020 aggactatgt ca~caagatt cttcagagcg atgttggcct ctgcctgttg ggcttccaag 1080 ccttggatat ccccaagcct gcgggacccc tctggatcct tggggacgtc tttttggggc 1140 cctatgtggc tg=ctttgac cgtggggaca aaaacgtcgg cccgcgcgtg ggactggcgc 1200 gtgctcagtc tc~ttcaaca gaccgggcag aaagaaggac tacgcaggcg cagttcttca 1260 aaagacgccc t;~~tagggt acaagctcac cgggccacag cagctatgct tctttccaat 1320 taaacaaact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1360 <210> 14 <211> 1712 <212> DNA
<213> Mus musculus <900> 19 tcttcgaaag cc;ggctgag gggaatcctg gacaggggaa tcctggacgt ggagatcgtg 60 agtcatggct gc=_cccgag acgctgatga gatccacaag gacgttcaga actactatgg 120 gaatgtactg aa~acatctg cagacctcca gactaatgct tgtgtcacgc gagccaagcc 180 ggtccccagc ta~atccggg aaagtctgca gaatgtacac gaagacgtta gttcgaggta 240 ttatggctgt gc~~tgactg ttcctgagcg gctggaaaac tgccgaattt tggatctggg 300 tagtgggagt gg~agggatt gctatgtgct tagccagctg gttggtgaga agggacatgt 360 caccggaata ga~atgactg aggtccaggt cgaagtggct aaaacctatc ttgaacacca 420 catggaaaaa tt=3gtttcc aggcacccaa tgtgactttt ctccacggcc gcatcgagaa 480 gttggcagag gc~gggatcc agagtgagag ctatggtatt gtcatatcca actgtgttat 540 caaccttgtt cc=?ataaac aacaagtcct ccaggaggtc tatcgagtgc cgaagcacgg 600 cggggagctc t~=~tcagtg acgtctatgc cagccttgaa gtgccagaag acatcaagtc 660 gcacaaagtt t~~=gggggg aatgcctggg aggcgctctg tactggaagg atcttgccat 720 Page 4 of 56 cattgcccaa aagattgc tctgccctcc acgtttggtc actgccgata tc ~actgt 780 tgaaaacaag gagctcgaag gggttcttgg tcactgtcgc tttgtgtctg ccacatttcg 840 cctcttcaaa ctccctaaga cagagccagc cgaaagatgc cgagttgttt acaatggagg 900 aatcaaggga catgaaaagg aactaatttt cgatgcaaat ttcacattca aggaaggcga 960 agctgttgca gtggatgagg agacggcagc tgtcctgaag aactcacgtt ttgctccgga 1020 ttttctcttc acacctgttg acgcctcgct gccagctccc caggggccgt tctgagttag 1080 agacaaaggt tctaatcaga gatccattca agcttgcaga ggactctgac aagatgaagc 1190 ccagacatgc acctgaaggc acgggaggct gctgtggcaa gaggaaaaac tgctagatct 1200 acagccagcg cggagcccac cgggctcaag agggtggcta aaggacagtc acagaggctt 1260 cttagcctgc tcttcgccag tgcacagatt atgtgaaggt ggcaaagcca ccacaagcta 1320 gaccactgct aagaataaga gtgactttta gaggatgtta attgaaggtt cacagcaaat 1380 cgcctgcttt tctatttctc tatctcagag ttctggtgcc acctagtggt cagaagtaga 1440 acttggaagc ccaaggttta ctcaaagggc caaaggcatc atcaacgttg tgagaattat 1500 cttccttctg gcctaccaca ggacacctct gggttcttct ctgtggttac caggaagcac 1560 agtacttact aaatttatgc taaccatgac aaaagattgt caactcaaat ttgctaggag 1620 tattctttag gttgctgtct gcaatttttt t=tctgtaac tgaatgaaaa agaaaacaaa 1680 taaaaaataa atttgacttc gaaaaaaaaa as 1712 <210> 15 <211> 1539 <212> DNA
<213> Mus musculus <400> 15 cggacgcgtg ggggagtctg tggagccccc ggtcaaagac ggcatcctct accagcagca 60 cgtaaagttt ggcaagaaat gctggcgcaa agtgtgggct ctgctgtatg cgggaggccc 120 atcaggggta gctcggctag aaagctggga cgtgcgtgat ggtggcctgg gaccagcagg 180 cgacaggtcc acagggccca gccgtcgagg ggaacgccgg gtcatacgct tggctgactg 290 tgtatctgtc ctgcctgcgg atggcgagag ctgtcccagg gacactggtg ccttcctgat 300 taccaccact gagcgaagcc acctgttggc tgcacagcac cgccagtcct gggtggaccc 360 catctgtcag ctggccttcc cgggtaccgg agaatgttcg tcaggatcag gacaggctga 920 gaatccaaaa aggggctttg ttcccatgga agaaaactct atctactcct cctggcagga 480 agtgaccgaa tttccggtga tcgtgcagaa gacagaagcc acctcccgct gccagctgaa 540 aggaccctac ctcctggtgc tgggccaaga tgacatccaa ctgagggaga catc~aagcc 600 ccaggcctgt tttagctggc cctaccgttt cctgcgcaag tacggctctg acaagggtgt 660 gttctcgttt gaggctggcc gccgctgtga ctcaggtgag ggcctttttg ccttcagtag 720 cccgcgtgcc ccagacatat gtggggttgt ggctgccgcc attgcccgcc agcgggagcg 780 tcttccagag ctggccatgt ccccaccctg ccccctgcct cgggccctct ccct~ccctc 890 cctagagccc cctggagagc ttcgggaggt g~ccccagga tttgagctgc ccactcccag 900 aaagctgcct ctaactgatc ccgggcctca aagcctacca ttgctgctca gccccaacac 960 aagaaggacc ggcatccggt ctctatgcgt ccgtgtgcaa gcagaccagc aagcacacag 1020 gcacggcgga gcatttctat gagaacgtgt gcatgctgga ggccagcctt gggctgacca 1080 atgggggtcc tgaagcccaa gagggccccc ttggtggccg cagccccctt gggcagcctt 1140 atctaccata acactgagga tctgagttgg ccgggctcgg cccaggacag caatctggaa 1200 gcccagtacc ggaggctgct ggaactggag ctggatgagg ccggaagcgc cggccgctct 1260 ggagcgcagg caggcatcaa ggccaagctg gtgaccctgc tgacccgtga acggaagaag 1320 ggccccgccc cctgtgaccg gccctgaagg cctgagcggc cagccactgc aggacagagg 1380 tgatcaccca agaccaggaa caacttcgaa cataacccgt ctactctgac ctgcagggac 1490 aagccaggtg gcccggggag gagccacact ctgccctacc tcctccctca gactgtacag 1500 attgaacagt aataaagctt gcctatcaac ttcaaaaaa 1539 <210> 16 <211> 3599 <212> DNA
<213> Mus abbotti <400> 16 agagacagcg tgatcccggc ctcccacggg gcagctttta ctgtctaggg aagaaatccc 60 caaagtccat ggagtctgaa gactctgtca agcctcgcta ggaaacctag gagttttaga 120 gggcacttgg caccggaagc tagccgggta g~cggagcct cacctggatt gagt=cacag 180 ctgcctagac aggctcagac taggtgctgg gcacctggga ggaggaggag acattagcag 290 caaaggctgt taacagaagt gcctgcctag ~cttggaggc aagacgctgc tgttcacagt 300 gcgagacgga ggtaggagta taatggctgt ccaggtgctg cggcagatgg tctacttcct 360 actgagtctg ttttctctgg tgcaaggtgc acacagtggc agcccccgag aagacttccg 420 Page 5 of 56 cttctgtggc cagcgga~ agacccaaca gagcaccctc cactatgatc as .:tcaga 480 gcctcacatc tttgtgtgga acacagagga gaccctcaca attcgtgccc ccttcctggc 590 agccccagat at~ccccgct tcttcccaga gcctagaggg ctctatcact tctgcctcta 600 ctggagtcgc cacactggga gactccactt gcgctatggc aagcatgact acctgcttag 660 tagccaagcc tccagactcc tctgcttcca gaaacaggaq cagagcctga agcagggagc 720 cccgctgatc gccacctctg tcagctcctg gcagattccc cagaacacca gcctgcctgg 780 ggctccgagc ttcatcttct ccttccacaa tgccccacac aaggtctccc acaatgcatc 890 tgtggacatg tgtgatctca agaaggaatt gcagcagctt agcaggtacc tgcagcaccc 900 tcaaaaggct gccaagcggc ccaccgcagc gttcatcagc cagcagttac agagcctgga 960 gtcaaagctg acctctgtga gcttcctggg agacacatta tcctttgagg aggaccgggt 1020 caatgctaca gtgtggaagc tgccacccac agccggtcta gaggatctgc atatccactc 1080 ccagaaggag gaggagcaga gtgaggtcca ggcatactcg ctgttgcttc cccgggccgt 1190 attccagcag accagaggcc gtcgccggga tgacgccaag aggctcctgg tagtagactt 1200 cagcagccaa gctttgttcc aggacaagaa ttctagccaa gtcctgggtg agaaggtctt 1260 gggtattgtc gtgcagaaca ccaaagtcac caacctctca gatccggtgg tactcacctt 1320 ccagcaccag cc~cagccaa aaaatgtgae tctgcagtgc gtgttctggg ttgaagaccc 1380 ggcatcaagc aacacaggga gctggagcag tgcaggctgc gagacagtga gcagagacac 1940 acagacatcc tgcctgtgca accacctgac ctactttgca gtgctgatgg tgtcatccac 1500 agaggtagaa gccactcaca aacactacct cacgctcctg tcctacgtgg gctgtgtcat 1560 ctctgctctg gcttgtgtct tcactatcgc tgcctacctc tgctccagga ggaagtcacg 1620 tgactacacc atcaaagtcc acatgaacct gctgtccgct gtcttcctgc tggacgtgag 1680 cttcctgctc agcgagcctg tggcactgac gggctccgaa gcagcctgtc gcaccagtgc 1790 catgttcctg cacttctccc tgcttgcctg cctctcctgg atgggcctcg agggctacaa 1800 tctctaccga ctggtggtgg aggtcttcgg tacctatgtg cccggctatc tgctcaagct 1860 gagcatcgtg ggctggggtt ttcctgtctt cctggtcact ctggtggcgt tggtggatgt 1920 gaataactac ggccccatta tcctagctgt gcgccggact ccggaacgtg tcacctaccc 1980 ctctatgtgc tggatccggg actccctggt gagctatgtc accaacctgg gcctcttcag 2090 tctggtgttc ctgttcaacc tggctatgct ggccaccatg gtggtgcaga tcctgcggct 2100 tcgcccgcac agccagaact ggccccacgt gctgaccctg ctgggcctca gcctggtcct 2160 tggcctcccc tgggccttgg tcttcttttc ctttgcttcc ggcaccttcc agcttgtcat 2220 cctctacctc ttcagcatca taacttccta ccaaggcttc ctcatcttcc tgtggtactg 2280 gtccatgcgg ttccaggccc aaggcggccc ctcccctctg aagaacaact cagacagcgc 2390 caaactcccc atcagctccg gcagcacctc ctccagccgc atctaagcca ccgccacacc 2400 tcccctccgg gaggacacat gcatggcgtc cgctcacgat gtctgtggcc cagtgctgtg 2960 cccacccagc ct~tgttggt tagtggcata ctagagaagg ccctggtcct tgaaggcgta 2520 gggctgttgc tctggtaggt agatacctag cttgccttgg ggacgactct ggtcctcaaa 2580 gggctcagaa gcacactgcc attctgtttg tggggccgtt tcagtctgga gctaaggcct 2690 tgtctttctg gccacctctg ggtccagctg ttgctgctgg gtgttgagac ctgcagaccc 2700 aagctggggt ta?atctcga aggaggctga cacatccggc ctgagacaca gctaactgtc 2760 ttgacttgct gctctgtctc tgtggtcacc atgcagatcc cgagggtggc actgggggta 2820 aatgttctgg gagaaggttt ggaggcagag caccttagga gctgagcatc tcccccagcc 2880 tttctgcaaa ccctcctctt cattccccat ccccaacccc tcctctgtgt tcccctaacc 2940 ctccacctga agcctggggt cctagaccaa tgctgtgatt tggggtggta gttcccagca 3000 gtttcctggt gccagctatc aacttctgtc tgttgtgtgg gctttggcct ctgactcagg 3060 gcaggtttct gtctgagccc tctctccaag ctgcctcacc tttgctcgca cctcagaggg 3120 acctccatct c~cctgaagc ctcctccctc tggcaagtac tgggatacag ccaccctttc 3180 aacccagcac tc~gaagacc aagacagccc cctctggtga cactggccaa gcttgatctt 3240 tttcctaaga agtggtcttc agatccccgc aggtcgctca gaagacactg ggctgcctag 3300 tgtgaattct gtcctactaa cgtacagtga gcagctcctc acccccaccc ccgcaaaagc 3360 tctcaccaag tc~tggagtg tcaggcaggg ggctggaaat ccaggaggac ttcctgcaaa 3420 aggcagcatt tcatcttgac ctcagccttc aggttgggga gaatgttctt tttaaatacc 3480 agttcatttg tc~~ttgata ttaaagctct ttatagagag tctggaaact gtaggcgatt 3540 gtcgagaaga gaaataaaaa tgagctgtta tctaatgcca tggcaaagca gcacaaaaa 3599 <210> 17 <211> 399 <212> DNA
<213> Mus musculus <400> 17 agcgacatgg ccccgcccgc gctccaggcc cagcctccag gcggctctca actgaggttc 60 c~gctgttcc tgctgctgtt gctgctgctg ctgtcatggc catcgcaggg ggacgccctg 120 ~caatgcctg aa~agcgacc ctccggccct gagtcccaac tcaacgccga cgagctacgg 180 ?gtcgcttcc agyacctgct gagccggctg catgccannc atagcgagag gactctaact 290 Page 6 of 56 ctgaaccaag tcctgac: ctgtcnggat actcantcca cangtgagat tc 3tccac 300 ggccagctgc t..ctccncgt caaccgggcg tcgctgantc anggtctccc cgaacctacc 360 ncgtgcancn agcgctgctc ctgctgacnn cnaangccg 399 <210> 18 <211> 900 <212> DNA
<213> Mus mus::ulus <900> 18 cggacgcgtg gggaaagcgg gtggggatat acgtgtgctg gtgccctaca attcgacagg 60 cgtcttggga gg;tcgacca ccttgcactg tactctgact tctaatgaga atgtgactat 120 cactcaaata ac:-tggatga agaangattc aggtggaccc cacgctcttg tggctgtctt 180 ccaccccaag a~gyggccca acatcaaaga gccagagagg gtgaaattct tggctgccca 240 acaggatctg aggaacgcat ctctggccat ctcgaactta agtgtnnaag acgaangcat 300 ctatgaatgt ca~attgcca cattccccag aagcagtana agcaccaatg cctggctgaa 360 ngtgcaagcc c~~ctnana acactgcana agccctggac 400 <210> 19 <211> 2313 <212> DNA
<213> Mus musculus <400> 19 cgaattcctg ggtcgaccca cgcgcccgag agggtagaca gaaaggcggg aaagggctgt 60 gaggtcaaat ggaccccatg gaactgaaca acgtcagcat cgaacccgac ggagacagct 120 gcagcgggga cagtattcag gacagctaca ccggcatgga aaactccgac aaggacgcca 180 tgaacagcca atttgctaat gaagatgccg aaagtcagaa gttcctgaca aatgggtttt 290 tagggaagaa gaagctagcc gattacgcgg atgagcatca ccctggaatg acttcctttg 300 gaatgtcctc atttaacctg agcaacgcca tcatgggcag tgggatctta ggcttgtcct 360 atgccatggc caa:.accggg atcatccttt ttataatcat gctgcttact gtggcaatac 920 tctcgctcta ctcggttcac cttttgctga agacagccaa ggaaggaggg tctctaatct 980 atgaaaaatt gggcgagaaa gcatttggat ggcctgggaa aattggagcc ttcatctcta 590 ttacaatgca gaacattgga gccatgtcaa gctacctctt catcattaag tacgaactgc 600 ctgaagtaat cagagcattc atgggacttg aagaaaacac tggggaatgg tacctcaacg 660 gcaactacct cgtcttattt gtgtccgtgg ggatcatcct cccgctctct ctccttaaaa 720 atttaggcta ccttggctac accagtggat tttctctctc ctgcatggtg tttttcgtca 780 gtgtggtgat tt~~aaaaaa ttccaaattc cctgccctct gcctgctctg gatcacaaca 890 acggaaatct gacgttcaac aacacacttc cgattcacat gatctcgctg cctaatgact 900 cggagagctc gggtgtgaac ttcatgatgg attacgctca ccacaaccca gctgggctgg 960 atgagaagca ggtcgcaggc cctcttcaca gcaatggcgt ggagtacgaa gcccagggtg 1020 ctgagaaatg ccaaccaaaa tactttgtgt tcaattcccg gacggcctat gcaatcccaa 1080 tcctggcttt tgcttttgtc tgccaccctg aggtccttcc catctacagc gagcttaaag 1190 atcgatcccg cagaaagatg cagacggtgt ccaacatttc catctcaggc atgctcgtca 1200 tgtaccttct tgcggccctc tttggttatc tgagcttcta cggggacgtt gaagacgagc 1260 tgctgcatgc tt~~agcaag gtctacacat ttgatacggc tcttctcatg gtgcgcctgg 1320 cagtcctggt ggcagtgaca ctgaccgtgc ccatcgtgct gttcccgatc cgtacttcgg 1380 tgatcacact gc~~tttcca aggaaaccct tcagctggct gaagcatttc gggatcgctg 1990 caatcatcat cgcactcaac aacatcctgg tcatcctcgt gcctaccatc aaatacatct 1500 ttggattcat aggggcttct tctgccacta tgctgatttt cattcttccg gctgcgtttt 1560 atctcaagct cgtcaagaaa gaacctctaa gatcacccca gaagattggg gctttggtct 1620 tccttgtgac tg?aattatt ttcatgatgg gaagcatggc gctcattata ctcgactgga 1680 tctacaaccc gccgaatccc aatcaccact aatcccgggg agacgcgtct ccactggaaa 1790 cagctgaaat tc~~tgaagg acattttagt tgtcttgatt gggatgttag tctgaggaat 1800 tagcaagatt ccaaagacgt ttttctagct ctatcagcac acattttaac ccaggccgtg 1860 cagtgcagtg tgtgatgccc gagttgtgtt tgcagcagct gtgcaagctg aagcctgttg 1920 gctgcgtgtg tt~gtcagca gacaatagcc tgtcccccca tggtcactcc acttctctcc 1980 acccccagat taacaggtaa ttctactctc agaacatcag acaaagacct cctggttggg 2090 atacttgtgg aagagaaaat tatgggtttt gttgggaatg gttttgttgg gaatggtgaa 2100 ggatgcatta aaaattctgt gcgaagtatc atcagttacg gccatctctc actctacacc 2160 aacactaagg gtc~gttgac tagctgaggc agggggatat cttgggctgt ccctgtgagg 2220 atcatgacgt atgacggttg ccagtataga gtacttcatt tcaatactca aggaatagtt 2280 tgcccaacct gcttattaca ccgagttagt gaa 2313 Page 7 of 56 <210> 20 <211> 3408 <212> DNA
<213> Mus musculus <900> 20 gcagctaccg gtccggaatt cccgggtcga cccacgcgtc ccacggctgc gagaagatca 60 cagaaggggg cggtgacgca ggaccaggac tcgcgcgtcc agcggagaag caggagaagc 120 cggcgacctt gc~ctctcag cctgatccct gtcttggcgg cctgaacatt cgcagctgga 180 gagatggcgt tcgtgaagag tggatggtta ct~cggcaga gcaccattct gaaacgctgg 290 aagaagaatt ggttcgacct gtggtcagac ggtcacctga tctactacga tgatcagact 300 cggcagagca tagaggataa ggtccacatg cccgtggact gcatcaatat ccgcacgggg 360 catgagtgcc gggacatcca gcctccagat gggaagccca gagactgtct gctgcagatc 420 gtttgccgag acgggaagac catcagtctc tgtgcagaga gcacagacga ttgcctggca 980 tggaagttta cactgcagga ttccagaaca aacacagctt acgttggttc agcaatcctg 540 tctgaagaga ctgcagtggc cgcgtccccg cctccctacg caacctatgc tacaccnacc 600 cctgaggtct ac7gctatgg tccatacagc ggcgcatacc ccgcaggaac tcaagttgtc 660 ~atgccgcca acgggcaggc atatgcagtg ccataccagt acccgtatgc aggagtttat 720 ggacaacagc ctgccaacca agtcatcatc cgcgagcggt accgagacaa tgacagtgac 780 ctggctctgg gcatgctcgc cggggcagcc accggcatgg ccctgggctc tctgttctgg 840 gtcttctaga gccttcaaca ttttctgtgc atagcttctg ttagtcctgt gtgcagtaat 900 ttgatttgca gggcatttct gtttgtgaca agtgtctttc ataataattt aaatagttct 960 tttgaaggtg gtaatctaat aattgtgact gacctgcatg gtaccacaaa gaaagcccga 1020 ggtatgctgt gagtgagagc ctgagtcctt ccgggtacta gcttgcacca agtctttctt 1080 agggactttt ggatggcttt atgtaaacac acccagttaa atgggcaatt tccgtccagt 1190 taggtgcagt gttgaattaa gggatggctt tccttgctat gccaatacta atactgctga 1200 tggaggaaga tgtgtgcaag tgtggtgagg agagtcacag cttctttaac tgtggattct 1260 cttctagacc cctgctgcgt gttaccctag gagctgtggg ctggtggctc ctgcaagact 1320 atggtgtgag gaccctgtaa cgtacctctt ggagcactta ggtacccctt gaagctccta 1380 ggtatcacca gcaggattgg ctgctcagga tgcagagggc caccccctcc ctttaaaaat 1990 tacgctccag taatctgccc agttttattt tcttgttatt cttctgtttg cttttcctgg 1500 ggatgattgg cattagtctg gagttaggaa ttgattcgag tgccggtggg tggaggcatg 1560 cagggagctg tccagcgacc tgctctcagt gtttgtttta ggtatattga ttgccagctc 1620 aggctgcaga gagcctatag agactatttt: tctacttgta aagaaagtat ggtgagggga 1680 attgaggaga gccttgtttg aatgttcctg cctcaggcct cctggggccc acactgcgtg 1740 gtcctgggga ggcttttcct cagcagtgga agggaggccc cgtggctgtc cagagtctca 1800 ggttttgagt gagaaatggg attgggtaga gcatctcagg gatttgttct aatccctcat 1860 gttatgggga tccagccgtg ttctcagtcc agacccgctc acctcagaag agcttaaaac 1920 atttctggtc cccaaatgtg tggcactctg agaagctcac aatctggctt tctaacgaaa 1980 atttgtattt ctaaaattag agaatacatg ttccacgcat ttaaaattta tgttctttca 2040 tgttttaaag ctcccaaatc cagctttgtg actggcatat tttagtttca aacagtaccc 2100 cggcacaaag gtgggatggc acagtgaagg ccccccgccc tctactttgc atagtcttgt 2160 ttctccaggg tgctcccagg aagcattcat tctgactttg ctcagcccag tgcatgcgtg 2220 ctgccttgcc gccgtgctgc tgggtagctc tttcttggtc agatcaagtc ttcaacagat 2280 ctccatgtga gacagttgcc aagtagatga ggtggtgccc atagtgcttt ctcgatactc 2390 cttggggacc tgttgacacc tgcccatttc cagctgacat ttgtttttct gtcatctctg 2400 atagatggga tatgtgacaa catggtacgg acgccgttca gtgtcgcttt aataagcatg 2460 atgctgattt tacatcctgt gctgtatgac tgccatttgc tcacagtgtc accattgcta 2520 aagctccgtg ctttacttac aaaacactaa aaccagtggt tagtgtttca cagtgatttt 2580 aattttagag ttagttactg gcattcctaa agccatagag tactgagtca catccctgaa 2640 gtacttttga aacagaattg tctcctactg tcccatgggt gtgccctgcc tgtctcctgg 2700 ccccaatggg gctagctgta ccaggcagcc atagttgagc ctgatcattc ctgtcaccag 2760 tttgacttga ttatataccc agaatggaat acattcttgg gcatctcagt tcctcagccc 2820 tgatcctcat agacgccacc ctttcgatgg cttttgcggc gtcacttgta cctcagtgag 2880 ~cctgcgatt cttgagttag aggggacgac ttgtccagca ttgaggaaca tgtctcctcc 2940 actgagactt aaatgatgat gcagggctgg aagaggctgg ctgctgacac tgcatcgtgg 3000 ctgatgtcat tgctctccta gttctttgat ttaagaacct ttcatatgga aggcctgagg 3060 ctccctcaga tc7~cccttg ccaagaaggc ctggcttagg tcattagtgc ccacagtagc 3120 cttctggagt gtagcaagtt cctgcgtttg agacagaatg gttcagattt attttctaca 3180 tctgttgttg accccatgca ccctctcatt ttgccttcca gtctacgtag atgaaagatg 3240 aaaggcagag gatgcagaca gtcttctttg tgattgcttc tgttattctg ttgcatctac 3300 ::gagcccgtt ttctccctgt ctgtgcatac agtatgttta taagtgaact tgttaaaata 3360 ~taaatgatc ac~aacccta aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3908 Page 8 of 56 <210> 21 <211> 878 <212> DNA
<213> Mus musculus <400> 21 aaatctcagc tgtgcagcct tctagaacct catgtctaca aagtgacaaa ctttcctcag 60 gcacaatcca tcatacagtg gatcaatcag tcagagtcag aggaagaact agtcaaaatt 120 acctcatttt ctgaattcat taacacctta aagaaaaccc acaaatacct aatggaagag 180 agtttcaaaa ctgagccccc agaaagagtg gaagaagcaa agagaatggc tacatatgaa 290 gtcaccacag ctctcagctc cttcttgaag tacctcagag aaacacagca gccagacatg 300 cagctgttgc tactctccat tgctactggt gtaggctatc agttggtaaa cagtatcttt 360 cagcatcttc tggggtgtga tgagttaaac ttcctcttgg atcaaacgga aaataacgaa 420 cataaatacc aagaactgaa aaatatttgc aattacagag cccaggcatt cttggtgctc 980 acagccctaa gagccacagt tgaaatcaca gatgtttcta cagaagagaa aggacaacgt 590 ttgacattaa tacaacaaca tatggggtca ctgttgtctg aagaagttgc acatgtcctc 600 acaaaacatg gagaacatca tgactgggaa aggctggaga atcatttgag attactcatt 660 gagggggact ataaagccac cacccattcc ttacaaatgg atgaagtaaa aaaacaattg 720 caaagtttnt gccatgaaaa gaaacagact tataaacaac aaggtaatga aaacagaaca 780 aaagaaatga tagaaaatgg acatttcctg gacttactcc aacgtttagg cctagacaat 890 tactatccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 87B
<210> 22 <211> 731 <212> DNA
<213> Mus musculus <900> 22 actagactct ggtagagaaa aaagtgcctc ggagtggctg ctgaacaacc tcatgactca 60 ccagagtgtg gagcttttca naaaaactca cttntaaacg tcaggaaaca gctcattcgt 120 tcntctaact tatttgtgat gcaagtggaa atagatgtnt atacagctct taaaaagtgg 180 atgttccttc agctggtgcc ntcctggaat ggggtctttg aaaacagctt ttgacaaaaa 290 cagatgtctg gttttccaag tggaaagaag actttgaaag gacgactttc cntgaaactg 300 aacagggaaa accagntcgc gcccntgtnc cgacatttaa ggc~acagta cattatcagt 360 gatctggctt ctgcaaggat cattgagcag gattctctgg taccttcaga atggctggcg 920 gcagtgtata aacagcagtg gctggctatg ctacgggctg aacaagacag tgaagtggcg 980 cctcaagaaa tcaataaaga agancttgag ggaaacagca tg~~~tgtgg tcgaaagctt 590 gccaaagatg gtgngtactg ctggcgctgg acacgcttca att.cggctt tgacctcctt 600 gtgncttaca ccaatcgata catcattttc caacgccata cnctgaacca gccatgttat 660 tggatctgtc agcttacacc tcgaaggagc atagcattta aatgcccttg gcttctttga 720 cagtagtggg a 731 <210> 23 <211> 400 <212> DNA
<213> Mus musculus <900> 23 gacatttaag gctacagtac attatcagtg atctggcttc tgcaaggatc attgagcagg 60 attctctggt accttcagaa tggctggcgg cagtgtataa acaccagtgg ctggctatgc 120 tacgggctga acaagacagt gaagtggggc ctcaagaaat caataaagaa gaacttgagg 180 gaaacagcat gaggtgtggt cgaaagcttg ccaaagatgg tgag:actgc tggcgctgga 240 cacgcttcaa tttcggcttt gacctccttg tgacttacac cap=cgatac atcattttcc 300 aacgccatac nctgaaccag ccatgttatt ggatctgtca gc~=acacct cgaagganca 360 tancatttaa atgcccttgg cttctttgac agtagtggga 900 <210> 24 <211> 2699 <212> DNA
<213> Mus musculus <900> 29 tgagcgcaac gcaattaatg tgagttagct cattcattag gcac:.ccagg ctttacactt 60 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taa~aatttc acacaggaaa 120 Page 9 of 56 cagctatgac catgatt~ ~ ccaagctcta atacgactca ctatagggaa ac, .ggtacg 180 cctgcaggta ccggtccgga attcccgggt cgacccacgc gtccgacggc tgcgagaaga 290 cgacagaagg gggcaccaca gaggacccct tcaacctaag gaaacaccca ggcttcgata 300 ggaccatgct gcagaggtgg cagaaaaggg agatcagcaa ctttgaatac ctcatgtacc 360 tcaacacact ggccggaagg acctacaatg actacatgca gtatcccgtg tttccctggg 420 tcctcgctga ctacacctca gagatgttga acttgacgaa tcccaagact ttccg;gatc 480 tttctaagcc aatgggggct cagaccaagg aaaggaagtt gaagtttacc cagaggttta 590 aagatgttga aaagattgaa ggagacatga ccgtgcagtg ccactactac acccactatt 600 cctcagccat cattgtcgct tcctacttgg tccgaatgcc accattcacg caggccttct 660 gctccttaca gggcggaagc tttgatgtgg ctgatagaat gttccacagt gtaaagagca 720 cgtgggagtc tgcctccaaa gagaacatga gcgatgtcag ggagctgaca cctgaattct 780 tctacctgcc cgagttttta accaactgta atgcagtgga gtttggctgc atgcaggatg 890 gaacgacact gggggatgtg cagcttcctc cctgggctga cggggatccg aggaaattca 900 tcagcttgca cagacaggct ctggaaagtg acttcgtcag cagcaacctc caccactgga 960 tagacctaat tt~tgggtat aagcagcagg ggccggctgc tgtagaggca gtgaacactt 1020 tccaccccta cttctacggt gatagaatag acctgggcag catcactgac ccgctgatca 1080 agagcaccat cctgggcttc atcagcaact ttggacaggt gcccaagcag atcttcacta 1190 aaccccaccc atccagaaac accacaggga aaaacccagg gcctggaaag gatgcttcca 1200 cccctgtagg cc=cccaggc cactcacagt ccttcctcca cagcctgcca gcactgagac 1260 cctctcaggt cacagtcaaa gatatgtacc ttttctctct agggtcggaa tcccccaaag 1320 gggccatcgg ccacatcgtc cctactgaga agtcaatcct ggcagtggag aagaacaagc 1380 tgctgatgcc ccctctctgg aacaggacct tcagctgggg ctttgatgac ttcagttgct 1490 gcctggggag ctacggctct gacaagatcc tgatgacctt tgagaacctg gctgcctggg 1500 gtccctgtct gtgcgctgta tgcccttccc ccacgatgat cgtcacatcc ggggccagcg 1560 cagtggtgtg catctgggag ctgagcctgg tcaaaggtcg cccgagaggt ctgaaactcc 1620 gacaggcctt gtatggacac actcaggcgg tcacatgtct gacagcctct gtcaccttca 1680 gcctcctggt gagcggatcc caggatcgca cttgtatcct gtgggacctg gaccacctct 1740 ctcgtgtggc ctgcctgcct gtccaccggg aaggcatctc agccattgcc atcagtgatg 1800 tctcgggaac cattgtctcc tgtgccggag cccacttgtc cctgtggaat gtcaatggac 1860 agcccctggc cagtattacc acagcctggg gcccagaagg aaccataacg tgctgctgca 1920 tagtagaggg gccagcgtgg gatgcaagcc acgtgatcat cacggggagt aaggacggaa 1980 tggttcggat ttggaagaca gaggacgtga agatgcctgt tcccaggcag gcagtgatgg 2040 aggagccctc cacggagccc ctaagcccca gaggtcacaa gtgggccaag aatcttgccc 2100 tgagccgaga gctggatgtc agtgttgctc tgagtggcaa gcccagcaag gcaagtcctg 2160 ctgtgacagc tc~ggccatc actaggaacc agagcaagct cttggttggc gatgagaagg 2220 gcgaatcttc tgctggtctg ctgatgggta ggagacaagg agctggaggg accgacctga 2280 aagcctggag ccctggggtc ggcagcaaca ggctacaggc acaagatgat gtgtagcctg 2390 ggctgcttaa ccagagcaag ttttgggggg gctccactcc acacagttct caaggagtcc 2400 ctgatggttt gcaccgtgta ccctaaacat gtctgtagtc tatgggactt ctgtaagaag 2960 gatcttggta gacactgatg ctggaaactg acgctgcatg ggaaatttct acgtggctca 2520 cttcaccaag gcttattgca ctgggaaaag aagagggggt gggtattggt tcatggaaac 2580 caccccactg tctttatttt attaaaactc cattttccga aaaaaaaaaa aaaaaaaaaa 2690 aaaaaaaaa 2649 <210> 25 <211> 999 <212> DNA
<213> Mus musculus <900> 25 ~ccattactt ccacctccca tgaggactgc acctgcggca gcgattttat gtgaacttgg 60 attactgtat ggaaatggag gagtgagaaa gtgtgggaat cataaaagaa ggcagaacca 120 aaagacagag ggatcttggg ccatgagtca cagcaaggaa agcctcccac caaacatctg 180 ~actggactc ctacaggaac aagaaatgaa ctatcgtgtt cagcccctga gacttgggct 290 ~aacatgatt ttcacattgt ctaccctacc taatagagca gagatgtaaa tattattctt 300 attttagagg tg~gatgcct cagctgcaat gggtgagaac tactcctcat ttattctctt 360 ~caaggcaat aaaagagaat ggaccaaaga cagtctgtca tcacatctag tcaaaagagc 920 ~aatgtcgca gtacaactct tcaaaagaaa aagaaaaaac aagaaaaaag taataaacag 980 stgtgttctg cttgaaaaa 999 <210> 26 <211> 686 <212> DNA
<213> Mus musculus Page 10 of 56 <900> 26 cqgggtcgcg c~~gaattaa gtgagtccat tcttgcacac cgagaaaata tgcgacagat 60 gataagaagt t~~~ctgaac cctttgggaa gagacttgct cagtatctct gatggtagan 120 ggagagctca ta~~cgtaga ggacataatg atggtgaaga ttctttgact catacagatg 180 tcagctcttt c~agacaatg gaccaaatgg tgtctaatat gagaaactat atgcagaaat 240 tagaaagaaa c:=cggtcaa ctttcagtgg atccaaatgg acattcattt tgttcttcct 300 cagttatgac tta~tccaaa ataggagatg aaccgccaaa ggtttttcat gcctcaactc 360 aaactcgtcg a~~tccagga agaataaagg aaaccaggaa agcnatgaga gattctgaca 920 gtgggactag aaaaaatggc tattggtcnt catatccatg aaccgagctc ntgttcntta 980 ataaagtccn nr.wanccnag aaagactggg agatgaaaaa aggtccaccc aggaanttca 590 tcccttatga aatgaaaatt gatgccccct gccttttgaa tgaaggaatt ggccaaattn 600 aaggtttttg aaattttcaa accccggaac aacccaatcc tnggaaaacc cctnnaaatg 660 aaaaaatttt t~gggccctt aaaaan 686 <210> z7 <211> 651 <212> DNA
<213> Mus mus~alus <900> 27 caaaannacn ty;.gtttagc aaataaaaac tgcccagtgg gacaancgtc gccgggaatc 60 gctgtcagta c~~ccgantt canaaatgcc tggctgttgg ganggncaaa gaaatggnnc 120 gcacagacag n: :aaaaggc cggagaagnc gnntgcccnc gaaaccgaag aaccacanga 180 nccctctccc ccttcgcccc cgttccangc gaacctgacn ancaaangaa nggagangan 290 acccagcana nc~ngcaant ctatganctc ctgactggct ccanggaaan catcccgggc 300 ngggcagaaa aaatccctgg gcnncgcaga actgccccaa gccgaccaag aactgcnnnn 360 tgaancagcn ntctnagaac tgnttgtccn nccannagca nacagaagat gaacatcgac 920 atntctgcct tc~~ctgcat ngcngccctg ggctatggnc acnnaaaaac acggcncngg 980 gaaccggaaa ar.ggaanaan gncnnccnga tngntgantg ncgcggagac acgngacttt 590 cnacnanggg gggggnannc gccccccttn nttgttccna ctgggggggg gaacnncccn 600 aaattcgngc c~~~nngggc ccnagggggg ggggggnccn ttttctttct n 651 <210> 28 <211> 683 <212> DNA
<213> Mus musc;:ius <900> 28 gggtctgttt ggactgcaga agcaaggggg tgatgtagcc catccttccc tttggagatg 60 ctgagggtgt tt~~tcctgc acccacagcc agggggatgc cactcctccc tccggcttga 120 cctgtttctc tgccgctacc tccctccccg tctcattccg ttgtctgtgg atggtcattg 180 cagtttaaga gcagaacaga tcttttactt tggccgcttg aaaagctagt gtacctcctc 240 tcagtgtttt gg~~tccatc tctcntcctc cantaccttg cttcttactg ataattttgc 300 ~ggaattcct aac a ttcaa tgacattttt tttaactact atattgattg tcctttaaaa 360 aagaaaagtg ca:atttatc caaaatgtgt atttcttata cgcttttctt tgttatacca 920 tttcctcagc t~~~ctcttt tatatttgta gganaaactc ccatgttatg gaatcccact 980 gtatgattta taaacagaca atatgtngag tgccttttgc aanaaaaagg gtgtgtttga 590 aatcatccgg ar.~~agccan gaaactgtct ccaaaggaaa cnccnacctc tctgttcctt 600 gctgttntgc tga=catccg ccanaaggtg cttccccccg aattttggtt tggttntgtt 660 tnccgaaaaa t~~=ttccgn ttn 683 <210> 29 <211> 624 <212> DNA
<213> Mus muscuius <900> 29 aatgcacctc c~=gtattcc cactttcgta ntcatttcgg ttctgatctt gtcaaaccca 60 gcctgaccgc t=~_gacgcc gggatggcct cgttactana cttttctttt taaggaantg 120 ctgttttttt t~~anggttt tcaaaacatt ttgaaaagca tttacttttt tgaccacgan 180 ccatganttt tcaaaaaaat ccggggttgt gtgggttttt ggtttttgtt ttaatttttg 240 gttgcgttgc c_~=tttttt taatggggtt ggccccatga aatgggtgcc ccactcactt 300 ~tctganatc gaacngactg tgaatccgct ctttgtcnga anctgaacaa nctgtggctt 360 Page I 1 of 56 ttttccaact c~gtgtg~ . tttctgaatg ttatgtggta agaccccgcg g5 ,tggcan 920 caactgccct ggaaccccan cccctgcntc catctgtngc tgntgcgccc cacantaaaa 480 cattcanacn t~cctgaaaa gttcttgaaa aaanttaatt anattgtccc nnttttactg 590 ggaaaaaatt anccntactc cnnccaattt tnttcttncn antgggctga aagaaatcct 600 tgttccccan ttttcctttg ggcn 624 <210> 30 <211> 691 <212> DNA
<213> Mus abbotti <400> 30 atcccagacc aggcaacaag gtcctttgag gtctataatg aaagatctgc attctgatga 60 caatgaggag gaatcagatg aagtggagga taacgacaat gactctgaaa tggagaggcc 120 tgtnaataga ggangcagcc gaagtcgcag agttagctta agtgatggca gcgatagtga 180 aagcagttct ccttcttcac ccctacatca cgaacctcca ccacccttac taaaaaccaa 290 caacaaccag a_tcttgaag tgaaaagtcc nataaagcaa agcaaatcag ataagcaaat 300 aaagaatggt gaatgtgaca aggcatacct agatgaactg gtngagcttc ccagaangtt 360 natgacattg agagaaagac acattctgca gcagatcgtg aaccttatng aagaaactgg 920 gacactttca tatcaccaac acaacatttg attttgatct ttgctcgctg gacaaaacca 980 cagtccgtta actacagagt tcctggaaac atctggaaca tcctgaagat ataaccactg 540 gatgcctcna gaactaatgt gtttnnnnnn nnnnngggtt nnnnnntttg ggtgtgaatt 600 ~ttgttcccg ttgtttaaat gaaaaccccc cnaatgatgc n 691 <210> 31 <211> 400 <212> DNA
<213> Mus musculus <900> 31 gcgagccgac ggagnggctc tacggtggat agcgtgttcc ggaacctatc cctcgaatta 60 gccgagtcag gcagagaggg ggcggggngt gcttccgccc ttgctaggag gggctgcatt 120 gcaggggaga cccagcggca gattctgtca cagacgaggg agaaggcgtg aggagacaaa 180 gccgtcacat ccgcgacagc ttccttcagc agcctcctcc tctccagtcc agagccgacc 240 cccgagcccc tgaggcatcc cttccgtctt cggaacaccc tagtcattca ttgctaacag 300 gggatcatga gggaccctgt aagtagccag tacagctcct ttcttttctg gangatgccc 360 atcccacaac t?gatctgtc ngagctggaa ggcctgggcc 900 <210> 32 <211> 900 <212> DNA
<213> Mus musculus <400> 32 acggctgcga gaagacgaca gaagggaagc taagggctgg tcgctgcgtc tgaacgccgg 60 gttggacgaa ccgctgtgcg cccttggcgg acgtnagcgg aaagaagatg gcggtgcagg 120 tggtgcaagc tgtgcangcg gttcatcttg antctgacgc tttcctantt tgtctcaacc 180 atgctctgag cacagaaaan gangaagtga tgggtctgtg tataggggan ttgaatgatg 290 acataaggag tcactccnaa tttacataca ctggaacgga aatgcgcaca gtccnagaaa 300 agatggatac catcagaatt gttcatatcc attctgtcat catcttgcgg cgttctgaca 360 aganaaanga ccgtgtnnaa atttctccan ancanctgtc 900 <210> 33 <211> 400 <212> DNA
<213> Mus musculus <900> 33 gatggcggtc t~~acaggag ttaaagttcc tcgtaatttt cgcttgttgg aagaacttga 60 agaaggacaa aaaggagtag gtgatggtac tgttagctgg ggccttgaag atgatgaaga 120 catgacactt acaaggtgga caggcatgat tattgggcca ccaaggacaa actatgaaaa 180 cagaatatat agcctgaaag tagaatgtgg atctaaatac ccagaagctc ctccatcagt 290 tagatttgta acaaaaatta atatgaatgg gatcaataat tccagtggaa tggtggatgc 300 acggagcata c~aatattag caaaatggca aaattcctat agcattaaag tcatacttca 360 Page 12 of 56 aganctaaga ctcttat~_ gtccaaagaa GG=atgaanc 400 <210> 34 <211> 900 <212> DNA
<213> Mus musculus <400> 39 acggctgcga gaagacgaca gaagggggag c_=ctgcgtc ccagggactc cagtacacca 60 ccatggcgga ttctgagcgt ctctcggccc ccygctgctg gttagcctgc accag~ttct 120 cgcgcaccaa aaagggaatt ctcctgtttg c~aanattat actgtgcctg gtgatcttga 180 tttgcttcag tgcatctaca acatcggcct ac~cctccct gtcggtgatt gagatgatct 240 gtgctgctgt cttacttgtc ttctacacgt ctgacctgca ctccaagata tcattcatca 300 actggccttg gactgacttc ttcagatccc t~ataacaac catcctgtac ctgatcacct 360 ccattgttgt ccttgtagaa agaagaagca g ~ccagagt 900 <210> 35 <211> 400 <212> DNA
<213> Mus musculus <900> 35 tacggctgcg agaagacgac agaaggggct ccgccttctt ccccacctcc ggctgccggc 60 gacaccggtc tttgcagtcc gggaccccgt gc7atcgtta gcccggtgca cgatgccccc 120 gaaaaaggga ggagatggaa ttaaaccgcc tccaattatt ggaagatttg gaacc~cact 180 gaaaattggt atcgttggat tgccaaatgt tgggaaatct accttcttca atgtattaac 290 caatagtcag gcttcancag aaaacttccc attctgcact attgatccta atgagagcag 300 agtgcctgtg ccagatgaga ggttcgactt tc~ttgccag taccataaac cancaagcaa 360 gatcctgctt tcctaaatgt aatggatatt gctggccttg 900 <210> 36 <211> 900 <212> DNA
<213> Mus musculus <400> 36 acggctgcga gaagacgacn gaaggggtcc ==~ttcctgc gcggcgtttc agtccctctt 60 gagttgggct gtcgtcnant cgcggcgana c~~gcgcacc gcagccatga cagaagctga 120 tgtnaatccg aangcctatc ccctcncana c::~ccacctc accaagaanc tgctggacct 180 tgttcnacag tcttgttnct acaancagct tc:gaaagga ccnatgaacc nccaaaaccc 240 tcaacagaag catctctgan ttcattgtga ~;~ngcanan ctgaaccttg gagat~~cct 300 gcacctccct ctnctgtcga aaaaaanaat g=~cctacct ntttgtncnn ccaacaggct 360 ttangaaagg cctntgggtt tccagccntc a=ccctgtct 900 <210> 37 <211> 399 <212> DNA
<213> Mus musculus <900> 37 gaaaggcgcc tagataccgc gatacttgcg g7tatcctgg agctcgactc tcctgttctt 60 gccacatttg actgaagaag gacaacgggg ~caaggtttg aggactgaca gattc~anac 120 ccaggcttcc tcngccccca naaagccacc a-:acctcaca tggaaccaaa ggccccatgc 180 ccanccgccg tcccctcaga gganangaaa t»cgtgttc ttgt.~.ggcgt cactg;cagc 290 gtggccgctc tgaagctgcc tctcctgnta t~~annctgt tggacgttcc tgngatgtgg 300 aagcgccgtt ctgacccggt tctccacatt gacctgcgga tgtcggctga cctca~gcta 360 gtggctcccc tcnatgcnna cactctgngg aa~t 399 <210> 38 <211> 315 <212> DNA
<213> Mus musculus <900> 38 Page 13 of 56 acggctgcga gaagacg, , gaangggggc aggatggctc ggtggtacag t~ .agatca 60 anangcatac cccactgagc aagctgatga agcctactgt gagaggcagg gcttgtcnat 120 nangcagatt cgattccggt ttgatggaca accaatcaat gaaacagaca ctccacccca 180 nctggagatg gaggatgagg acaccnttga tgtattccag cagcagacag gaggatcanc 240 ctcccgaggg agcgtcccac acccaaccgt tgtcctgacc tgtgctattg agcagtgacc 300 atgctnccac accca 315 <210> 39 <211> 400 <212> DNA
<213> Mus musculus <900> 39 acggctgcga gaagacgaca gaaggggaga gcatcatggc gctaagcggt cgactggcat 60 tggccgcgct cagactgtgg ggtccgggag gatgtatctg anctgcttcg gaccanancc 120 tgcagtaggg tggtggtcat ggtgggggcc ggcatcanca cacccagtgg catcccggac 180 ttcagatccc caggggagcg gcctctacag caaccttcag cagtatgaca tcccgtaccc 290 tgaagccatc tttgaacttg gctttttctt tcacaacccc aagccctttt tcatgttggc 300 caaggactgt nccctgngca ctacaggcca atgtcactca ctacttcctg aagctcctcc 360 acgacangga ccgcttctgc gctctntaca canaaatc.ga 900 <210> 90 <211> 349 <212> DNA
<213> Mus musculus <900> 40 ggccatcaan cgtgctgttg gtcatggccg actcctcaaa gatggacccc tctaccgcct 60 caacactaag gcagccagtg ctgaagggaa agagggctgt gagtcgctct cgtgtttacc 120 tcccgtgtca ctgcttccac acganaaaga caagccggtt gctgaaccaa tccccatctg 180 tagcttctgt cttggtacaa aagaacagaa r_cgggagaag cagcccgagg aactcgtctc 240 ctgcgcggac tgcggcaata ncggtcatcc atcgtgttta aagttctccc canagctnac 300 agtgagagtg aagccttacn gtggcttnca ttgantgtaa aacatcact 399 <210> 91 <211> 397 <212> DNA
<213> Mus musculus <900> 91 cccacgcgtc cgatctcctc cagggccacc aagcacctct gaagagccat gttccaagct 60 gccggagccg cccaggccac cccctctcat gaagccaaag gcagcagtgg cagcagcacg 120 gtacagcggt ctaagtcctt tagcttgcgg gctcaggtga aggagacctg tgcagcctgc 180 cagaagactg tgtacccgat ggagcggctg gtggcagaca agctcatttt ccacaactct 290 tgtttctgtt gcaaacactg ccacaccaaa ctcagcctgg gcagttatgc tgcaatgcac 300 ggtgaatttt actgcagacc tcactttcag cagctgttta agagtaaagg caactacgat 360 gaaggttttc gtaaacagca caaggactct gggccac 397 <210> 92 <211> 158 <212> PRT
<213> Mus musculus <400> 42 Met Phe Gln Ala Ala Gly Ala Ala Gln Ala Thr Pro Ser His Glu Ala Lys Gly Ser Ser Gly Ser Ser Thr Val Gln Arg Ser Lys Ser Phe Ser Leu Arg Ala Gln Val Lys Glu Thr Cys Ala Ala Cys Gln Lys Thr Val ~Ir Pro Met Glu Arg Leu 'Jal Ala Asp Lys Leu Ile ?he His Asn Ser Page 14 of 56 Cys Phe Cys Cys Lys His Cys His Thr Lys Leu Ser Leu Gly Ser Tyr Ala Ala Met :?is Gly Glu Phe Tyr Cys Arg Pro His Phe Gln Gln Leu Phe Lys Ser Lys Gly Asn Tyr Asp Glu Gly Fhe Gly Arg Lys Gln His Lys Glu Leu Trp Ala His Lys Glu Val Asp Ser Gly Thr Lys Thr Ala Asp Pro Phe Asn Thr His Ser Leu Pro Ala His Gly Leu Pro Leu Gly Ser Gly Lys G_u Ile Asn Pro Gly Ala Arg Gly Gly Arg Gly <210> 43 <211> 1360 <212> DNA
<213> Mus musculus <400> 93 agctctagtc cccagagatg tcgccactac tgctgctgct gctgtgcctg ctgctgggga 60 atttggagcc tgaggaggcc aaactgatcc gtgtccctct tcaacgaatc caccttggac 120 acagaatctt aaacccactg aatggatggg aacagctggc agagctttct aggacctcca 180 cctctggtgg caacccctcc tttgtgcctc tctccaagtt catgaacacc cagtattttg 290 gaactattgg tttgggaacg cctcctcaga atttcaccgt tgtctttgac acgggttctt 300 ccaacttgtg ggttccgtcc acgagatgtc atttcttcag tttggcatgc tggtttcacc 360 atcgctttaa tcccaaggcc tccagctcct tcaggcccaa tgggaccaag tttgccattc 920 agtatgggac cgggcggctg agcggaatcc tgagccagga caatctgact atcgggggga 480 tccacgatgc ttttgtgaca tttggagagg ctctgtggga gcccagcctg atctttgctt 540 tagcccactt tgatgggatc ctgggcctcg gcttccccac tctggctgtg ggcggagttc 600 agcctccgct ggatgcgatg gtggagcaag ggctgctgga gaaacccgtc ttctcctttt 660 acctcaacag ggattctgaa gggtctgatg ggggagagct ggtcctaggg ggctcagacc 720 ccgctcacta cgtacctccc ctcaccttca taccagtcac catccctgcc tactggcagg 780 tccacatgga gagtgtgaag gtcggcacag ggctgagcct ctgtgcccag ggctgcagtg 890 ccatcctaga cacaggcaca tccctcatca caggacctag tgaggagatc cgggccttga 900 ataaagccat tgggggatat cccttcctga atgggcagta cttcattcag tgttccaaga 960 cgccaacgct tccccctgtc tccttccacc ttggtggagt ctggtttaac ctcacaggcc 1020 aggactatgt catcaagatt cttcagagcg atgttggcct ctgcctgttg ggcttccaag 1080 ccttggatat ccccaagcct gcgggacccc tctggatcct tggggacgtc tttttggggc 1140 cctatgtggc tgtctttgac cgtggggaca aaaacgtcgg cccgcgcgtg ggactggcgc 1200 gtgctcagtc tccttcaaca gaccgggcag aaagaaggac tacgcaggcg cagttcttca 1260 aaagacgccc tggttagggt acaagctcac cgggccacag cagctatgct tctttccaat 1320 taaacaaact aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1360 <210> 44 <211> 419 <212> PRT
<213> Mus musculus <400> 99 :4et Ser Pro Les Leu Leu Leu Leu Leu Cys Leu Leu Leu Gly Asn Leu Glu Pro Glu G~:: Ala Lys Leu Ile Arg Val Pro Leu Gln Arg Ile His Leu Gly His ::r? Ile Leu Asn Pry Leu Asn Gly Trp Glu Gln Leu Ala Page 15 of 56 Glu Leu Ser Arg Thr Ser Thr Ser Gly ply Asn Pro Ser Phe Val Pro Leu Ser Lys Phe Met Asn Thr Gln Tyr Phe Gly Thr Ile Gly Leu Gly Thr Pro Pro Gln Asn Phe Thr Val Val Phe Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Thr Arg Cys His Phe Phe Ser Leu Ala Cys Trp Phe His His Arg Phe Asn Pro Lys Ala Ser Ser Ser Phe Arg Pro Asn Gly Thr Lys Phe Ala Ile Gln Tyr Gig :hr Gly Arg Leu Ser Gly Ile Leu Ser Gln Asp Asn Leu Thr Ile Gly Gly Ile His Asp Ala Phe Val Thr Phe Gly Glu Ala Leu Trp Glu Pro Ser Leu Ile Phe Ala Leu Ala His Phe Asp Gly Ile Leu Gly Leu Gly Phe Pro Thr Leu Ala Val Gly Gly Val Gln Pro Pro Leu Asp Ala Met Val Glu Gln Gly Leu Leu Glu Lys Pro Val Phe Ser Phe Tyr Leu Asn Arg Asp Ser Glu Gly Ser Asp Gly Gly Glu Leu Val Leu Gly Gly Sir Asp Pro Ala His Tyr Val Pro Pro Leu Thr Phe Ile Pro Val Thr Ile Pro Ala Tyr Trp Gln Val His 295 250 255.
Met Glu Ser Val Lys Val Gly Thr Gll Leu Ser Leu Cys Ala Gln Gly Cys Ser Ala Ile Leu Asp Thr Gly Thr Ser Leu Ile Thr Gly Pro Ser Glu Glu Ile Arg Ala Leu Asn Lys Ala Ile Gly Gly Tyr Pro Phe Leu Asn Gly Gln Tyr Phe Ile Gln Cys Ser Lys Thr Pro Thr Leu Pro Pro Val Ser Phe His Leu Gly Gly Val Trp Phe Asn Leu Thr Gly Gln Asp Tyr Val Ile Lys Ile Leu Gln Ser Asc Val Gly Leu Cys Leu Leu Gly 390 39~ 350 Phe Gln Ala Leu Asp Ile Pro Lys Prc Ala Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Leu Gly Pro Tyr VG: Ala Val Phe Asp Arg Gly Asp Page 16 of 56 Lys Asn Val Gly Pro Arg Val Gly Leu Ala Arg Ala Gln Ser Arg Ser 385 390 395 y00 Thr Asp Arg Ala Glu Arg Arg Thr Thr Gln Ala Gln Phe Phe Lys Arg Arg Pro Gly <210> 95 <211> 3338 <212> DNA
<213> Mus musculus <400> 95 cggtgacgca ggaccaggac tcgcgcgtcc agcggagaag caggagaagc cggcgacctt 60 gcgctctcag cctgatccct gtcttggcgg cctgaacatt cgcagctgga gagatggcgt 120 tcgtgaagag tggatggtta cttcggcaga gcaccattct gaaacgctgg aagaagaatt 180 ggttcgacct gtggtcagac ggtcacctga tctactacga tgatcagact cggcagagca 290 tagaggataa ggtccacatg cccgtggact gcatcaatat ccgcacgggg catgagtgcc 300 gggacatcca gcctccagat gggaagccca gagactgtct gctgcagatc gtttgccgag 360 acgggaagac catcagtctc tgtgcagaga gcacagacga ttgcctggca tggaagttta 920 cactgcagga ttccagaaca aacacagctt acgttggttc agcaatcctg tctgaagaga 980 ctgcagtggc cgcgtccccg cctccctacg caacctatgc tacaccnacc cctgaggtct 590 acggctatgg tccatacagc ggcgcatacc ccgcaggaac tcaagttgtc tatgccgcca 600 acgggcaggc atatgcagtg ccataccagt acccgtatgc aggagtttat ggacaacagc 660 ctgccaacca agtcatcatc cgcgagcggt accgagacaa tgacagtgac ctggctctgg 720 gcatgctcgc cggggcagcc accggcatgg ccctgggctc tctgttctgg gtcttctaga 780 gccttcaaca ttttctgtgc atagcttctg ttagtcctgt gtgcagtaat ttgatttgca 890 gggcatttct gtttgtgaca agtgtctttc ataataattt aaatagttct tttgaaggtg 900 gtaatctaat aattgtgact gacctgcatg gtaccacaaa gaaagcccga ggtatgctgt 960 gagtgagagc ctgagtcctt ccgggtacta gcttgcacca agtctttctt agggactttt 1020 ggatggcttt atgtaaacac acccagttaa atgggcaatt tccgtccagt taggtgcagt 1080 gttgaattaa gggatggctt tccttgctat gccaatacta atactgctga tggaggaaga 1140 tgtgtgcaag tgtggtgagg agagtcacag cttctttaac tgtggattct cttctagacc 1200 cctgctgcgt gttaccctag gagctgtggg ctggtggctc ctgcaagact atggtgtgag 1260 gaccctgtaa cgtacctctt ggagcactta ggtacccctt gaagctccta ggtatcacca 1320 gcaggattgg ctgctcagga tgcagagggc caccccctcc ctttaaaaat tacgctccag 1380 taatctgccc agttttattt tcttgttatt cttctgtttg cttttcctgg ggatgattgg 1940 cattagtctg gagttaggaa ttgattcgag tgccgqtggg tggaggcatg cagggagctg 1500 tccagcgacc tgctctcagt gtttgtttta ggtatattga ttgccagctc aggctgcaga 1560 gagcctatag agactatttt tctacttgta aagaaagtat ggtgagggga attgaggaga 1620 gccttgtttg aatgttcctg cctcaggcct cctggggccc acactgcgtg gtcctgggga 1680 ggcttttcct cagcagtgga agggaggccc cgtggctgtc cagagtctca ggttttgagt 1740 gagaaatggg attgggtaga gcatctcagg gatttgttct aatccctcat gttatgggga 1800 tccagccgtg ttctcagtcc agacccgctc acctcagaag agcttaaaac atttctggtc 1860 cccaaatgtg tggcactctg agaagctcac aatctggctt tctaacgaaa atttgtattt 1920 ctaaaattag agaatacatg ttccacgcat ttaaaattta tgttctttca tgttttaaag 1980 ctcccaaatc cagctttgtg actggcatat tttagtttca aacagtaccc cggcacaaag 2090 gtgggatggc acagtgaagg ccccccgccc tctactttgc atagtcttgt ttctccaggg 2100 tgctcccagg aagcattcat tctgactttg ctcagcccag tgcatgcgtg ctgccttgcc 2160 gccgtgctgc tgggtagctc tttcttggtc agatcaagtc ttcaacagat ctccatgtga 2220 gacagttgcc aagtagatga ggtggtgccc atagtgcttt ctcgatactc cttggggacc 2280 tgttgacacc tgcccatttc cagctgacat ttgtttttct gtcatctctg atagatggga 2390 tatgtgacaa catggtacgg acgccgttca gtgtcgcttt aataagcatg atgctgattt 2900 tacatcctgt gctgtatgac tgccatttgc tcacagtgtc accattgcta aagctccgtg 2460 ctttacttac aaaacactaa aaccagtggt tagtgtttca cagtgatttt aattttagag 2520 ttagttactg gcattcctaa agccatagag tactgagtca catccctgaa gtacttttga 2580 aacagaattg tctcctactg tcccatgggt gtgccctgcc tgtctcctgg ccccaatggg 2640 gctagctgta ccaggcagcc atagttgagc ctgatcattc ctg:caccag tttgacttga 2700 ttatataccc agaatggaat acattcttgg gcatctcagt tcctcagccc tgatcctcat 2760 agacgccacc ctttcgatgg cttttgcggc gtcacttgta cct:.agtgag tcctgcgatt 2820 Page 17 of 56 cttgagttag aggggac,. ttgtccagca ttgaggaaca tgtctcctcc ac .agactt 2880 aaatgatgat gcagggctgg aagaggctgg ctgctgacac tgcatcgtgg ctgatgtcat 2990 tgctctccta gttctttgat ttaagaacct ttcatatgga aggcctgagg ctccctcaga 3000 tcgtcccttg ccaagaaggc ctggcttagg tcattagtgc ccacagtagc cttctggagt 3060 gtagcaagtt cctgcgtttg agacagaatg gttcagattt attttctaca tctgttgttg 3120 accccatgca ccc=ctcatt ttgccttcca gtctacgtag atgaaagatg aaaggcagag 3180 gatgcagaca gtcttctttg tgattgcttc tgttattctg ttgcatctac cgagcccgtt 3240 ttctccctgt ctgtgcatac agtatgttta taagtgaact tgttaaaata ttaaatgatc 3300 actaacccta aaaaaaaaaa aaaaaaaaaa aaaaaaaa 3338 <210> 96 <211> 221 <212> PRT
<213> Mus musculus <900> 46 Met Ala Phe Vai Lys Ser Gly Trp Leu Leu Arg Gln Ser Thr Ile Leu Lys Arg Trp Lys Lys Asn Trp Phe Asp Leu Trp Ser Asp Gly His Leu Ile Tyr Tyr Asp Asp Gln Thr Arg Gln Ser Ile Glu Asp Lys Val His Met Pro Val Asp Cys Ile Asn Ile Arg Thr Gly His Glu Cys Arg Asp Ile Gln Pro Pro Asp Gly Lys Pro Arg Asp Cys Leu Leu Gln Ile Val Cys Arg Asp Gly Lys Thr Ile Ser Leu Cys Ala Glu Ser Thr Asp Asp Cys Leu Ala Trp Lys Phe Thr Leu Gln Asp Ser Arg Thr Asn Thr Ala Tyr Val Gly Ser Ala Ile Leu Ser Glu Glu Thr Ala Val Ala Ala Ser Pro Pro Pro Tyr Ala Thr Tyr Ala Thr Xaa Thr Pro Glu Val Tyr Gly Tyr Gly Pro Tyr Ser Gly Ala Tyr Pro Ala Gly Thr Gln Val Val Tyr Ala Ala Asn Gly Gln Ala Tyr Ala Val Pro Tyr Gln Tyr Pro Tyr Ala Gly Val Tyr Gly Gln Gln Pro Ala Asn Gln Val Ile Ile Arg Glu Arg Tyr Arg Asp Asn Asp Ser Asp Leu Ala Leu Gly Met Leu Ala Gly Ala Ala Thr Gly Met Ala Leu Gly Ser Leu Phe Trp Val Phe <210> 47 <211> 2396 <212> DNA
<213> Mus musc;:lus Page 18 of 56 <400> 97 caccacagag gaccccttca acctaaggaa acacccag~~ ttcgatagga ccatgctgca 60 gaggtggcag aaaagggaga tcagcaactt tgaatacc-c atgtacctca acacactggc 120 cggaaggacc tacaatgact acatgcagta tcccgtgttt ccctgggtcc tcgctgacta 180 cacctcagag atgttgaact tgacgaatcc caagactt=~ cgggatcttt ctaagccaat 240 gggggctcag accaaggaaa ggaagttgaa gtttacccag aggtttaaag atgttgaaaa 300 gattgaagga gacatgaccg tgcagtgcca ctactacacc cactattcct cagccatcat 360 tgtcgcttcc tacttggtcc gaatgccacc attcacgcag gccttctgct ccttacaggg 420 cggaagcttt gatgtggctg atagaatgtt ccacagtgta aagagcacgt gggagtctgc 980 ctccaaagag aacatgagcg atgtcaggga gctgacacc~ gaattcttct acctgcccga 590 gtttttaacc aactgtaatg cagtggagtt tggctgcat? caggatggaa cgacactggg 600 ggatgtgcag cttcctccct gggctgacgg ggatccgagg aaattcatca gcttgcacag 660 acaggctctg gaaagtgact tcgtcaqcag caacctccac cactggatag acctaatttt 720 tgggtataag cagcaggggc cggctgctgt agaggcagt? aacactttcc acccctactt 780 ctacggtgat agaatagacc tgggcagcat cactgacccy ctgatcaaga gcaccatcct 890 gggcttcatc agcaactttg gacaggtgcc caagcagatc ttcactaaac cccacccatc 900 cagaaacacc acagggaaaa acccagggcc tggaaagg~t gcttccaccc ctgtaggcct 960 cccaggccac tcacagtcct tcctccacag cctgccagc~ ctgagaccct ctcaggtcac 1020 agtcaaagat at~taccttt tctctctagg gtcggaatc~ cccaaagggg ccatcggcca 1080 catcgtccct actgagaagt caatcctggc agtggagaag aacaagctgc tgatgccccc 1190 tctctggaac aggaccttca gctggggctt tgatgact== agttgctgcc tggggagcta 1200 cggctctgac aagatcctga tgacctttga gaacctggct gcctggggtc cctgtctgtg 1260 cgctgtatgc ccttccccca cgatgatcgt cacatccggg gccagcgcag tggtgtgcat 1320 ctgggagctg agcctggtca aaggtcgccc gagaggtct3 aaactccgac aggccttgta 1380 tggacacact caggcggtca catgtctgac agcctctgtc accttcagcc tcctggtgag 1990 cggatcccag gatcgcactt gtatcctgtg ggacctggac cacctctctc gtgtggcctg 1500 cctgcctgtc caccgggaag gcatctcagc cattgccatc agtgatgtct cgggaaccat 1560 tgtctcctgt gccggagccc acttgtccct gtggaatgtc aatggacagc ccctggccag 1620 tattaccaca gcctqgggcc cagaaggaac cataacgtgc tgctgcatag tagaggggcc 1680 agcgtgggat gcaagccacg tgatcatcac ggggagtaag gacggaatgg ttcggatttg 1790 gaagacagag gacgtgaaga tgcctgttcc caggcaggca gtgatggagg agccctccac 1800 ggagccccta agccccagag gtcacaagtg ggccaagaat cttgccctga gccgagagct 1860 ggatgtcagt gttgctctga gtggcaagcc cagcaaggca agtcctgctg tgacagctct 1920 ggccatcact aggaaccaga gcaagctctt ggttggcgat gagaagggcg aatcttctgc 1980 tggtctgctg atgggtagga gacaaggagc tggagggacc gacctgaaag cctggagccc 2090 tggggtcggc agcaacaggc tacaggcaca agatgatgtg tagcctgggc tgcttaacca 2100 gagcaagttt tgggggggct ccactccaca cagttctcaa ggagtccctg atggtttgca 2160 ccgtgtaccc taaacatgtc tgtagtctat gggacttct; taagaaggat cttggtagac 2220 actgatgctg gaaactgacg ctgcatggga aatttctacg tggctcactt caccaaggct 2280 tattgcactg ggaaaagaag agggggtggg tattggttca tggaaaccac cccactgtct 2390 ttattttatt aaaactccat tttccgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2396 <210> 98 <211> 693 <212> PRT
<213> Mus musculus <900> 48 Thr Thr Glu Asp Pro Phe Asn Leu Arg Lys His Pro Gly Phe Asp Arg Thr Met Leu Gln Arg Trp Gln Lys Arg Glu lle Ser Asn Phe Glu Tyr Leu Met Tyr Leu Asn Thr Leu Ala Gly Arg Thr Tyr Asn Asp Tyr Met Gln Tyr Pro Val Phe Pro Trp Val Leu Ala Asp Tyr Thr Ser Glu Met Leu Asn Leu Thr Asn Pro Lys Thr Phe Arg Asp Leu Ser Lys Pro Met Gly Ala Gln ~!:r Lys Glu Arg Lys Leu Lys Fhe Thr Gln Arg Phe Lys Page 19 of 56 WO 00/i 1168 PCT/US99/19052 Asp Val Glu Lys Ile Glu Gly Asp Met '"hr Val Gln Cys His Tyr Tyr Thr His Tyr Ser Ser Ala Ile Ile Val Ala Ser Tyr Leu Val Arg Met Pro Pro Phe Thr Gln Ala Phe Cys Ser Leu Gln Gly Gly Ser Phe Asp Val Ala Asp Arg Met Phe His Ser Val Lys Ser Thr Trp Glu Ser Ala Ser Lys Glu Asn Met Sex Asp Val Arg Glu Leu Thr Pro Glu Phe Phe Tyr Leu Pro Glu Phe Leu Thr Asn Cys Asn Ala Val Glu Phe Gly Cys Met Gln Asp Gly Thr Thr Leu Gly Aso Val Gln Leu Pro Pro Trp. Ala Asp Gly Asp Pro Arg Lys Phe Ile Ser Leu His Arg Gln Ala Leu Glu Ser Asp Phe Val Ser Ser Asn Leu His His Trp Ile Asp Leu Ile Phe Gly Tyr Lys Gln Gln Gly Pro Ala Ala Val Glu Ala Val Asn Thr Phe His Pro Tyr Phe Tyr Gly Asp Arg Ile Asp Leu Gly Ser Ile Thr Asp Pro Leu Ile Lys Ser Thr Ile Leu Gly Phe Ile Ser Asn Phe Gly Gln Val Pro Lys Gln Ile Phe Thr Lys Pro His Pro Ser Arg Asn Thr Thr Gly Lys Asn Pro Gly Pro Gly Lys Asp Ala Ser Thr Pro Val Gly Leu Pro Gly His Ser Gln Ser Phe Leu His Ser Leu Pro Ala Leu Arg Pro Ser Gln Val Thr Val Lys Asp Met Tyr Leu Phe Ser Leu Gly Ser Glu Ser Pro Lys Gly Ala Ile Gly His Ile Val Pro Thr Glu Lys Ser Ile Leu Ala Val Glu Lys Asn Lys Leu Leu Met Pro Pro Leu Trp Asn Arg Thr Phe Ser Trp Gly Phe Asp Asp Phe Ser Cys Cys Leu Gly Ser Tyr Gly Ser Asp Lys Ile Leu Met Thr Phe Glu Asn Leu Ala Ala Trp Gly Pro Cys Leu Cys Ala Val Cys Pro Ser Pro Thr Met lle Val Thr Ser 920 42~ 930 Page 20 of 56 Gly Ala Ser Ala Val Val Cys Ile Trp Glu Leu Ser Leu Val Lys ply Arg Pro Arg Gly Leu Lys Leu Arg Gln Ala Leu Tyr Gly His Thr ~ln Ala Val Thr Cys Leu Thr Ala Ser Val Thr Phe Ser Leu Leu Val Ser Gly Ser Gln Asp Arg Thr Cys Ile Leu Trp Asp Leu Asp His Leu Ser Arg Val Ala Cys Leu Pro Val His Arg Glu Gly Ile Ser Ala Ile Ala Ile Ser Asp Val Ser Gly Thr Ile Val Ser Cys Ala Gly Ala His Leu Ser Leu Trp Asn Val Asn Gly Gln Pro Leu Ala Ser Ile Thr Thr Ala Trp Gly Pro Glu Gly Thr Ile Thr Cys Cys Cys Ile Val Glu Gly Pro Ala Trp Asp Ala Ser His Val Ile Ile Thr Gly Ser Lys Asp Gly Met Val Arg Ile Trp Lys Thr Glu Asp Val Lys Met Pro Val Pro Arg Gln Ala Val Met Glu Glu Pro Ser Thr Glu Pro Leu Ser Pro Arg Gly His Lys Trp Ala Lys Asn Leu Ala Leu Ser Arg Glu Leu Asp Val Ser val Ala Leu Ser Gly Lys Pro Ser Lys Ala Ser Pro Ala Val Thr Ala ~eu Ala Ile Thr Arg Asn Gln Ser Lys Leu Leu Val Gly Asp Glu Lys Gly Glu Ser Ser Ala Gly Leu Leu Met Gly Arg Arg Gln Gly Ala Gly Gly Thr Asp Leu Lys Ala Trp Ser Pro Gly Val Gly Ser Asn Arg Leu Gln Ala Gln Asp Asp Val <210> 49 <211> 2363 <212> DNA
<213> Mus musculus <900> 49 cagaggcagg caggacaggc actgcasgca ctgagagcct tgcagtcaga agggaagcac 60 asacaggaag aggcagtgag gagaaaagga gcasaactga gacaagcaat ggagatccct 120 gaggagtgct ggccaacggc tgaagtgtcc ctaaaagaca tcactgaaat aatggagaga 180 catctcagtc acatggaacg gaccctgtct cacagtcaaa agctctcaga tggagacctg 290 gtaagatggg catctggagg gctggtcctg cagggaattt ataagaccaa ccacccaaga 300 Page 21 of 56 agcctgattc agaagag, agagctactc agtgtcccta agcagttctc a~ gctggc 360 ccagtacatg gcacagagat aaaaacaaag gaattttcat cttttcaaga acaagccatg 420 tttacacaga ctatagagag ggtgggtttc agttcaaccc ccttagttaa aggagaaggt 480 tggggactta gtctagaagc tggtatgggt cacaacaaac agacagaatc tgaagataac 590 taccagtccc attcaaagca aacttatttt tgctcagcca ggttcagcta catcccattg 600 gccacctgcc attttcacat caatgatctt gaactctccc aggctgctct ccaggaacta 660 aaaagtattg aagaaatcct ggagcagact acacaccacc gagatggact acccttactg 720 aggcacaggg ctaaaaactt tttccacagg tttggctctc atgctaacca aggccctgtg 780 cacctggggg gaatctactg ctggaaagcc atttcagaag gtttcaaaag tgagcacttg 890 gctgatgtaa agcagcaagc agaagagtct ttgaatattt acattatggg cagttatagt 900 ggctttggag ttaaagttgg tgcgagtgta aatataacaa attcaaaatc aaaaacagca 960 ttttacagta aaactcatct aaactcgcaa accaaggtac aactatctgt agccaagata 1020 ggtggaccag cagaagcaga tggaattgcc cagtggacag ctggccttgt agctagcaat 1080 caaacctggt ctcttattga taggaaactg cagttggtac ctatttggga cattatcctg 1190 tccagtcaca gaactgaatt taagaatgct cttcaactgg ctaactgcct caaagaccac 1200 tacactgctc tga~tgaact agctgcccag attcaagaag gggaagaatt tctgactgct 1260 agaaaagaay ctaagctttt cctaaagaat gtgaaaggct gggaggtttc tgatcctgaa 1320 gaacagctta ggaagttagt agattttatg caaacattga gtcaaaaaat aaaaagttat 1380 gacatttgga ttaacacatg cctcatagat tgggatctgc aaaattttct aataaacatt 1990 gtcaacttct gcaaaaattc acccacttat aaaactcact ttattaaatc tcagttgtgc 1500 agccttctag aacctcatgt ctacaaagtg acaaactttc ctcaggcaca atccatcata 1560 cagtggatca atcagtcaga gtcagaggaa gaactagtca aaattacctc attttctgaa 1620 ttcattaaca ccttaaagaa aacccacaaa tacctaatgg aagagagttt caaaactgag 1680 cccccagaaa gagtggaaga agcaaagaga atggctacat atgaagtcac cacagctctc 1740 agctccttct tgaagtacct cagagaaaca cagcagccag acatgcagct gttgctactc 1800 tccattgcta ctggtgtagg ctatcagttg gtaaacagta tttttcagca tcttctgggg 1860 tgtgatgagt taaacttcct cttggatcaa atggaaaata acgaacataa ataccaagaa 1920 ctgaaaaata tttgcaatta cagagcccag gcattcttgg tgctcacagc cctaagagcc 1980 acagttgaaa tcacagatgt ttctacagaa gagaaaggac aacgtttgac attaatacaa 2090 caacatatgg ggtcactgtt gtctgaagaa gttgcacatg tcctcacaaa acatggagaa 2100 catcatgact gggaaaggct ggagaatgat ttgagattac tcattgaggg ggactataaa 2160 gccaccaccc attccttaca aatggatgaa gtaaaaaaac aattgcaaag tttntgccat 2220 gaaaagaaac agacttataa acaacaaggt aatgaaaaca gaacaaaaga aatgatagaa 2280 aatggacatt tcctggactt actccaacgt ttaggcctag acaattacta tccaaaaaaa 2340 aaaaaaaaaa aaaaaaaaaa aaa 2363 <210> 50 <211> 778 <212> PRT
<213> Mus musculus <900> 50 Gln Arg Gln Aia Gly Gln Ala Leu Xaa Ala Leu Arg Ala Leu Gln Ser Glu Gly Lys Hi_-- Xaa Gln Glu Glu Ala Val Arg Arg Lys Gly Ala Xaa Leu Arg Gln Aia Met Glu Ile Pro Glu Glu Cys Trp Pro Thr Ala Glu Val Ser Leu Lye Asp Ile Thr Glu Ile Met Glu Arg His Leu Ser His Met Glu Arg T!:r Leu Ser His Ser Gln Lys Leu Ser Asp Gly Asp Leu 65 ?0 75 80 Val Arg Trp Ala Ser Gly Gly Leu Val Leu Gln Gly Ile Tyr Lys Thr Asn His Pro Arg Ser Leu Ile Gln Lys Arg Glu Glu Leu Leu Ser 'Jal 10:. 105 110 Pro Lys Gln P::e Ser Leu Ala Gly Pro Val His Gly Thr Glu Ile Lys Page 22 of 56 Thr Lys Glu Phe Ser Ser Phe Gln Glu Gln Ala Met Phe Thr Gln Thr Ile Glu Arg Val Gly Phe Ser Ser Thr Pro Leu Val Lys Gly Glu Gly Trp Gly Leu Ser Leu Glu Ala Gly Met Gly His Asn Lys Gln Thr Glu Ser Glu Asp Asn Tyr Gln Ser His Ser Lys Gln Thr Tyr Phe Cys Set Ala Arg Phe Ser Tyr Ile Pro Leu Ala Thr Cys His Phe His Ile Asn Asp Leu Glu Leu Ser Gln Ala Ala Leu Gln Glu Leu Lys Ser Ile Glu Glu Ile Leu Glu Gln Thr Thr His His Arg Asp G1y Leu Pro Leu Leu Arg His Arg Aia Lys Asn Phe Phe His Arg Phe Gly Ser His Ala Asn Gln Gly Pro Val His Leu Gly Gly Ile Tyr Cys Trp Lys Ala Ile Ser Glu Gly Phe Lys Ser Glu His Leu Ala Asp Val Lys Gln Gln Ala Glu Glu Ser Leu Asn Ile Tyr Ile Met Gly Ser Tyr Ser Gly Phe Gly Val Lys Val Gly Ala Ser Val Asn Ile Thr Asn Ser Lys Ser Lys Thr Ala Phe Tyr Ser Lys Thr His Leu Asn Ser Gln Thr Lys Val Gln Leu Ser Val Ala Lys Ile Gly Gly Pro Ala Glu Ala Asp Gly Ile Ala Gln Trp Thr Ala Gly Leu Val Ala Ser Asn Gln Thr Trp Ser Val Ile Asp Arg Lys Leu Gln Leu Val Pro Ile Trp Asp Ile Ile Leu Ser Ser His Arg Thr Glu Phe Lys Asn Ala Leu Gln Leu Ala Asn Cys Leu Lys Asp His Tyr Thr Ala Leu Thr Glu Leu Ala Ala Gln Ile Gln Glu Gly Glu Glu Phe Leu Thr Ala Arg Lys Glu Ala Lys Leu Phe Leu Lys Asn Val Lys Gly Trp Glu Val Ser Asp Pro Glu Glu Gln Leu Arg Lys Leu Val Asp Phe Met Gln Thr Leu Ser Gln Lys Ile Lys Ser Tyr Asp Ile Trp Ile Page 23 of 56 WO 00/11 lb8 PCTNS99/19052 Asn Thr Cys Leu Ile Asp Trp Asp Le~~ ~ln Asn Phe Leu Ile Asn .le Val Asn Phe Cys Lys Asn Ser Pro Th= :yr Lys Thr His Phe Ile :.ys Ser Gln Leu Cys Ser Leu Leu Glu Prc His Val Tyr Lys Val Thr Asn 500 50~ 510 Phe Pro Gln Ala Gln Ser Ile Ile G1.~. Trp Ile Asn Gln Ser Glu Ser Glu Glu Glu Leu Val Lys Ile Thr Ser Phe Ser Glu Phe Ile Asn Thr Leu Lys Lys Thr His Lys Tyr Leu Mss ~lu Glu Ser Phe Lys Thr Glu Pro Pro Glu Arg Val Glu Glu Ala Lys Arg Met Ala Thr Tyr Glu Val Thr Thr Ala Leu Ser Ser Phe Leu Lys Tyr Leu Arg Glu Thr Gln Gln Pro Asp Met Gln Leu Leu Leu Leu Ser Ile Ala Thr Gly Val Gly Tyr Gln Leu Val Asn Ser Ile Phe Gln His Leu Leu Gly Cys Asp Glu Leu Asn Phe Leu Leu Asp Gln Met Glu Asn Asn Glu His Lys Tyr Gln Glu Leu Lys Asn Ile Cys Asn Tyr Arg Ala Gln Ala Phe Leu Val Leu Thr Ala Leu Arg Ala Thr Val Glu Ile Th= Asp Val Ser Thr Glu Glu Lys Gly Gln Arg Leu Thr Leu Ile Gln Gln His Met Gly Ser Leu Leu Ser Glu Glu Val Ala His Val Leu Thr Lys His Gly Glu His His Asp ':'rp Glu Arg Leu Glu Asn Asp Leu Arg Leu Leu Ile Glu Gly Asp Tyr ~ys Ala Thr Thr His Ser Leu Gln Met Asp Glu Val Lys Lys Gln Leu Gln Ser Xaa Cys His Glu Lys Lys Gln Th= Tyr Lys Gln Gln Gly Asn Glu Asn Arg Thr Lys Glu Met Ile Glu Asn Gly His Phe Leu Asp Leu ~eu Gln Arg Leu Gly Leu Asp Asn Tyr Tyr Pro <210> 51 <211> 1712 Page 24 of 56 WO 00/1116$ PCT/US99/19052 <z12> DNA
<213> Mus musculus <900> 51 tcttcgaaag ccgggctgag gggaatcctg gacaggggaa tcctggacgt ggagatcgtg 60 agtcatggct gcttcccgag acgctgatga gatccacaag gacgttcaga actactatgg 120 gaatgtactg aagacatctg cagacctcca gactaatgct tgtgtcacgc gagccaagcc 180 ggtccccagc tacatccggg aaagtctgca gaatgtacac gaagacgtta gttcgaggta 290 ttatggctgt ggtctgactg ttcctgagcg gctggaaaac tgccgaattt tggatctggg 300 tagtgggagt ggcagggatt gctatgtgct tagccagctg gttggtgaga agggacatgt 360 caccggaata gacatgactg aggtccaggt cgaagtggct aaaacctatc ttgaacacca 920 catggaaaaa tttggtttcc aggcacccaa tgtgactttt ctccacggcc gcatcgagaa 980 gttggcagag gctgggatcc agagtgagag ctatggtatt gtcatatcca actgtgttat 590 caaccttgtt cctgataaac aacaagtcct ccaggaggtc tatcgagtgc tgaagcacgg 600 cggggagctc tatttcagtg acgtctatgc cagccttgaa gtgccagaag acatcaagtc 660 gcacaaagtt ttatgggggg aatgcctggg aggcgctctg tactggaagg atcttgccat 720 cattgcccaa aagattgggt tctgccctcc acgtttggtc actgccgata tcattactgt 780 tgaaaacaag gagctcgaag gggttcttgg tgactgtcgc tttgtgtctg ccacatttcg 890 cctcttcaaa ctccctaaga cagagccagc cgaaagatgc cgagttgttt acaatggagg 900 aatcaaggga catgaaaagg aactaatttt cgatgcaaat ttcacattca aggaaggcga 960 agctgttgca gtggatgagg agacggcagc tgtcctgaag aactcacgtt ttgctccgga 1020 ttttctcttc acacctgttg acgcctcgct gccagctccc caggggccgt tctgagttag 1080 agacaaaggt tctaatcaga gatccattca agcttgcaga ggactctgac aagatgaagc 1190 ccagacatgc acctgaaggc acgggaggct gctgtggcaa gaggaaaaac tgctagatct 1200 acagccagcg cggagcccac cgggctcaag agggtggcta aaggacagtc acagaggctt 1260 cttagcctgc tcttcgccag tgcacagatt atgtgaaggt ggcaaagcca ccacaagcta 1320 gaccactgct aagaataaga gtgactttta gaggatgtta attgaaggtt cacagcaaat 1380 cgcctgcttt tctatttctc tatctcagag ttctggtgcc acctagtggt cagaagtaga 1440 acttggaagc ccaaggttta ctcaaagggc caaaggcatc atcaacgttg tgagaattat 1500 cttccttctg gcctaccaca ggacacctct gggttcttct ctgtggttac caggaagcac 1560 agtacttact aaatttatgc taaccatgac aaaagattgt caactcaaat ttgctaggag 1620 tattctttag gttgctgtct gcaatttttt tctctgtaac tgaatgaaaa agaaaacaaa 1680 taaaaaataa atttgacttc gaaaaaaaaa as 1712 <210> 52 <211> 336 <212> PRT
<213> Mus musculus <900> 52 Met Ala Ala Ser Arg Asp Ala Asp Glu Ile His Lys Asp Val Gln Asn Tyr Tyr Gly Asn Va1 Leu Lys Thr Ser Ala Asp Leu Gln Thr Asn Ala Cys Val Thr Arg Ala Lys Pro Val Pro Ser Tyr Ile Arg Glu Ser Leu Gln Asn Val His Glu Asp Val Ser Ser Arg Tyr Tyr Gly Cys Gly Leu Thr Val Pro Glu Arg Leu Glu Asn Cys Arg Ile Leu Asp Leu Gly Ser Gly Ser Gly Arg Asp Cys Tyr Val Leu Ser Gln Leu Val Gly Glu Lys Gly His Val Thr Gly Ile Asp Met Thr Glu Val Gln Val Glu Val Ala Lys Thr Tyr Leu Glu His His Met Glu Lys Phe Gly Phe Gln Ala Pro 115 120 :25 Page 25 of 56 Asn Val Thr Phe Les is Gly Arg Ile Glu Lys Leu Ala Glu 3 Gly Ile Gln Ser Glu Ser Tyr Gly Ile Val Ile Ser Asn Cys Val Ile Asn Leu Val Pro Asp Lys Gln Gln Val Leu Gln Glu Val Tyr Arg Val Leu Lys His Gly Gly Glu Leu Tyr Phe Ser Asp Val Tyr Ala Ser Leu Glu Val Pro Glu Asp Ile Lys Ser His Lys Val Leu Trp Gly Glu Cys Leu Gly Gly Ala Leu Tyr Trp Lys Asp Leu Ala Ile Ile Ala Gln Lys Ile Gly Phe Cys Pro Pro Arg Leu Val Thr Ala Asp Ile Ile Thr Val Glu Asn Lys Glu Leu Glu Gly Val Leu Gly Asp Cys Arg Phe Val Ser Ala Thr Phe Arg Leu Phe Lys Leu Pro Lys Thr Glu Pro Ala Glu Arg Cys Arg Val Val Tyr Asn Gly Gly Ile Lys Gly His Glu Lys Glu Leu Ile Phe Asp Ala Asn Phe Thr Phe Lys Glu Gly Glu Ala Val Ala Val Asp Glu Glu Thr Ala Ala Val Leu Lys Asn Ser Arg Phe Ala Pro Asp Phe Leu Phe Thr Pro Val Asp Ala Ser Leu Pro Ala Pro Gln Gly Pro Phe <210> 53 <211> 3599 <212> DNA
<213> Mus musculus <900> 53 agagacagcg tgatcccggc ctcccacggg gcaqctttta ctgtctaggg aagaaatccc 60 caaagtccat ggagtctgaa gactctgtca agcctcgcta ggaaacctag gagttttaga 120 gggcacttgg caccggaagc tagccgggta ggcggagcct cacctggatt gagttcacag 180 ctgcctagac aggctcagac taggtgctgg gcacctggga ggaggaggag acattagcag 290 caaaggctgt taacagaagt gcctgcctag gcttggaggc aagacgctgc tgttcacagt 300 gcgagacgga ggtaggagta taatggctgt ccaggtgctg cggcagatgg tctacttcct 360 actgagtctg ttt=ctctgg tgcaaggtgc acacagtggc agcccccgag aagacttccg 420 cttctgtggc cagcggaacc agacccaaca gagcaccctc cactatgatc aatcttcaga 480 gcctcacatc tttgtgtgga acacagagga gaccctcaca attcgtgccc ccttcctggc 590 agccccagat attccccgct tcttcccaga gcctagaggg ctctatcact tctgcctcta 600 ctggagtcgc cacactggga gactccactt gcgctatggc aagcatgact acctgcttag 660 tagccaagcc tccagactcc tctgcttcca gaaacaggag cagagcctga agcagggagc 720 cccgctgatc gccacctctg tcagctcctg gcagattccc cagaacacca gcctgcctgg 780 ggctccgagc tt~atcttct ccttccacaa tgccccacac aaggtctccc acaatgcatc 890 tgtggacatg tgtgatctca agaagqaatt gcagcagctt agcaggtacc tgcagcaccc 900 Page 26 of 56 tcaaaaggct gccaagcc ccaccgcagc gttcatcagc cagcagttac a5 ;ctgga 960 gtcaaagctg acctctgtga gcttcctggg agacacatta tcctttgagg aggaccgggt 1020 caatgctaca gtgtggaagc tgccacccac agccggtcta gaggatctgc atatccactc 1080 ccagaaggag gaggagcaga gtgaggtcca ggcatactcg ctgttgcttc cccgggccgt 1140 attccagcag accagaggcc gtcgccggga tgacgccaag aggctcctgg tagtagactt 1200 cagcagccaa gctttgttcc aggacaagaa ttctagccaa gtcctgggtg agaaggtctt 1260 gggtattgtc gtgcagaaca ccaaagtcac caacctctca gatccggtgg tactcacctt 1320 ccagcaccag cctcagccaa aaaatgtgac tctgcagtgc gtgttctggg ttgaagaccc 1380 ggcatcaagc agcacaggga gctggagcag tgcaggctgc gagacagtga gcagagacac 1990 acagacatcc tgcctgtgca accacctgac ctactttgca gtgctgatgg tgtcatccac 1500 agaggtagaa gccactcaca aacactacct cacgctcctg tcctacgtgg gctgtgtcat 1560 ctctgctctg gcttgtgtct tcactatcgc tgcctacctc tgctccagga ggaagtcacg 1620 tgactacacc atcaaagtcc acatgaacct gctgtccgct gtcttcctgc tggacgtgag 1680 cttcctgctc agcgagcctg tggcactgac gggctccgaa gcagcctgtc gcaccagtgc 1790 catgttcctg cacttctccc tgcttgcctg cctctcctgg atgggcctcg agggctacaa 1800 tctctaccga ctggtggtgg aggtcttcgg tacctatgtg cccggctatc tgctcaagct 1860 gagcatcgtg ggctggggtt ttcctgtctt cctggtcact ctggtggcgt tggtggatgt 1920 gaataactac ggccccatta tcctagctgt gcgccggact ccggaacgtg tcacctaccc 1980 ctctatgtgc tggatccggg actccctggt gagctatgtc accaacctgg gcctcttcag 2090 tctggtgttc ctgttcaacc tggctatgct ggccaccatg gtggtgcaga tcctgcggct 2100 tcgcccgcac agccagaact ggccccacgt gctgaccctg ctgggcctca gcctggtcct 2160 tggcctcccc tgggccttgg tcttcttttc ctttgcttcc ggcaccttcc agcttgtcat 2220 cctctacctc ttcagcatca taacttccta ccaaggcttc ctcatcttcc tgtggtactg 2280 gtccatgcgg ttccaggccc aaggcggccc ctcccctctg aagaacaact cagacagcgc 2390 caaactcccc atcagctccg gcagcacctc ctccagccgc atctaagcca ccgccacacc 2900 tcccctccgg gaggacacat gcatggcgtc cgctcacgat gtctgtggcc cagtgctgtg 2460 cccacccagc ctttgttggt tagtggcata ctagagaagg ccctggtcct tgaaggcgta 2520 gggctgttgc tctggtaggt agatacctag cttgccttgg ggacgactct ggtcctcaaa 2580 gggctcagaa gcacactgcc attctgtttg tggggccgtt tcagtctgga gctaaggcct 2690 tgtctttctg gccacctctg ggtccagctg ttgctgctgg gtgttgagac ctgcagaccc 2700 aagctggggt tagatctcga aggaggctga cacatccggc ctgagacaca gctaactgtc 2760 ttgacttgct gctctgtctc tgtggtcacc atgcagatcc cgagggtggc actgggggta 2820 aatgttctgg gagaaggttt ggaggcagag caccttagga gctgagcatc tcccccagcc 2880 tttctgcaaa ccctcctctt cattccccat ccccaacccc tcctctgtgt tcccctaacc 2940 ctccacctga agcctggggt cctagaccaa tgctgtgatt tggggtggta gttcccagca 3000 gtttcctggt gccagctatc aacttctgtc tgttgtgtgg gctttggcct ctgactcagg 3060 gcaggtttct gtctgagccc tctctccaag ctgcctcacc tttgctcgca cctcagaggg 3120 acctccatct ctcctgaagc ctcctccctc tggcaagtac tgggatacag ccaccctttc 3180 aacccagcac tctgaagacc aagacagccc cctctggtga cactggccaa gcttgatctt 3240 tttcctaaga agtggtcttc agatccccgc aggtcgctca gaagacactg ggctgcctag 3300 tgtgaattct gtcctactaa cgtacagtga gcagctcctc acccccaccc ccgcaaaagc 3360 tctcaccaag tcctggagtg tcaggcaggg ggctggaaat ccaggaggac ttcctgcaaa 3920 aggcagcatt tcatcttgac ctcagccttc aggttgggga gaatgttctt tttaaatacc 3980 agttcatttg tcttttgata ttaaagctct ttatagagag tctggaaact gtaggcgatt 3590 gtcgagaaga gaaataaaaa tgagctgtta tctaatgcca tggcaaagca gcacaaaaa 3599 <210> 54 <211> 687 <212> PRT
<213> Mus musculus <400> 59 Met Ala Val Gln Val Leu Arg Gln Met 'lal Tyr Phe _eu Leu Ser Leu i 5 10 15 Phe Ser Leu Val Gln Gly Ala His Ser Gly Ser Prc Arg Glu Asp Phe Arg Phe Cys Gly Gln Arg Asn Gln Thr Gln Gln Ser _~:r Leu His Tyr Asp Gln Ser Ser Glu Pro His Ile Phe 'Ja1 Trp Asn -hr Glu Glu Thr Page 27 of 56 Leu Thr Ile Arg Ala ro Phe Leu Ala Ala Pro Asp Ile Pro x. Phe Phe Pro Glu Pro Arg Gly Leu Tyr His Phe Cys Leu Tyr Trp Ser Arg His Thr Gly Arg Leu His Leu Arg Tyr Gly Lys His Asp Tyr Leu Leu Ser Ser Gln Ala Ser Arg Leu Leu Cys Phe Gln Lys Gln Glu Gln Ser Leu Lys Gln Gly Ala Pro Leu Ile Ala Thr Ser Val Ser Ser Trp Gln Ile Pro Gln Asn Thr Ser Leu Pro Gly Ala Pro Ser Phe Ile Phe Ser Phe His Asn Ala Pro His Lys Val Ser His Asn Ala Ser Val Asp Met Cys Asp Leu Lys Lys Glu Leu Gln Gln Leu Ser Arg Tyr Leu Gln His Pro Gln Lys Ala Ala Lys Arg Pro Thr Ala Ala Phe Ile Ser Gln Gln Leu Gln Ser Leu Glu Ser Lys Leu Thr Ser Val Ser Phe Leu Gly Asp Thr Leu Ser Phe Glu Glu Asp Arg Val Asn Ala Thr Val Trp Lys. Leu Pro Pro Thr Ala Gly Leu Glu Asp Leu His Ile His Ser Gln Lys Glu Glu Glu Gln Ser Glu Val Gln Ala Tyr Ser Leu Leu Leu Pro Arg Ala Val Phe Gln Gln Thr Arg Gly Arg Arg Arg Asp Asp Ala Lys Arg Leu Leu Val Val Asp Phe Ser Ser Gln Ala Leu Phe Gln Asp Lys Asn Ser Ser Gln Val Leu Gly Glu Lys Val Leu Gly Ile Val Val Gln Asn Thr Lys Val Thr Asn Leu Ser Asp Pro Val Val Leu Thr Phe Gln His Gln Pro Gln Pro Lys Asn Val Thr Leu Gln Cys Val Phe Trp Val Glu Asp Pro Ala Ser Ser Ser Thr Gly Ser Trp Ser Ser Ala Gly Cys Glu Thr Val Ser Arg Asp Thr Gln Thr Ser Cys Lau Cys Asn His Leu Thr Tyr Phe Ala Val Leu Met Val Ser Ser Thr Glu Val Glu Ala Thr His Lys His Tyr Leu Thr Leu Leu Ser Tyr Val Gly Cys Val .le Ser Ala Leu Page 28 of 56 9C. 910 4 Ala Cys Val P'.~.e Thr Ile Ala Ala Tyr Leu Cys Ser Arg Arg Lys Ser Arg Asp Tyr Ti:= Ile Lys Val His Met Asn Leu Leu Ser Ala Val Phe Leu Leu Asp Vai Ser Phe Leu Leu Ser Glu Pro Val Ala Leu Thr Gly Ser Glu Ala Ala Cys Arg Thr Ser Ala Met Phe Leu His Phe Ser Leu Leu Ala Cys L2~ Ser Trp Met Gly Leu Glu Gly Tyr Asn Leu Tyr Arg Leu Val Val G1~ 'Jal Phe Gly Thr Tyr Val Pro Gly Tyr Leu Leu Lys Leu Ser Ile Vai Gly Trp Gly Phe Pro Val Phe Leu Val Thr Leu Val Ala Leu Val Asp Val Asn Asn Tyr Gly Pro Ile Ile Leu Ala Val Arg Arg Thr Pro Glu Arg Val Thr Tyr Pro Ser Met Cys Trp Ile Arg Asp Ser Leu Val Ser Tyr Val Thr Asn Leu Gly Leu Phe Ser Leu Val Phe Leu Phe Asn Leu Ala Met Leu Ala Thr Met Val Val Gln Ile Leu Arg Leu Arg Pro His Ser Gln Asn Trp Pro His Val Leu Thr Leu Leu Gly Leu Ser Leu Vas Leu Gly Leu Pro Trp Ala Leu Val Phe Phe Ser Phe Ala Ser Gly T::r Phe Gln Leu Val Ile Leu Tyr Leu Phe Ser Ile Ile Thr Ser Tyr Glr. Gly Phe Leu Ile Phe Leu Trp Tyr Trp Ser Met Arg Phe Gln Ala Glr. Gly Gly Pro Ser Pro Leu Lys Asn Asn Ser Asp Ser Ala Lys Leu Pry Ile Ser Ser Gly Ser Thr Ser Ser Ser Arg Ile <210> 55 <211> 3590 <212> DNA
<213> Mus musculus <900> 55 ctcagggaag gcccatcttc cgggtggagg gggaagcggc gtgactggag tggaaatttt 60 tcccaacaca ac:=ctcgga ggcaacatat tggaagggac tcggggaggc cggagtccaa 120 atggaagtqg ct~aaagaaa cttctcgccc tgctgattct gagccccgcg tcgtgccgcg 180 cgccctcaat ta~~~catcg acctgtggtc ttgacagaac attcttcaca atccaaaaga 290 Page 29 of 56 aaaagcagac tggttcgtgtttgacgg ctatgatagc tgcagtgagg ac iiagtag'~3'00 cagctccagc tctgaggaga gtgaagaaga agttgctcc= ttaccttcca atctcccaat 360 catcaagaat aatggacaag tctacacata cccagatgg= aaatctggca tggctacctg 920 tgagatgtgt gggatggtcg gtgtgcgaga tgctttttac tctaaaacga aacgtttctg 480 cagtgtttcc tgttcaagaa gttactcgtc aaactctaag aaggcaagca ttctggccag 540 acttcagggt aagcctccaa caaagaaagc aaaagtcct= caaaaacaac ctttagttgc 600 taaattagct gcctatgccc agtatcaagc taccttgcaa aatcaagcaa agacaaaagc 660 aggcaattct gcaatctctg tggaaggctt cagctggggt aactacatca acagcaacag 720 cttcatagca gctccagtgg cctgttttaa gcatgcacct atggggacct gctggggtga 780 tatctcggaa aatgtaagga tagaagttcc caatacagac tgcagtctac ctaccaaagt 840 cttctggatt gctggaatta taaaattagc aggttataa~ gcccttttga gatatgaagg 900 atttgaaaat gattcttctc tggacttctg gtgcaatata tgtgggtctg atattcatcc 960 agttggttgg tgtgcagcta gtggaaaacc tctcgttcct cctagaactg ttcaacataa 1020 atatacaaac tggaaagctt ttctagtaaa aagacttact ggtgccaaaa cacttcctcc 1080 tgatttctca caaaaggttt ctgagagtat gcaatatcct ttcaaacctt gcatgagagt 1190 agaagtagtt gacaagaggc atttatgtcg aacaagagta gcagtggtgg aaagtgtaat 1200 tggaggacga ctacggctgg tgtatgaaga gagtgaagat ggaacagacg acttctggtg 1260 ccacatgcac agccccttaa tccaccatat tggatggtcG agaagcatag gccatcgatt 1320 caagagatca gatattacga agaaacagga cggacatttc gatacacctc cacacttatt 1380 tgctaaggta aaagaagtag accagagtgg agaatggttc aaagaaggaa tgaaattgga 1440 agctatagac ccattaaatc tttctacaat atgtgttgcc accattagaa aggtgctggc 1500 tgatggattc ctgatgattg ggattgatgg ctcagaagca gcagatggat ctgactggtt 1560 ctgttatcat gcaacctctc cttccatttt ccctgtgggt ttctgtgaaa ttaacatgat 1620 agaactgact ccacccagag gttacacaaa acttcctttt aaatggtttg actacctcag 1680 ggaaaccggc tccattgcag caccagtaaa actatttaat aaggatgttc caaaccacgg 1790 attccggtta ggaatgaaat tagaagctgt agatctcatg gagccacggt taatatgtgt 1800 agccacagtt actcgaatta ttcaccatct cttgaggata cattttgatg gttgggaaga 1860 agagtatgac cagtgqgtag actgtgagtc ccctgacctc tatcctgtag ggtggtgtca 1920 gttaactgga tatcaactac agcctccagc atcacagtca tcaagagaaa gccaatcagc 1980 ttcttcaaaa cagaagaaaa aggctaagtt tcagcaatac aaaggacata agaaaatgac 2040 cacgtcacag ctgaaggagg agctgctgga cggggaggac tatagcttcc tccatggagc 2100 atctgaccag gaaagcaatg gctctgccac cgtctacatc aaacaagagc catgaggcga 2160 ctcggaaacc atgggcaggc ggggctgttt acaggactga tttggaatca gccagctgta 2220 tagcgggcta ttctactggg acattttgct aaacacagaa aaaagttcag ttccagattt 2280 ttcaggtggg ggggaaacta ttttggtggg ggggcaattt ttcaatttat aaagacggac 2340 aatttttgtg ttgtatttga agcttttgaa agaattttgt aatattttcc aagtttggat 2400 ttatgtgcat tgttaacaag aactgaaatt ataactttt= tggtaagata aaagtttagg 2460 tagcaggatt gaaggaaatg attaagaagg atatagttgt aaatgcacat gaactgtcat 2520 tacaaatgaa ccttcttggt acctgttggg agattttttg gattttagag ttaggccagt 2580 cacatctcca gcttcctttg ctgcgaaaat atgcaactga acccctcaca gagggctcat 2690 caccatgtag atcacggaag gggtcattaa ttgtgctcgc tgacgtttta ttgcagccca 2700 tttaactgtt tgtacagaaa ctttttcatt ctgctaaaat ttatttggag ttgtatatga 2760 aactaggaga actctggata cttttatatg ctctccttca cgtaaagatg attaaaattg 2820 tctaacacta aagtgttaaa ctggaatggt tgacaaagta cacaagatct cagtctacat 2880 aaagggttgg gggaaattag tattttccta agtttattct gttttccttg ctagaaaaat 2940 cccataaaag ggatattctt gtcagacctc tgccatttct tccatctgtg agagagcaac 3000 gtgatgtaac cacacctgag agcaggtact gtcctgtgtt acaaaagaat cacatggaaa 3060 attgttaacc aaaataagtg tagatttcag aatcattcca gctctactct taactgtcct 3120 gaatttgtta gaaactgatt tgaaagaaat gtttcttaaa attccataca cacacacaca 3180 catattatga attgtcttta attgtagtca gaatttatta taacaatatt tgatttggac 3290 cattttaaac attccctatt ttaaaattca tacggcttcc ttaagaagta gaatgaaagg 3300 gtaaattggt gacatgtttg ctctctgcat tttctaacct tctaccgaat tgtgactgac 3360 tcagagagct ctagcattta ccagtgaggt tcagaaacta aactctcagg aattcattgc 3420 atctgttgta gaagtgcttc tgggtctgag cctggcctcc tcagagtggt aatactgccc 3480 acttcctctg gaaaataggc agggctaatg agaaactaat caaatgatta acctctgcgg 3540 cccttcagcc tttggaatgc taaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3590 <210> 56 <211> 631 <212> PRT
<213> Mus musculus <400> 56 Met Phe Asp Gly Tyr Asp Ser Cys Ser Glu Asp Thr Ser Ser Ser Ser Page 30 of 56 1 ; to Ser Ser Glu Glu Ser Glu Glu Glu Val Ala Pro Leu Pro Ser Asn Leu Pro Ile Ile Lys Asn Asn Gly Gln Val Tyr Thr Tyr Pro Asp Gly Lys Ser Gly Met Ala Thr Cys Glu Met Cys Gly Met Val Gly Val Arg Asp Ala Phe Tyr Ser Lys Thr Lys Arg Phe Cys Ser Val Ser Cys Ser Arg Ser Tyr Ser Ser Asn Ser Lys Lys Ala Ser Ile Leu Ala Arg Leu Gln Gly Lys Pro Pro Thr Lys Lys Ala Lys Val Leu Gln Lys Gln Pro Leu Val Ala Lys Leu Ala Ala Tyr Ala Gln Tyr Gln Ala Thr Leu Gln Asn Gln Ala Lys Thr Lys Ala Gly Asn Ser Ala Ile Ser Val Glu Gly Phe Ser Trp Gly Asn Tyr Ile Asn Ser Asn Ser Phe Ile Ala Ala Pro Val Ala Cys Phe Lys His Ala Pro Met Gly Thr Cys Trp Gly Asp Ile Ser Glu Asn Val Arg Ile Glu Val Pro Asn Thr Asp Cys Ser Leu Pro Thr Lys Val Phe Trp Ile Ala Gly Ile Ile Lys Leu Ala Gly Tyr Asn Ala Leu Leu Arg Tyr Glu Gly Phe Glu Asn Asp Ser Ser Leu Asp Phe Trp Cys Asn Ile Cys Gly Ser Asp Ile His Pro Val Gly Trp Cys Ala Ala Ser Gly Lys Pro Leu Val Pro Pro Arg Thr Val Gln His Lys Tyr Thr Asn Trp Lys Ala Phe Leu Val Lys Arg Leu Thr Gly Ala Lys Thr Leu Pro Pro Asp Phe Ser Gln Lys Val Ser Glu Ser Met Gln Tyr Pro. Phe Lys Pro Cys Met Arg Val Glu Val Val Asp Lys Arg !:is Leu Cys Arg Thr Arg Val Ala Val Val Glu Ser val Ile Gly Gly Arg Leu Arg Leu Val Tyr Glu Glu Ser Glu Asp Gly Thr Asp Asp Phe ~rp Cys His Met His Ser Pro Leu Ile His His Ile Gly Trp Ser Arg Ser Ile Gly His Page 31 oC56 Arg Phe Lys Arg Ser Asp Ile Thr Lys Lys Gln Asp Gly His Phe Asp Thr Pro Pro His Leu Phe Ala Lys Val Lys Glu Val Asp Gln Ser Gly Glu Trp Phe Lys Glu Gly Met Lys Leu Glu Ala Ile Asp Pro Leu Asn Leu Ser Thr Ile Cys Val Ala Thr Ile Arg Lys Val Leu Ala Asp. Gly Phe Leu Met Ile Gly Ile Asp Gly Ser Glu Ala Ala Asp Gly Ser Asp Trp Phe Cys Tyr His Ala Thr Ser Pro Ser Ile Phe Pro Val Gly Phe Cys Glu Ile Asn Met Ile Glu Leu Thr Pro Pro Arg Gly Tyr Thr Lys Leu Pro Phe Lys Trp Phe Asp Tyr Leu Arg Glu Thr Gly Ser Ile Ala Ala Pro Val Lys Leu Phe Asn Lys Asp Val Pro Asn His Gly Phe Arg Val Gly Met Lys Leu Glu Ala Val Asp Leu Met Glu Pro Arg Leu Ile Cys Val Ala Thr Val Thr Arg Ile Ile His Arg Leu Leu Arg Ile His Phe Asp Gly Trp Glu Glu Glu Tyr Asp Gln Trp Val Asp Cys Glu Ser Pro Asp Leu Asn Pro Val Gly Trp Cys Gln Leu Thr Gly Tyr Gln Leu Gln Pro Pro Ala Ser Gln Ser Ser Arg Glu Ser Gln Ser Ala Ser Ser Lys Gln Lys Lys Lys Ala Lys Ser Gln Gln Tyr Lys Gly His Lys Lys Met Thr Thr Ser Gln Leu Lys Glu Glu Leu Leu Asp Gly Glu Asp Tyr Ser Phe Leu His Gly Ala Ser Asp Gln Glu Ser Asn Gly Ser Ala Thr Val Tyr Ile Lys Gln Glu Pro <210> 57 <211> 1650 <212> DNA
<213> Mus musculus <900> 57 ggggcttcac tctccagcct tggagagcag tcagggaagg cctccaggag gaagcagcta 60 cc~gtggcag ggcagagcag tgcctgtacc agtctcccgg aacttcagaa ctgcggtggc 120 Page 32 of 56 catggagtct gtggagc~ cggtcaaaga cggcatcctc taccagcagc ac taagtt 180 tggcaagaaa tgctggcgca aagtgtgggc tctgctgtat gcgggaggcc catcaggggt 290 agctcggcta gaaagctggg acgtgcgtga tggtggcctg ggaccagcag gcgacaggtc 300 cacagggccc agccgtcgag gggaacgccg ggtcatacgc ttggctgact gtgtatctgt 360 cctgcctgcg gatggcgaga gctgtcccag ggacactggt gccttcctga ttaccaccac 420 tgagcgaagc cacctgttgg ctgcacagca ccgccagtcc tgggtggacc ccatctgtca 480 gctggccttc ccgggtaccg gagaatgttc gtcaggatca ggacaggctg agaatccaaa 590 aaggggcttt gttcccatgg aagaaaactc tatctactcc tcctggcagg aagtgaccga 600 atttccggtg atcgtgcaga agacagaagc cacctcccgc tgccagctga aaggacccta 660 cctcctggtg ctgggccaag atgacatcca actgagggag acatccaagc cccaggcctg 720 ttttagctgg ccctaccgtt tcctgcgcaa gtacggctct gacaagggtg tgttctcgtt 780 tgaggctggc cgccgctgtg actcaggtga gggccttttt gccttcagta gcccgcgtgc 840 cccagacata tgtggggttg tggctgccgc cattgcccgc cagcgggagc gtcttccaga 900 gctggccatg tccccaccct gccccctgcc tcgggccctc tccctgccct ccctagagcc 960 ccctggagag cttcgggagg tggccccagg atttgagctg cccactccca gaaagctgcc 1020 tctaactgat cccgggcctc aaagcctacc attgctgctc agccccaaca caagaaggac 1080 cggcatccgg tctctatgcg tccgtgtgca agcagaccag caagcacaca ggcacggcgg 1190 agcatttcta tgagaacgtg tgcatgctgg aggccagcct tgggctgacc aatgggggtc 1200 ctgaagccca agagggcccc cttggtggcc gcagccccct tgggcagcct tatctaccat 1260 aacactgagg atctgagttg gccgggctcg gcccaggaca gcaatctgga agcccagtac 1320 cggaggctgc tggaactgga gctggatgag gccggaagcg ccggccgctc tggagcgcag 1380 gcaggcatca aggccaagct ggtgaccctg ctgacccgtg aacggaagaa gggccccgcc 1440 ccctgtgacc ggccctgaag gcctgagcgg ccagccactg caggacagag gtgatcaccc 1500 aagaccagga acaacttcga acataacccg tctactctga cctgcaggga caagccaggt 1560 ggcccgggga ggagccacac tctgccctac ctcctccctc agactgtaca gattgaacag 1620 taataaagct tgcctatcaa cttcaaaaaa 1650 <210> 58 <211> 359 <212> PRT
<213> Mus musculus <900> 58 Met Glu Ser Val Glu Pro Pro Val Lys Asp Gly Ile Leu Tyr Gln Gln His Val Lys Phe Gly Lys Lys Cys Trp Arg Lys Val Trp Ala Leu Leu Tyr Ala Gly Gly Pro Ser Gly Val Ala Arg Leu Glu Ser Trp Asp Val Arg Asp Gly Gly Leu Gly Pro Ala Gly Asp Arg Ser Thr Gly Pro Ser Arg Arg Gly Glu Arg Arg Val Ile Arg Leu Ala Asp Cys Val Ser Val Leu Pro Ala Asp Gly Glu Ser Cys Pro Arg Asp Thr Gly Ala Phe Leu Ile Thr Thr Thr Glu Arg Ser His Leu Leu Ala Ala Gln His Arg Gln Ser Trp Val Asp Pro Ile Cys Gln Leu Ala Phe Pro Gly Thr Gly Glu Cys Ser Ser Gly Ser Gly Gln Ala Glu Asn Pro Lys Arg Gly Phe Val Pro Met Glu Glu Asn Ser Ile Tyr Ser Ser Trp Gln Glu Val Thr Glu Phe Pro Val Ile Val Gln Lys Thr Glu Ala Thr Ser Arg Cys Gln Leu Page 33 of 56 16~ 170 1 Lys Gly Pro Tyr Leu Leu Val Leu Gly Gln Asp Asp Ile Gln Leu Arg Glu Thr Ser Lys Pro Gln Ala Cys Phe Ser Trp Pro Tyr Arg Phe Leu Arg Lys Tyr Gly Ser Asp Lys Gly Val Phe Ser Phe Glu Ala Gly Arg Arg Cys Asp Ser Gly Glu Gly Leu Phe Ala Phe Ser Ser Pro Arg Ala Pro Asp Ile Cys Gly Val Val Ala Ala Ala Ile Ala Arg Gln Arg Glu Arg Leu Pro Glu Leu Ala Met Ser Pro Pro Cys Pro Leu Pro Arg Ala Leu Ser Leu Pro Ser Leu Glu Pro Pro Gly Glu Leu Arg Glu Val Ala Pro Gly Phe Glu Leu Pro Thr Pro Arg Lys Leu Pro Leu Thr Asp Pro Gly Pro Gln Ser Leu Pro Leu Leu Leu Ser Pro Asn Thr Arg Arg Thr Gly Ile Arg Ser Leu Cys Val Arg Val Gln Ala Asp Gln Gln Ala His Arg His Gly Gly Ala Phe Leu Glu Arg Val His Ala Gly Gly Gln Pro Trp Ala Asp Gln Trp Gly Ser <210> 59 <211> 1750 <212> DNA
<213> Mus musculus <400> 59 ggcctccgac atattgcccg caggagctgc ggcggtgagc ggagagcgcc gggggaagga 60 gatgggagga cgaagaggtc ccaacaggac atcttactat cgaaacccgc tctgtgagcc 120 aggatcatca ggggcctctg gtggaggcca ctcttctagt gcatccgtga gcagcgtccg 180 ttctcgaagc aggaccactt ctgggacggg cctctccagc cctccgctgg ccgcgcagac 290 cgttgtgcct ctacagcact gcaagatccc cgagctgccc gtccaggcca gcattctgtt 300 cgagctacag ctcttcttct gtcagctcat agccctgttc gtgcactaca tcaacatcta 360 taagacagtc tggtggtatc caccctcgca cccaccctcc cacacttccc tgaacttcca 920 cctgatcgac ttcaacttgc tgatggtgac cgccattgtt ctgggccgcc gattcatcgg 980 gtccatcgtg aaggaggctt ctcagagggg gaaaggtctc cctcttccgc tccatcctgc 540 tgttcctcac ccgcttcacg ttctcacggc gacaggctgg agtctgtgcc ggtccctcat 600 ccacctcttc aggacctact ccttcctgaa cctcctgttc ctctgctatc cgtttgggat 660 gtacattccg ttcctgcagc tgaactatga tcttcgcaag acgaacctct tcacccacat 720 ggcttccatg ggaccccgag aggcagtcag tggcctggca aggagtcggg actactttct 780 gacactgcgg gagacttgga agcagcatac acgacagcta tatggcccgg aagccatgcc 890 cacccatgcc tgctgcttgt cacccagcct cattcgcaat gaagttgagt tcctcaaaat 900 ggacttcaat tggcgaatga aggaagtact tgtcagctcc atgctgagtg cctactatgt 960 ggcctttgta cctgtgtggt ttgtgaagaa tacacattac tatgacaagc gctggtcctg 1020 tgagctcttc ctgctggtgt ccatcagtac ctccgtgatc ctcatgcagc acttgttgcc 1080 tgccagctac tgtgacctgc tgcacaaggc cgccgcccac ctggcgtgct ggcagaaggt 1140 Page 34 of 56 ggacccagctctgtgctcatgtgc~~ca gccaccgtggactgaagagtgc 3t~gcc1200 acagggtgtgctggtgaaac acagca~gaa gcagtgggccactacaacgt1260 tgtctacaaa ggcatcccctccgatgtctc ccactt~~gc ttttcagcaaccccctgcgg1320 ttccatttct atcctcaacatccttttgct gctggacggt tctaccagctgtactcctta1380 gccgtcattg atgtcctcagagaagtggca ccagaccatc tcatcctcttcagcaactac1990 tccctggccc tatgccttcttcaagctgct ccgggac~gc gcaaagctactcctactcag1500 ctggtattgg ccagcccccagaggacctgg accaccggtt ccagggtgatcctccaaaca1560 ctcctgagtc acatccgggcttcacccagg ggcttcc~gg ttggggttgggggggaggga1620 ccaagggctc tacaaaaaacaaaaacaaaa ccaccagagctttgtatttt1680 aacaacaaaa aaaaaccaac tgttacgtactgtttctttc tttgat~att gaaaaaagtcttatttttat1790 gatgtggtaa actccaaaaa 1750 <210>

<211>

<212>
PRT

<213>
Mus musculus <400>

Met Gly Arg Gly Fro r.sn Tyr Tyr Asn Pro Gly Arg Arg Thr Ser Arg Leu Cys Pro Ser Ser Gly Ala Gly Gly Ser Ser Glu Gly Ser Gly His Ser Ala Val Ser Val Arg Ser Arg Thr Ser Gly Ser Ser Arg Ser Thr Thr Gly Ser Pro Pro Leu Ala Thr Val Pro Leu Leu Ser Ala Gln Val Gln His Lys Pro Glu Leu Pro Ala Ser Leu Phe Cys Ile Val Gln Ile Glu Leu Leu Phe Cys Gln Leu Leu Phe His Tyr Gln Phe Ile Ala Val Ile Asn Tyr Thr Val Trp 'rrpPro Ser Pro Fro Ile Lys Tyr Pro His Ser His Ser Asn Phe !?is Phe Asn Leu Met Thr Leu Leu Ile Asp Leu '!al Ile Leu Gly Arg Arg Gly Ser Val Lys Thr Val Phe Ile Ile Ala Glu Ala Gln Gly Lys Gly Leu Pro Leu Pro Ala Ser Arg Pro Leu His Val Pro Pro His Val Leu 'rhrGly Trp Leu Cys His Leu Ala Thr Ser Arg Ser Ile Leu Phe Arg 'ChrPhe Leu Leu Leu Leu His Tyr Ser Asn Phe Leu 'ryr Phe Gly Met Tyr Phe Leu Leu Asn Cys Pro Ile Pro Gln Tyr Asp Arg Thr Asn Leu Fhe Met Ala Met Gly Leu Lys Thr His Ser Pro Arg Ala Ser Gly Leu Ala Arg Asp Phe Leu Glu Val Arg Ser Tyr '"hr Glt: Trp Lys Gln Hip Gln Leu Gly ro Leu Thr Thr Arg Tyr Arg Page 35 of 56 24; 250 Glu Ala Met Pro Thr His Ala Cys Cys Leu Ser Pro Ser Leu Ile ~.zg Asn Glu Val Glu Phe Leu Lys Met Asp Phe Asn Trp Arg Met Lys ~lu Val Leu Val Ser Ser Met Leu Ser Ala Tyr Tyr Val Ala Phe Val 2ro Val Trp Phe Vai Lys Asn Thr His Tyr Tyr Asp Lys Arg Trp Ser ~ys Glu Leu Phe Leu Leu Val Ser Ile Ser Thr Ser Val Ile Leu Met. ~ln His Leu Leu Fro Ala Ser Tyr Cys Asp Leu Leu His Lys Ala Ala :-~la His Leu Ala Cys Trp Gln Lys Val Asp Pro Ala Leu Cys Ser Asn 'lal Leu Gln Pro Pzo Trp Thr Glu Glu Cys Met Trp Pro Gln Gly Val Leu Val Lys His Ser Lys Asn Val Tyr Lys Ala Val Gly His Tyr Asn 'lal Ala Ser Pro Pro Met Ser Pro Thr Ser Ala Ser Ile Ser Phe Ser Ala Thr Pro Cys Gly Ser Ser Thr Ser Phe Cys Cys Trp Arg Val Pro Ser Leu Ser Thr Se. Cys Thr Pro <210> 61 <211> 3071 <212> DNA
<213> Mus musculus <900> 61 gtgaaagcag cagtgcgcct ctgctccctt cagagcacag cctggtgtca aggtc~aggt 60 tccaccggct gctgctgtca ccgcagggga gtctagcccc tcccagaagg agacacagaa 120 gaatggccat ctcaactggt ttgttcctgc tgctggggct ccttggccag ccctgggcag 180 gggctgctgc tgattcacag gctgtggtgt gcgaggggac tgcctgctat acagcccatt 240 ggggcaagct gagtgccgct gaagcccagc atcgctgcaa tgagaatgga ggcaatcttg 300 ccaccgtgaa gagtgaggag gaggcccggc atgttcagca agccctgact cagctcctga 360 agaccaaggc acccttggaa gcaaagatgg gcaaattctg gatcgggctc cagcgagaga 920 agggcaactg tacgtaccat gatttgccaa tgaggggctt cagctgggtg ggtggtggag 980 aggacacagc ttattcaaac tggtacaaag ccagcaagag ctcctgtatc tttaaacgct 540 gtgtgtccct catactggac ctgtccttga cacctcaccc cagccatctg cccaagtggc 600 atgagagtcc ctgtgggacc cccgaagctc caggtaacag cattgaaggt ttcctgtgca 660 agttcaactt caaaggcatg tgtaggccac tggcgctggg tggtccaggg cgggtgacct 720 ataccacccc tttccaggcc actacctcct ctctggaggc tgtgcctttt gcctctgtag 780 ccaatgtagc tt7tggggat gaagctaaga gtgaaaccca ctatttccta tgcaatgaaa 890 agactccagg aatatttcac tggggcagct caggcccact ctgtgtcagc cccaagtttg 900 gttgcagttt caacaacggg ggctgccagc aggattgctt cgaaggtggc gatggctcct 960 tccgctgcgg ctgccggcct ggatttcgac tgctggatga tctagtaact tgtgcctcca 1020 ggaacccctg caa~tcaaac ccatgcacag gaggtggcat gtgccattct gtaccactca 1080 ~tgaaaacta cac~tgccgt tgtcccagcg gctaccagct ggactctagc caagtgcact 1140 Page 36 of 56 gtgtggatat agatgag~. caggactccc cctgtgccca ggattgtgtc a~ ~tctag 1200 ggagcttcca c~g~gaatgt tgggttggtt accaacccag tggccccaag gaagaggcct 1260 gtgaagatgt g~atgagtgt gcagctgcca actcgccctg tgcccaaggc tgcatcaaca 1320 ctgatggctc L~~ctactgc tcctgtaaag agggctatat tgtgtctggg gaagacagta 1380 cccagtgtga g=atatagat gagtgttcgg acgcaagggg caatccatgt gattccctgt 1990 gcttcaacac agatggttcc ttcaggtgtg gctgcccgcc aggctgggag ctggctccca 1500 atggggtctt t=gtagcagg ggcactgtgt tttctgaact accagccagg cctccccaaa 1560 aggaagacaa cgatgacaga aaggagagta ctatgcctcc tactgaaatg cccagttctc 1620 ctagtggctc taaggatgtc tccaacagag cacagacaac aggtctcttc gtccaatcag 1680 atattcccac tgcctctgtt ccactagaaa tagaaatccc tagtgaagta tctgatgtct 1740 ggttcgagtt gggcacatac ctccccacga cctccggcca cagcaagccg acacatgaag 1800 attctgtgtc tgcacacagt gacaccgatg ggcagaacct gcttctgttt tacatcctgg 1860 ggacggtggt ggccatctca ctcttgctgg tgctggccct agggattctc atttatcata 1920 aacggagagc caagaaggag gagataaaag agaagaagcc tcagaatgca gccgacagct 1980 attcctgggt tccagagcga gcagagagcc aagccccgga gaatcagtac agcccaacac 2040 cagggacaga ct7ctgaaga ctatgtggcc ttagagacag ctgccactac cttcagagct 2100 accttcttag a~gaggggga agccacatca ttctgaatga cttgactgga ctctcagcaa 2160 aaaaattgtg caccttccac ttaagaacct ggtggcttgg gataggcagg tattttcttg 2220 gtgcctttga ta=3tctggg ggtgaaagct gtgtgttggt ttgtcattgt ggggagtttt 2280 gtggatattg acagacctca ctcaaacacc cttttcaaat ccaatagcaa ctggttcctc 2390 tggttcctaa ttagggggaa aggagtcaga ggggtgggac agggtggggg gatggggctt 2900 caaagttttt tcttatcact tgatttatca tcgaaggagt tactggtgct aattacaatg 2960 gaaacagttc c~~tccatca caggacagac acacctcaat cctccatggg gtcaacaact 2520 atataccccc agtgacccct taggcaagga cttgttgaga actgcatcac attttgacct 2580 gttctcaaca gtacccatct atttcaggtg ggatctctgg acctttcctc cttcccatct 2690 tgtctgcaat gtggcaaatg gcttcttttt gcatttttac tccgccccca ccccaagctg 2700 aagttcattt gcagatcagc gattaagtct gaattgtgtg gtggtcagtc ttgtttcctt 2760 ttgtcagggg ttattgtaaa tgttagtaat ttcgcctcaa gccctcagta agaacataaa 2820 tattttaaaa tatgtgcgtt tgaaatctgt ttcatgcatc ctggaactgt gggatgctca 2880 ggcaagagtg ac=ttagtct ttcagtgaat gttgcccaga atgtgggtag ggaaggctca 2990 caggttactc tcctccttag agctacaaca taacattctg aggggagtca cagggttgcc 3000 tttaaaaagt g~gagctatg tcatgctttg agctttctgt taagcacctc tcctaataaa 3060 ctctgaaaaa a 3071 <210> 62 <211> 649 <212> PRT
<213> Mus musculus <900> 62 Met Ala Ile Ser Thr Gly Leu Phe Leu Leu Leu Gly Leu Leu Gly Gln Pro Trp Ala G?y Ala Ala Ala Asp Ser Gln Ala Val Val Cys Glu Gly Thr Ala Cys :fir Thr Ala His Trp Gly Lys Leu Ser Ala Ala Glu Ala Gln His Arg Cys Asn Glu Asn Gly Gly Asn Leu Ala Thr Val Lys Ser Glu Glu Glu ~:~_a Arg His Val Gln Gln Ala Leu Thr Gln Leu Leu Lys Thr Lys Ala F=o Leu Glu Ala Lys Met Gly Lys Phe Trp Ile Gly Leu Gln Arg Glu ~;~s Gly Asn Cys Thr Tyr His Asp Leu Pro Met Arg Gly Phe Ser Trp '.'al Gly Gly Gly Glu Asp Thr Ala Tyr Ser Asn Trp Tyr Page 37 of 56 Lys Ala Ser Lys Se. 2r Cys =.e Phe Lys Ar? Cys Val Ser I Ile Leu Asp Leu Ser Leu Thr Pro His Pro Ser His Leu Pro Lys Trp His Glu Ser Pro Cys Gly Thr Pro Glu Ala Pro G1~ Asn Ser Ile Glu Gly Fhe Leu Cys Lys Phe Asn Phe Lys Gly Met Cys Arg Pro Leu Ala Leu Gly Gly Pro Gly Arg Val Thr :'yr Thr Thr Prc Phe Gln Ala Thr Thr Ser Ser Leu Glu Ala Val Pro Fhe Ala Ser Val Ala Asn Val Ala Cys Gly Asp Glu Ala Lys Ser Glu Thr His Tyr Phe Leu Cys Asn Glu Lys 225 230 235 24p Thr Pro Gly Ile Phe His Trp Gly Ser Ser Gly Pro Leu Cys Val Ser Fro Lys Phe Gly Cys Ser Phe Asn Asn Gly Gly Cys Gln Gln Asp Cys 260 265 27p Fhe Glu Gly Gly Asp Gly Ser Phe Arg Cys Gly Cys Arg Pro Gly Phe Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn Pro Cys Ser Ser Asn Pro Cys Thr Gly Gly Gly Met Cys His Ser Val Pro Leu Ser Giu Asn Tyr Thr Cys Arg Cys Fro Ser Gly Tyr Gln Leu Asp Ser Ser Gln Val His Cys Val Asp Ile Asp Glu Cys Gln Asp Ser Pro Cys Ala Gln Asp Cys Val Asn Thr Leu Gly Ser Phe His Cys Glu Cys Trp Val Gly Tyr Gln Pro Ser Gly Pro Lys Glu Glu Ala Cys Glu Asp Val Asp Glu Cys Ala Ala Ala Asn Ser Pro Cys Ala Gln Gly Cys Ile Asn Thr Asp Gly Ser Phe Tyr Cys Ser Cys Lys Glu Gly Tyr Ile Val Ser Gly Glu Asp Ser Thr Gln Cys Glu Asp Ile Asp Glu Cys Ser Asp Ala Arg Giy Asn Pro Cys Asp Ser Leu Cys Phe Asn Thr Asp ~ly Ser Phe Arg 935 490 ;q5 Cys Gly Cys Pro Pro Gly Trp Glu Leu Ala Fro Asn ~iy Val Phe Cys Ser Arg Gly Thr Val Phe Ser Glu Leu Fro Ala Arg ,ro Pro Gln Lys Page 38 of 56 Glu Asp Asn Asp Asp Arg Lys Glu Ser Thr Met Pro Pro Thr Glu :!et Pro Ser Ser Pro Ser Gly Ser Lys Asp Val Ser Asn Arg Ala Gln .hr Thr Gly Leu Phe Val Gln Ser Asp Ile Pro Thr Ala Ser Val Pro Leu Glu Ile Glu Ile Pro Ser Glu Val Ser Asp Val Trp Phe Glu Leu aly Thr Tyr Leu Pro Thr Thr Ser Gly His Ser Lys Pro Thr His Glu asp 595 550 555 ,60 Ser Val Ser Ala His Ser Asp '"hr Asp Gly Gln Asn Leu Leu Leu the Tyr Ile Leu Gly Thr Val Val Ala Ile Ser Leu Leu Leu Val Leu i~la Leu Gly Ile Leu Ile Tyr His Lys Arg Arg Ala Lys Lys Glu Glu ile Lys Glu Lys Lys Pro Gln Asn Ala Ala Asp Ser Tyr Ser Trp Val ?ro Glu Arg Ala Glu Ser Gln Ala Pro Glu Asn Gln Tyr Ser Pro Thr Pro Gly Thr Asp Cys <210> 63 <211> 999 <212> DNA
<213> Mus musculus <900> 63 cccattactt ccacctccca tgaggactgc acctgcggca gcgattttat gtgaacttgg 60 attactgtat ggaaatggag gagtgagaaa gtgtgggaat cataaaagaa ggcagaacca 120 aaagacagag ggatcttggg ccatgagtca cagcaaggaa agcctcccac caaacatctg 180 cactggactc ctacaggaac aagaaatgaa ctatcgtgtt cagcccctga gactt~ggct 290 taacatgatt ttcacattgt ctaccctacc taatagagca gagatgtaaa tattattctt 300 attttagagg tgtgatgcct cagctgcaat gggtgagaac tactcctcat ttattctctt 360 ccaaggcaat aaaagagaat ggaccaaaga cagtctgtca tcacatctag tcaaaagagc 920 taatgtcgca gtacaactct tcaaaagaaa aagaaaaaac aagaaaaaag taataaacag 980 atgtgttctg cttgaaaaa qgg <210> 69 <211> 89 <212> PRT
<213> Mus musculus <900> 64 Met Arg Thr Ala Pro Ala Ala Ala Ile Leu Cys Glu ~eu Gly Leu ~eu Tyr Gly Asn Gly Gly Val Arg Lys Cys Gly Asn His ys Arg Arg ~ln Page 39 of 56 Asn Gln Lys T::r G?u Gly Ser Try Ala Met Ser His Ser Lys Glu Ser Leu Pro Pro Asn .le Cys Thr Gly Leu Leu Gln Glu Gln Glu Met Asn Tyr Arg Val Gln ?ro Leu Arg Leu Gly Leu Asn Met Ile Phe Thr Lee:

Ser Thr Leu Pro As.~. Arg Ala Glu Met '~ 5 <210> 65 <211> 3857 <212> DNA
<213> Mus muscul;a <900> 65 agagggtaga cagaaaggcg ggaaagggct gtgaggtcaa atggacccca tggaactgaa 60 caacgtcagc atcgaacccg acggagacag ctgcagcggg gacagtattc aggacagcta 120 caccggcatg gaaaactccg acaaggacgc catgaacagc caatttgcta atgaagatgc 180 cgaaagtcag aagttcctga caaatgggtt tttagggaag aagaagctag ccgattacgc 290 ggatgagcat caccctggaa tgacttcctt tggaatgtcc tcatttaacc tgagcaacgc 300 catcatgggc agtgggatct taggcttgtc ctatgccatg gccaacaccg ggatcatcct 360 ttttataatc atgctgct~a ctgtggcaat actctcgctc tactcggttc accttttgct 420 gaagacagcc aaggaaggag ggtctctaat ctatgaaaaa ttgggcgaga aagcatttgg 480 atggcctggg aaaattggag ccttcatctc tattacaatg cagaacattg gagccatgtc 590 aagctacctc ttcatcatta agtacgaact gcctgaagta atcagagcat tcatgggact 600 tgaagaaaac actggggaat ggtacctcaa cggcaactac ctcgtcttat ttgtgtcc~t 660 ggggatcatc ctcccgctct ctctccttaa aaatttaggc taccttggct acaccagtgg 720 attttctctc tcctgcatgg tgtttttcgt cagtgtggtg atttacaaaa aattccaaat 780 tccctgccct ctgcc~cctc tggatcacaa caacggaaat ctgacgttca acaacacact 840 tccgattcac atgatc;ccc tgcctaatga ctcggagagc tcgggtgtga acttcatgat 900 ggattacgct caccacaacc cagctgggct ggatgagaag caggtcgcag gccctctt~a 960 cagcaatggc gtggag=acg aagcccaggg tgctgagaaa tgccaaccaa aatactt=y~ 1020 gttcaattcc cggacg7cct atgcaatccc aatcctggct tttgcttttg tctgccaccc 1080 tgaggtcctt cccatc=aca gcgagcttaa agatcgatcc cgcagaaaga tgcagacgy~ 1140 gtccaacatt tccatc~cag g~a=3ctcgt ca=gtacctt cttgcggccc tctttggt=a 1200 tctgagcttc tacggggacg ttcaagacga gctgctgcat gcttacagca aggtctaca~ 1260 atttgatacg gctcttctca tggtgcgcct ggcagtcctg gtggcagtga cactgaccgt 1320 gcccatcgtg ctgttcccya tcc;=acttc ggtgatcaca ctgctgtttc caaggaaac~ 1380 cttcagctgg ctgaagca_~ tcgggatcgc tgcaatcatc atcgcactca acaacatcc= 1990 ggtcatcctc gtgcctacca tcaaatacat ctttggattc ataggggctt cttctgccac 1500 tatgctgatt ttcattc:;c cggctgcgtt ttatctcaag ctcgtcaaga aagaacctct 1560 aagatcaccc cagaaaa~:g gggctttggt cttccttgtg actggaatta ttttcatgat 1620 gggaagcatg gcgctcat~a tactcgactg gatctacaac ccgccgaatc ccaatcacca 1680 ctaat~ccgg ggagacgc.;t ctccactgga aacagctgaa attgtctgaa ggacattt=a 1790 gttg=cttga ttggga=yet agtctgagga attagcaaga ttccaaagac gtttttctag 1800 ctctatcagc acacatttta acccaggccg tgcagtgcag tgtgtgatgc ccgagttgtg 1860 tttgcagcag ctgtgcaayc tgaagcctgt tggctgcgtg tgttggtcag cagacaatag 1920 cctg=ccccc catggtcac~ ccacttctct ccacccccag attaacaggt aattctactc 1980 tcagaacatc agacaaagac ctcctggttg ggatacttgt ggaagagaaa attatgggtt 2090 ttgttgggaa tggtttt7tt gggaatggtg aaggatgcat taaaaattct gtgcgaagta 2100 tcatcagtta cggccatc_c tcactctaca ccaacactaa gggtcggttg actagctgag 2160 gcagggggat atcttgg;~t gtccctgtga ggatcatgac gtatgacggt tgccagtata 2220 gagtac~tca tttcaa~~_= caaggaatag tttgcccaac ctgcttatta caccgagtta 2280 gtgaat;ttc attct~=_=a ttt~ttggta tggcaaatgt tcaaacatct cactaaacat 2340 agaggggggt tattta~~_= atcatgaaac aactaccaaa aagtaatggt ttttaaca~c 2900 tgccttttca tgttgtttct gatatctttg ttccactttg tctttgaaca aggcttcccc 2960 tctcg=cttc cgttct~a~t ct~cgtaat~ ttcagaaaaa ccacagtccg tgtgggagac 2520 acactacccc agtatt;ttt gatacatctc tatttgataa acattcagtg caggaaac_g 2580 tgattttgct atatgtt:7t gtacataatc tcattctgca gttatcagaa cgtt.gacata 2690 tgggacattg gatttt=a~= ttttacatat g~aggttttt tttcttcaca gacaaaatg= 2700 Page 40 of 36 ttatatcatc agggtggggc agggaattaa gctggtgggc tcaaaaatcc atgggtactt 2760 atctgtccat tggagacatc taaaaattaa agtcaaagtt gtgcatagtt cagtaatgct 2820 cttcactgtt tacaagacta taatcatctc agcaaagtag cgaaggaagg gtttgtaaat 2880 aattttcagt gctaacaatg gtctttgaat ttaacatgtc tagaataggg tttagttcat 2990 tttagtttac atcggaactt ggtgacattc atttgccatt aatagaacat cgtgtggtta 3000 ggaataaatg caccaacatg acccaaaacc acattgctca catccatgga cgcttacacg 3060 ggatggagtt cagactccgc ccactgccta gaatctctgg cattgacttc tttttttcaa 3120 ttactggttg tatattttac atttcagtaa aaagccaagt ctgaaatgca agtatcctac 3180 aggtagtgat atactgtcac accaaagcag tttttcaaca gtgtgcctaa gaaacatata 3290 tttgtttact gtgtcatttt ctggacctta agactataca atggtgtgct ttgtgataga 3300 aattgagcat tactagaaac taccataact tcggagttat tatcatttgg gagggaaaaa 3360 aatcaccaaa taggatcttt aagatgtctt tctattttca ttcatccaag aaataaatca 3920 agatagaaag cagaaagggt gtgctgcaaa tattaaatta ttattttatc tgaacatatt 3980 aatgtgaata taaaagatta atccaaaaat attggaaacc tgtgaaatct gtacgtattt 3590 cggatcttta cacttagagc agctaacctg gaagtatcca cttgtgtagt tagatattca 3600 agagcaatat cacagtgcca aagacttcat tcacacagca ccgatcattt gtgtggccgt 3660 tttcaggatt agagttcaca cagactccag ggcagatact cagaataccg ttttctggta 3720 aatttaaccc atttgtaaac agatacaaat tttattttct tacatcattt ataagacggt 3780 tcaatgtact ggggggattt tttttattac agactgtgta ttggtatata ataaataaac 3840 ttttcaaatg aaaaaaa 3857 <210> 66 <211> 547 <212> PRT
<213> Mus musculus <900> 66 Met Asp Pro Met Glu Leu Asn Asn Val Ser Ile Glu Pro Asp Gly Asp I 5 10 15' Ser Cys Ser Gly Asp Ser I'_e Gln Asp Ser Tyr Thr Gly Met Glu Asn Ser Asp Lys Asp Ala Met Asn Ser Gln Phe Ala Asn Glu Asp Ala Glu Ser Gln Lys Phe Leu Thr As: Gly Phe Leu Gly Lys Lys Lys Leu Ala 50 5~ 60 Asp Tyr Ala Asp Glu His H_s Pro Gly Met Thr Ser Phe Gly Met Ser Ser P~.e Asn Leu Ser Asn A1a Ile Met Gly Ser Gly Ile Leu Gly Leu 85 90 g5 Ser Tyr Ala Met Ala Asn Thr Gly Ile Ile Leu Phe Ile Ile Met Leu Leu Thr Val Ala Ile Leu Se. Leu Tyr Ser Val His Leu Leu Leu Lys Thr Ala Lys Glu Gly Gly Se_- Leu Ile Tyr Glu Lys Leu Gly Glu Lys 130 13~ 190 Ala Phe Gly Trp Pro Gly Lys Ile Gly Ala Phe Ile Ser Ile Thr Met 195 150 155 i60 Gln Asn Ile Gly Ala Met Se= Ser Tyr Leu Phe Ile Ile Lys Tyr Glu Leu ?_-o Glu Val Ile Arg Ala Phe Met Gly Leu Glu Glu Asn Thr Gly Glu Trp Tyr Leu Asn Gly Asn Tyr Leu Val Leu Phe Val Ser Val Gly Page 4 t of 56 Ile Ile Leu Pro Leu Ser Leu Leu Lys Asn Leu Gly Tyr Leu Gly Tyr Thr Ser Gly Phe Ser Leu Ser Cys Met Val Phe Phe Val Ser Val Val Ile Tyr Lys Lys Phe Gln Ile Pro Cys Pro Leu Pro Ala Leu Asp His is n Asn Gly Asn Leu Thr Phe Asn Asn Thr Leu Pro Ile His Met Ile Ser Leu Pro Asn Asp Ser Glu Ser Ser Gly Val Asn Phe Met Met Asp Tyr Ala His His Asn Pro Ala Gly Leu Asp Glu Lys Gln Val Ala Gly Pro Leu His Ser Asn Gly Val Glu Tyr Glu Ala Gln Gly Ala Glu Lys Cys Gln Pro Lys Tyr Phe Val Phe Asn Ser Arg Thr Ala Tyr Ala Ile Pro Ile Leu Ala Phe Ala Phe Val Cys His Pro Glu Val Leu Pro Ile Tyr Ser Glu Leu Lys Asp Arg Ser Arg Arg Lys Met Gln Thr Val Ser Asn Ile Ser Ile Ser Gly Met Leu Val Met Tyr Leu Leu Ala Ala Leu Phe Gly Tyr Leu Ser Phe Tyr Gly Asp Val Glu Asp Glu Leu Leu His rla Tyr Ser Lys Val Tyr Thr Phe Asp Thr Ala Leu Leu Met Val Arg Leu Ala Val Leu Val Ala Val Thr Leu Thr Val Pro Ile Val Leu Phe Pro Ile Arg Thr Ser Val Ile Thr Leu Leu Phe Pro Arg Lys Pro Phe Ser Trp Leu Lys His Phe Gly Ile Ala Ala Ile Ile Ile Ala Leu Asn Asn Ile Leu Val Ile Leu Val Pro Thr Ile Lys Tyr Ile Phe Gly Phe Ile Gly Ala Ser Ser Ala Thr Met Leu Ile Phe Ile Leu Pro Ala Ala Phe Tyr Leu Lys Leu Val Lys Lys Glu Pro Leu Arg Ser Pro Gln Lys Ile Gly Ala Leu Val Phe Leu Val Thr Gly Ile Ile Phe Met Met Gly Ser Met Ala Leu Ile Ile Leu Asp Trp Ile Tyr Asn Pro Pro Asn Pro Page 42 of 56 Asn His His <210> 67 <211> 1070 <212> DNA
<213> Mus musculus <900> 67 agcgacatgg ccccgcccgc gctccaggcc cagcctccag gcggctctca actgaggttc 60 ctgctgttcc tgctgctgtt gctgctgctg ctgtcatggc catcgcaggg ggacgccctg 120 gcaatgcctg aacagcgacc ctccggccct gagtcccaac tcaacgccga cgagctacgg 180 ggtcgcttcc aggacctgct gagccggctg catgccaacc agagccgaga ggactcgaac 240 tcagaaccaa gtcctgaccc agctgtccgg atactcagtc cagagqtgag attggggtcc 300 cacggccagc tgctactccg cgtcaaccgg gcgtcgctga gtcagggtct ccccgaagcc 360 taccgcgtgc accgagcgct gctcctgctg acgccgacgg cccgcccctg ggacatcact 420 aggcccctga agcgtgcgct cagcctccgg ggaccccgtg ctcccgcatt acgcctgcgc 480 ctgacgccgc ctccggacct ggctatgctg ccctctggcg gcacgcagct ggaactgcgc 540 ttacgggtag ccgccggcag ggggcgccga agcgcgcatg cgcacccaag agactcgtgc 600 ccactgggtc cggggcgctg ctgtcacttg gagactgtgc aggcaactct tgaagacttg 660 ggctggagcg actgggtgct gtccccgcgc cagctgcagc tgagcatgtg cgtgggcgag 720 tgtccccacc tgtatcgctc cgcgaacacg catgcgcaga tcaaagcacg cctgcatggc 780 ctgcagcctg acaaggtgcc tgccccgtgc tgtgtcccct ccagctacac cccggtggtt 840 cttatgcaca ggacagacag tggtgtgtca ctgcagactt atgatgacct ggtggcccgg 900 ggctgccact gcgcttgagc accgggccct gctcctcacc tacactcccc ttcaaggatg 960 ctatttatat ttgtatttat taatattatt aatttattgg ggtcgggctg ggtggatgga 1020 ttgtgtattt atttaaaact ctgctaataa aggtgagctt ggtttcaaaa 1070 <210> 68 <211> 298 <212> PRT
<213> Mus musculus <400> 68 Met Ala Pro Pro Ala Leu Gln Ala Gln Pro Pro Gly Gly Ser Gln Leu Arg Phe Leu Leu Phe Leu Leu Leu Leu Leu Leu Leu Leu Ser Trp Fro 5er Gln Gly Asp Ala Leu Ala Met Pro Glu Gln Arg Pro Ser Gly Fro Glu Sex Gln Leu Asn Ala Asp Glu Leu Arg Gly Arg Phe Gln Asp Leu Leu Ser Arg Leu His Ala Asn Gln Ser Arg Glu Asp Ser Asn Ser Glu Pro Ser Pro Asp Pro Ala Val Arg Ile Leu Ser Pro Glu Val Arg Leu Gly Ser His Gly Gln Leu Leu Leu Arg Val Asn Arg Ala Ser Leu Ser Gln Gly Leu Pro Glu Ala Tyr Arg Val His Arg Ala Leu Leu Leu ~eu Thr Pro Thr Ala Arg Pro Trp Asp Ile Thr Arg Pro Leu Lys Arg ~la :.eu Ser Leu Arg Gly Pro Arg Ala Pro Ala Leu Arg ieu Arg Leu hr Page 43 of 56 195 50 155 .60 Pro Pro Pro Asp Leu Ala Met Leu Pro Ser Gly Gly Thr Gln Leu ~~u Leu Arg Leu Arg Val Ala Ala Gly Arg Gly Arg Arg Ser Ala His .la His Pro Arg Asp Ser Cys Pro Leu Gly Pro Gly Arg Cys Cys His _eu Glu Thr Val Gin Ala Thr Leu Glu Asp Leu Gly Trp Ser Asp Trp '.'al Leu Ser Pro Arg Gln Leu Gln Leu Ser Met Cys Val Gly Glu Cys =ro His Leu Tyr A_g Ser Ala Asn Thr His Ala Gln Ile Lys Ala Arg _au His Gly Leu Gln Pro Asp Lys Val Pro Ala Pro Cys Cys Val Pro Ser Ser Tyr Thr Pro Val Val Leu Met His Arg Thr Asp Ser Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Val Ala Arg <210> 69 <211> 134 <212> PRT
<213> Mus musculus <400> 69 Ala Lys Tyr Va_ Cys Leu Ala Asn Lys Asn Cys Pro Val Asp Lys :-.rg Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys Leu Ala '.'al Gly Met Val Lys Glu Val Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Gln Glu Pro Ser Pro Pro Ser Pro Pro Phe Gln Ala Asn Pro Asp Tyr Gln Met Ser Gly Asp nsp Thr Gln His I~.:e Gln Gln Phe Tyr Asp Leu Leu Thr Gly Ser Met Flu Ile Ile Arg Gay Trp Ala Glu Lys Ile Pro Gly Phe Ala Asp Leu ro Lys Ala Asp G:.n Asp Leu Leu Phe Glu Ser Ala Phe Leu Glu Leu .he Val Leu Arg Leu Ala Tyr Page 44 of 56 <210> 70 <211> 190 <212> PRT
<213> Mus musculus <900> 70 Ser Gln Thr Arg Gln Gln Gly Pro Leu Arg Ser Ile Met Lys Asp Leu His Ser Asp Asp Asn Glu Glu Glu Ser Asp Glu Val Glu Asp Asn Asp Asn Asp Ser Glu Met Glu Arg Pro Val Asn Arg Gly Xaa Ser Arg Ser Arg Arg Val Ser Leu Ser Asp Gly Ser Asp Ser Glu Ser Ser Ser Ala Ser Ser Pro Leu His His Glu Pro Pro Pro Pro Leu Leu Lys Thr Asn Asn Asn Gln Ile Leu Glu Val Lys Ser Pro Ile Lys Gln Ser Lys Ser Asp Lys Gln Ile Lys Asn Gly Glu Cys Asp Lys Ala Tyr Leu Asp Glu Leu Val Glu Leu Pro Arg Xaa Xaa Met Thr Leu Arg Glu Arg His Ile Leu Gln Gln Ile Val Asn Leu Xaa Glu Glu Thr Gly <210> 71 <211> 89 <212> PRT
<213> Mus musculus <900> 71 Met Arg Thr Ala Pro Ala Ala Ala Ile Leu Cys Glu Leu Gly Leu Leu Tyr Gly Asn Gly Gly Val Arg Lys Cys Gly Asn His Lys Arg Arg Gln Asn Gln Lys Thr Glu Gly Ser Trp Ala Met Ser His Ser Lys Glu Ser Leu Pro Pro Asn Ile Cys Thr Gly Leu Leu Gln Glu Gln Glu Met Asn Tyr Arg Val Gln Pro Leu Arg Leu Gly Leu Asn Met Ile Phe Thr Leu Ser Thr Leu Pro Asn Arg Ala Glu Met <210> 72 <211> 3121 <212> DNA
<213> Homo sapiens Page 45 of 56 WO 00/11168 PC'TlUS99/19052 <400> 72 gaaagcagca gtgcgcctct gctcccttca gagcacagcc tggtgtcaag gtccaggttc 60 caccggctgc tgctgtcacc gcaggggagt ctagcccctc ccagaaggag acacagaaga 120 atggccatct caactggttt gttcctgctg ctggqgctcc ttggccagcc ctgggcaggg 180 gctgctgctg attcacaggc tgtggtgtgc gaggggactg cctgctatac agcccattgg 290 ggcaagctga gtgccgctga agcccagcat cgctgcaatg agaatggagg caatcttgcc 300 accgtgaaga gtgaggagga ggcccggcat gttcagcaag ccctgactca gctcctgaag 360 accaaggcac ccttggaagc aaagatgggc aaattctgga tcgggctcca gcgagagaag 920 ggcaactgta cgtaccatga tttgccaatg aggggcttca gctgggtggg tggtggagag 480 gacacagctt attcaaactg gtacaaagcc agcaagagct cctgtatctt taaacgctgt 540 gtgtccctca tactggacct gtccttgaca cctcacccca gccatctgcc caagtggcat 600 gagagtccct gtgggacccc cgaagctcca ggtaacagca ttgaaggttt cctgtgcaag 660 ttcaacttca aaggcatgtg taggccactg gcgctgggtg gtccagggcg ggtgacctat 720 accacccctt tccaggccac tacctcctct ctggaggctg tgccttttgc ctctgtagcc 780 aatgtagctt gtggggatga agctaagagt gaaacccact atttcctatg caatgaaaag 890 actccaggaa tatttcactg gggcagctca ggcccactct gtgtcagccc caagtttggt 900 tgcagtttca acaacggggg ctgccagcag gattgcttcg aaggtggcga tggctccttc 960 cgctgcggct gccggcctgg atttcgactg ctggatgatc tagtaacttg tgcctccagg 1020 aacccctgca gctcaaaccc atgcacagga ggtggcatgt gccattctgt accactcagt 1080 gaaaactaca cttgccgttg tcccagcggc taccagctgg actctagcca agtgcactgt 1190 gtggatatag atgagtgcca ggactccccc tgtgcccagg attgtgtcaa cactctaggg 1200 agcttccact gtgaatgttg ggttggttac caacccagtg gccccaagga agaggcctgt 1260 gaagatgtgg atgagtgtgc agctgccaac tcgccctgtg cccaaggctg catcaacact 1320 gatggctctt tctactgctc ctgtaaagag ggctatattg tgtctgggga agacagtacc 1380 cagtgtgagg atatagatga gtgttcggac gcaaggggca atccatgtga ttccctgtgc 1440 ttcaacacag atggttcctt caggtgtggc tgcccgccag gctgggagct ggctcccaat 1500 ggggtctttt gtagcagggg cactgtgttt tctgaactac cagccaggcc tccccaaaag 1560 gaagacaacg atgacagaaa ggagagtact atgcctccta ctgaaatgcc cagttctcct 1620 agtggctcta aggatgtctc caacagagca cagacaacag gtctcttcgt ccaatcagat 1680 attcccactg cctctgttcc actagaaata gaaatcccta gtgaagtatc tgatgtctgg 1740 ttcgagttgg gcacatacct ccccacgacc tccggccaca gcaagccgac acatgaagat 1800 tctgtgtctg cacacagtga caccgatggg cagaacctgc ttctgtttta catcctgggg 1860 acggtggtgg ccatctcact cttgctggtg ctggccctag ggattctcat ttatcataaa 1920 cggagagcca agaaggagga gataaaagag aagaagcctc agaatgcagc cgacagctat 1980 tcctgggttc cagagcgagc agagagccaa gccccggaga atcagtacag cccaacacca 2090 gggacagact gctgaagact atgtggcctt agagacagct gccactacct tcagagctac 2100 cttcttagat gagggggaag ccacatcatt ctgaatgact tgactggact ctcagcaaaa 2160 aaattgtgca ccttccactt aagaacctgg tggcttggga taggcaggta ttttcttggt 2220 gcctttgata tgtctggggg tgaaagctgt gtgttggttt gtcattgtgg ggagttttgt 2280 ggatattgac agacctcact caaacaccct tttcaaatcc aatagcaact ggttcctctg 2390 gttcctaatt agggggaaag gagtcagagg ggtgggacag ggtgggggga tggggcttca 2900 aagttttttc ttatcacttg atttatcatc gaaggagtta ctggtgctaa ttacaatgga 2960 aacagttcct ttccatcaca ggacagacac acctcaatcc tccatggggt caacaactat 2520 atacccccag tgacccctta ggcaaggact tgttgagaac tgcatcacat tttgacctgt 2580 tctcaacagt acccatctat ttcaggtggg atctctggac ctttcctcct tcccatcttg 2690 tctgcaatgt ggcaaatggc ttctttttgc atttttactc cgcccccacc ccaagctgaa 2700 gttcatttgc agatcagcga ttaagtctga attgtgtggt ggtcagtctt gtttcctttt 2760 gtcaggggtt attgtaaatg ttagtaattt cgcctcaagc cctcagtaag aacataaata 2820 ttttaaaata tgtgcgtttg aaatctgttt catgcatcct ggaactgtgg gatgctcagg 2880 caagagtgac tttagtcttt cagtgaatgt tgcccagaat gtgggtaggg aaggctcaca 2990 ggttactctc ctccttagag ctacaacata acattctgag gggagtcaca gggttgcctt 3000 taaaaagtgg gagctatgtc atgctttgag ctttctgtta agcacctetc ctaataaact 3060 ctgaaaaaat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3120 a 3121 <210> 73 <211> 644 <212> PRT
<213> Homo sapiens <400> 73 Met Ala Ile Ser Thr Gly Leu Phe Leu Leu Leu Gly ~eu Leu Gly Gln i 5 10 15 Page 46 of S6 Pro Trp Ala Gly Al la Ala Asp Ser Gln Ala Val Val Cys C Cly Thr Ala Cys Tyr Thr Ala His Trp Gly Lys Leu Ser Ala Ala Glu ~la Gln His Arg Cys Asn Glu Asn Gly Gly Asn Leu Ala Thr Val Lys Ser Glu Glu Glu Ala Arg His Val Gln Gln Ala Leu Thr Gln Leu Leu Lys Thr Lys Ala Pro Leu Glu Ala Lys Met Gly Lys Phe Trp Ile Gly Leu Gln Arg Glu Lys Gly Asn Cys Thr Tyr His Asp Leu Pro Met Arg Giy Phe Ser Trp Val Gly Gly Gly Glu Asp Thr Ala Tyr Ser Asn Trp =yr Lys Ala Ser Lys Sex Ser Cys Ile Phe Lys Arg Cys Val Ser Leu Ile Leu Asp Leu Ser Leu Thr Pro His Pro Ser His Leu Pro Lys Trp His Glu Ser Pro Cys Gly Thr Pro Glu Ala Pro Gly Asn Ser Ile Glu Gly Phe Leu Cys Lys Phe Asn Phe Lys Gly Met Cys Arg Pro Leu Ala Leu Gly Gly Pro Gly Arg Val Thr Tyr Thr Thr Pro Phe Gln Ala Thr Thr Ser Ser Leu Glu Ala Val Pro Phe Ala Ser Val Ala Asn Val Ala Cys Gly Asp Glu Ala Lys Ser Glu Thr His Tyr Phe Leu Cys Asn Glu Lys Thr Pro Gly Ile Phe His Trp Gly Ser Ser Gly Pro Leu Cys Val Ser Pro Lys Phe Gly Cys Ser Phe Asn Asn Gly Gly Cys Gln Gln Asp Cys Phe Glu Gly Gly Asp Gly Ser Phe Arg Cys Gly Cys Arg Pro Gly Phe Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn Pro Cys Ser Ser Asn Pro Cys Thr Gly Gly Gly Met Cys His Ser Val Pro Leu Ser Glu Asn Tyr Thr Cys Arg Cys Pro Ser Gly Tyr Gln Leu Asp Ser Ser Gln Val His Cys Val Asp Ile Asp Glu Cys Gln Asp Ser Pro Cys Fla Gln Asp Cys Val Asn Thr Leu Gly Ser Phe His Cys Glu Cys Trp '!al Page 47 of 56 Gly Tyr Gln Pro Ser Gly Pro Lys Glu Glu Ala Cys Glu Asp Val hsp Glu Cys Ala Ala Ala Asn Ser Pro Cys Ala Gln Gly Cys Ile Asn Thr Asp Gly Ser Phe Tyr Cys Ser Cys Lys Glu Gly Tyr Ile Val Ser Gly Glu Asp Ser T!:r Gln Cys Glu Asp Ile Asp Glu Cys Ser Asp Ala Arg Gly Asn Pro Cys Asp Ser Leu Cys Phe Asn Thr Asp Gly Ser Phe Arg Cys Gly Cys P=c Pro Gly Trp Glu Leu Ala Pro Asn Gly Val Phe Cys Ser Arg Gly T::r Val Phe Ser Glu Leu Pro Ala Arg Pro Pro Gln Lys Glu Asp Asn Asp Asp Arg Lys Glu Ser Thr Met Pro Pro Thr Glu Met Pro Ser Ser Prc Ser Gly Ser Lys Asp Val Ser Asn Arg Ala Gln Thr Thr Gly Leu Phe Val Gln Ser Asp Ile Pro Thr Ala Ser Val Pro Leu Glu Ile Glu Ile Pro Ser Glu Val Ser Asp Val Trp Phe Glu Leu Gly Thr Tyr Leu P=~ Thr Thr Ser Gly His Ser Lys Pro Thr His Glu Asp Ser Val Ser Ala His Ser Asp Thr Asp Gly Gln Rsn Leu Leu Leu Phe Tyr Ile Leu Gi; Thr Val Val Ala Ile Ser Leu Leu Leu Val Leu Ala Leu Gly Ile Le~~ Ile Tyr His Lys Arg Arg Ala Lys Lys Glu Glu Ile Lys Glu Lys Lys Pro Gln Asn Ala Ala Asp Ser Tyr Ser Trp Val Pro Glu Arg Ala G_~a Ser Gln Ala Pro Glu Asn Gln Tyr Ser Pro Thr Pro Gly Thr Asp Cys <210> 79 <211> 27 <212> DNA
<2I3> Artific;al Sequence <220>
<223> Descrip~=o~ of Artificial Sequence: Primer Page 48 of 56 <400> 79 tggtgtcgac gcagagtagc 27 ggccgct <210> 75 <211> 19 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence: Primer Artificial <900> 75 ggcccgggcc ggcc 14 <210> 76 <211> 17 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence: Primer Artificial <400> 76 tcgaggccgc ccgggcc 17 <210> 77 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence: Primer Artificial <400> 77 cagccctcac tccttctc 18 <210> 78 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence: Primer Artificial <400> 78 ggtggggtct ttcattcc 18 <210> 79 <211> 18 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence: Primer Artificial <400> 79 ttcagcaagc cctgactc 18 <210> 80 <211> 18 <212> DNA

<213> Artificial Sequence Page 49 of Sb <2zo>
<223> Description of Artific_a= Sequence: Primer <900> 80 gccaccttcg aagcaatc 18 <210> 81 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> B1 gagcggtaca ggagaatg 18 <210> 82 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 82 gcccacccaa ccaaatca . 18 <210> 83 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <900> 83 accgcgttct tctgtaac 18 <210> 89 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <900> 89 cagctaacag caggatcc 18 <210> 85 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <900> $5 gacccaggct gcccc 15 <210> 86 <211> 15 <212> DNA
Page 50 of 56 <213> Artificial S ence <220>

<223> Description of Sequence:Primer Artificial <400> 86 ggtactcttg ttgag 15 <210> 87 <211> 39 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence:Primer Artificial <400> 87 gagggtacca aa~=tcgtgg gaag 34 tccgcaacca <210> 88 <211> 36 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence:Primer Artificial <400> 88 ctcagatctg aattcggcct gaggag 36 gtccctcggt <210> 89 <211> 35 <212> DNA

<213> Artificial Sequence <z2o>

<223> Description of Sequence:Primer Artificial <400> 89 gacaagatct cagctgaata ctgtg 35 gcgacccacc <210> 90 <211> 41 <212> DNA

<213> Artificial Sequence <220>

<223> Description of Sequence:Primer Artificial <900> 90 gcatctagag cggccgctca tcggtgagga 91 ggcctgtccc g <210> 91 <211> 37 <212> DNA

<213> Artificial Sequence <220>

<223> Descript'_on of Sequence:Primer Artificial <400> 91 gcatctagag cggccgcgaa atctcct 37 cgctgcttag <210> 92 Page 51 of 56 <211> 131 <212> PRT
<213> Mus musculus <900> 92 Glu Val Phe Leu Asn Pro Leu Gly Arg Asp Leu Leu Ser Ile Ser Asp Gly Arg Xaa Arg Ala Ile His Arg Arg Gly His Asn Asp Gly Glu Asp Ser Leu Thr His Thr Asp Val Ser Ser Phe Gln Thr Met Asp Gln Met Val Ser Asn Met Arg Asn Tyr Met Gln Lys Leu Glu Arg Asn Thr Gly Gln Leu Ser Val Asp Pro Asn Gly His Ser Phe Cys Ser Ser Ser Val Met Thr Tyr Ser Lys Ile Gly Asp Glu Pro Pro Lys Val Phe His Ala Ser Thr Gln Thr Arg Arg Ala Pro Gly Arg Ile Lys Glu Thr Arg Lys Ala Met Arg Asp Ser Asp Ser Gly Thr Arg Lys His Gly Tyr Trp Ser Ser Tyr Pro <210> 93 <211> 131 <212> PRT
<213> Mus musculus <400> 93 Arg Ser Phe Ser Glu Pro Phe Gly Arg Asp Leu Leu Ser Ile Ser Asp 1 5 10 15' Gly Arg Gly Arg Ala His Asn Arg Arg Gly His Asn Asp Gly Glu Asp Ser Leu Thr His Thr Asp Val Ser Ser Phe Gln Thr Met Asp Gln Met Val Ser Asn Met Arg Asn Tyr Met Gln Lys Leu Glu Arg Asn Phe Gly Gln Leu Ser Val Asp Pro Asn Gly His Ser Phe Cys Ser Ser Ser Val Met Thr Tyr Ser Lys Ile Gly Asp Glu Pro Pro Lys Val Phe Gln Ala Ser Thr Gln Thr Rrg Arg Ala Pro Gly Gly Ile Lys Glu Thr Arg Lys Ala Met Arg Asp Ser Asp Ser Gly Leu Glu Lys Met Ala Ile Gly His His Ile His Page 52 of 56 <210> 94 <211> 159 <212> PRT
<213> Mus musculus <900> 99 Ala Lys Tyr Val Cys Leu Ala Asn Lys Asn Cys Pro Val Asp Lys Arg Arg Arg Asn Arg Cys Gln Tyr Cys Arg Phe Gln Lys Cys Leu Ala Val Gly Met Val Lys Glu Xaa Val Arg Thr Asp Ser Leu Lys Gly Arg Arg Gly Arg Leu Pro Ser Lys Pro Lys Ser Pro Gln Glu Pro Ser Pro Pro Ser Pro Pro Val Ser Leu Ile Ser Ala Leu Val Arg Ala His Val Asp Ser Asn Pro Ala Met Thr Ser Leu Asp Tyr Ser Arg Phe Gln Ala Asn Pro Asp Tyr Gln Met Ser Gly Asp Asp Thr Gln His Ile Gln Gln Phe Tyr Asp Leu Leu Thr Gly Ser Met Glu Ile Ile Arg Gly Trp Ala Xaa Xaa Ile Pro Gly Phe Ala Asp Leu Pro Lys Ala Asp Gln Asp Leu Leu Phe Glu Ser Ala Phe Leu Glu Leu Phe Val Leu Azg Leu Ala Tyr <210> 95 <211> 140 <212> PRT
<213> Mus musculus <900> 95 Ser Gln Thr Arg Gln Gln Gly Pro Leu Arg Ser Ile Met Lys Asp Leu 1 5 10 15' His Ser Asp Asp Asn Glu Glu Glu Ser Asp Glu Val Glu Asp Asn Asp Asn Asp Ser Glu Met Glu Arg Pro Val Asn Arg Gly Gly Ser Arg Ser Arg Arg Val Ser Leu Ser Asp Gly Ser Asp Ser Glu Ser Ser Ser Ala Ser Ser Pro Leu His His Glu Pro Pro Pro Pro Leu Leu Lys Thr Asn Asn Asn Gln Ile Leu Glu Val Lys Ser Pro Ile Lys Gln Ser Lys Ser Page 53 of 56 Asp Lys Gln Ile Ly_ sn Gly G";u Cys Asp Lys Ala Tyr Leu . Glu Leu Val Glu Leu His Arg Arg :.eu Met Thr Leu Arg Glu Arg His Ile Leu Gln Gln Ile Val Asn Leu Ile Glu Glu Thr Gly <210> 96 <211> 652 <212> PRT
<213> Mus musculus <900> 96 Met Ala Thr Ser Met Gly Leu Leu Leu Leu Leu Leu Leu Leu Leu Thr Gln Pro Gly Ala Gly Thr Gly Ala Asp Thr Glu Ala Val Val Cys Val Gly Thr Ala Cys Tyr Thr Ala His Ser Gly Lys Leu Ser Ala Ala Glu Ala Gln Asn His Cys Asn Gln Asn Gly Gly Asn Leu Ala Thr Val Lys Ser Lys Glu Glu Ala Gln His Val Gln Arg Val Leu Ala Gln Leu Leu Arg Arg Glu Ala Ala Leu Thr Ala Arg Met Ser Lys Phe Trp Ile Gly Leu Gln Arg Glu Lys Gly Lys Cys Leu Asp Pro Ser Leu Pro Leu Lys Gly Phe Ser Trp Val Gly Gly Gly Glu Asp Thr Pro Tyr Ser Asn Trp His Lys Glu Leu Arg Asn Ser Cys Ile Ser Lys Arg Cys Val Ser Leu Leu Leu Asp Leu Ser Gln Pro Leu Leu Pro Asn Arg Leu Pro Lys Trp Ser Glu Gly Pro Cys Gly Ser Pro Gly Ser Pro Gly Ser Asn Ile Glu Gly Phe Val Cys Lys Phe Ser Phe Lys Gly Met Cys Arg Pro Leu Ala Leu Gly Gly Pro Gly Gln Val Thr Tyr Thr Thr Pro Phe Gln Thr Thr Ser Ser Ser Leu Glu Ala Val Pro Phe Ala Ser Ala Ala Asn Val~ Ala Cys Gly Glu Gly Asp Lys Asp Glu Thr Gln Ser His Tyr Phe Leu Cys Lys Glu Lys Ala Pro Asp Val Phe Asp Trp Gly Ser Ser Gly Pro Leu Page 54 of 56 Cys Val Ser Pro Ly. yr Gly Cys Asn Phe Asn Asn Gly Gly ~ :iis Gln Asp Cys Phe Glu Gly Gly Asp Gly Ser Phe Leu Cys Gly Cys Arg Pro Gly Arg Arg Leu Leu Asp Asp Leu Val Thr Cys Ala Ser Arg Asn Pro Cys Ser Ser Ser Pro Cys Arg Gly Gly Ala Thr Cys Val Leu Gly Pro His Gly Lys Asn Tyr Thr Cys Arg Cys Pro Gln Gly Tyr Gln Leu Asp Ser Ser G'_n Leu Asp Cys Val Asp Val Asp Glu Cys Gln Asp Ser Pro Cys Ala Glz Glu Cys Val Asn Thr Pro Gly Gly Phe Arg Cys Glu Cys Trp Val Gly Tyr Glu Pro Gly Gly Pro Gly Glu Gly Ala Cys Gln Asp Val Asp Glu Cys Ala Leu Gly Arg Ser Pro Cys Ala Gln Gly Cys Thr Asn Thr Asp Gly Ser Phe His Cys Ser Cys Glu Glu Gly Tyr Val Leu Ala Gly Glu Asp Gly Thr Gln Cys Gln Asp Val Asp Glu Cys Val Gly Pro Gly Gly Pro Leu Cys Asp Ser Leu Cys Phe Asn Thr Gln Gly Ser Phe His Cys Gly Cys Leu Pro Gly Trp Val Leu Ala Pro Asn Gly Val Ser Cys Thr Met Gly Pro Val Ser Leu Gly Pro Pro Ser Gly Pro Pro Asp Glu Glu Asp Lys Gly Glu Lys Glu Gly Ser Thr Val Pro Arg Ala Ala Thr Ala Ser Pro Thr Arg Gly Pro Glu Gly Thr Pro Lys Ala Thr Pro Thr T':r Ser Arg Pro Ser Leu Ser Ser Asp Ala Pro Ile Thr Ser Ala Pro Leu Lys Met Leu Ala Pro Ser Gly Ser Ser Gly Val Trp Arg Glu Pro Ser Ile His His Ala Thr Ala Ala Ser Gly Pro Gln Glu Pro Ala Gly G'_y Asp Ser Ser Val Ala Thr Gln Asn Asn Asp Gly Thr Asp Gly Gln Lvs Leu Leu Leu Phe Tyr Ile Leu Gly Thr Val Val nla -_le Leu Leu Te;.i Leu Ala Leu Ala Leu Gly Leu Leu Val Tyr Arg Lys Page 55 of 56 595 600 6'05"' Arg Arg Ala ys Arg Glu Glu Lys Lys Glu Lys Lys Pro Gln Asn Ala Ala Asp Ser Tyr Ser Trp Val Pro Glu Arg Ala Glu Ser Arg Ala Met Glu Asn Gln Tyr Ser Pro Thr Pro Gly Thr Asp Cys Page 56 of 56

Claims (50)

WHAT IS CLAIMED IS:

1. An isolated nucleic acid derived from an isolated hematopoietic stem cell, the isolated nucleic acid comprising the following characteristics:
- specifically expressed in the hematopoietic stem cell; and - encoding a hematopoietic stem cell - specific protein.

2. An isolated nucleic acid of claim 1, the isolated nucleic acid further comprising the following characteristic:
- capable of hybridizing under standard conditions with a sequence selected from the group consisting of SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.:3, SEQ.ID.No.: 4, SEQ.ID.No.:5, SEQ.ID.No.:6, SEQ.ID.No.:7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.:10, SEQ.ID.No.:11, SEQ.ID.No.:12, SEQ.ID.No.:
13, SEQ.ID.No.: 14, SEQ.ID.No.:15, SEQ.ID.No.:16, SEQ.ID.No.: 17 SEQ.ID.No.:18, SEQ.ID.No.:19, SEQ.ID.No.: 20SEQ.ID.No.:21, SEQ.ID.No.:
SEQ.ID.No.:22, SEQ.ID.No.:23, SEQ.ID.No.: 24, SEQ.ID.No.:25, SEQ.ID.No.: 26 SEQ.ID.No.:27,SEQ.ID.No.:28, SEQ.ID.No.:29,SEQ.ID.No.:30 SEQ.ID.No.:
31, SEQ.ID.No.: 32. SEQ.ID.No.:33, SEQ.ID.No.:34, SEQ.ID.No.: 35 SEQ.ID.No.: 36, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.:
40, SEQ.ID.No.: 41, SEQ.ID.No.:42. and SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.:: 53, SEQ.ID.No.:
55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a portion thereof.

3. The isolated nucleic acid of claim 1, the isolated nucleic acid further comprising the characteristic of encoding a protein capable of modulating hematopoietic stem cell activity.

4. The isolated nucleic acid of claim 3, wherein the activity is selected from the group consisting of hematopoietic stem cell differentiation and hematopoietic stem cell replication.

5. The isolated nucleic acid of claim 3, wherein the protein is selected from the group consisting of a growth factor, a transcription factor, a splicing factor, a capping factor, a transport protein, a translation factor, and a replication factor

6. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises the nucleotide sequence of SEQ.ID.No.: 72, an analog thereof, or a portion thereof.

7. The isolated nucleic acid of claim 1, wherein the hematopoietic stem cell is a primitive hematopoietic stem cell.

8. The isolated nucleic acid of claim 7, wherein the primitive hematopoietic stem cell is selected from the group consisting of an umbilical cord cell, a bone marrow cell and a fetal liver cell.

9. The isolated nucleic acid of claim 7, wherein the primitive hematopoietic stem cell is selected from the group consisting of a AFT024 cell, a 2012 cell and a 2018 cell.

10. A composition comprising the nucleic acid of claim 1, wherein the nucleic acid comprises one selected from the group consisting of SEQ.ID.No.: 1, SEQ.ID.No.:
2, SEQ.ID.No.: 3, SEQ.ID.No.: 4, SEQ.ID.No.: 5, SEQ.ID.No.: 6, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.: 14, SEQ.ID.No.: 15, SEQ.ID.No.: 16, SEQ.ID.No.:
17, SEQ.ID.No.: 18, SEQ.ID.No.: 19, SEQ.ID.No.: 20, SEQ.ID.No.: 21, SEQ.ID.No.:
22, SEQ.ID.No.: 23, SEQ.ID.No.: 24, SEQ.ID.No.: 25, SEQ.ID.No.: 26, SEQ.ID.No.:
27, SEQ.ID.No.: 28, SEQ.ID.No.: 29, SEQ.ID.No.: 30, SEQ.ID.No.: 31, SEQ.ID.No.:
32, SEQ.ID.No.: 33, SEQ.ID.No.: 34, SEQ.ID.No.: 35, SEQ.ID.No.: 36, SEQ.ID.No.:
37, SEQ.ID.No.: 38, SEQ.ID.No.: 39, SEQ.ID.No.: 40, SEQ.ID.No.: 41, SEQ.ID.No.:
42, SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.:
51, SEQ.ID.No.: 53, SEQ.ID.No.: 55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.:
61, SEQ.ID.No.: 63, SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a portion thereof.

11. The nucleic acid of claim 1, wherein the nucleic acid is selected from the group consisting of DNA, RNA and cDNA.

12. A vector comprising the nucleic acid of claim 1.

13. The vector of claim 12, wherein the vector comprises viral or plasmid DNA.

14. An expression vector comprising the nucleic acid of claim 1 and a regulatory element.

15. A host vector system which comprises the expression vector of claim 12 in a suitable host.

16. The host vector system of claim 15, wherein the suitable host is selected from the group consisting of a bacterial cell, a eukaryotic cell, a mammalian cell and an insect cell.

17. An isolated hematopoietic stem cell specific protein or a portion thereof encoded by the nucleic acid of claim 1.

18. The isolated hematopoietic stem cell of claim 16, further comprising the following characteristic:
- capable of modulating hematopoietic stem cell activity.

19. The isolated hematopoietic stem cell of claim 18, wherein the activity is selected from the group consisting of hematopoietic stem cell differentiation and hematopoietic stem cell replication.

20. The protein of claim 17, wherein the protein is selected from the group consisting of a growth factor, a transcription factor, a splicing factor, a capping factor, a transport protein, a translation factor, and a replication factor.

21. The protein of claim 17, the protein having substantially the same amino acid sequence as one selected from the group consisting of SEQ.ID.No.: 42, SEQ.ID.No.:

44, SEQ.ID.No.: 46, SEQ.ID.No.: 48, SEQ.ID.No.: 50, SEQ.ID.No.: 52, SEQ.ID.No.:
54, SEQ.ID.No.: 56, SEQ.ID.No.: 58, SEQ.ID.No.: 60, SEQ.ID.No.: 62, SEQ.ID.No.:
64, SEQ.ID.No.: 66, SEQ.ID.No.: 68, SEQ.ID.No.: 70, SEQ.ID.No.: 71, and SEQ.ID.No.: 73.

22. A nucleic acid probe capable of specifically hybridizing with the nucleic acid of claim 1 under standard conditions.

23. An antibody capable of specifically binding to the protein of claim 17 without substantially cross-reacting with a non-stem cell specific protein or homologs thereof under conditions permissive to antibody binding.

24. A cell capable of producing the antibody of claim 23.

25. A method for identifying the presence of a primitive hemopoietic stem cell in a sample comprising:
(a) contacting the sample with the antibody of claim 23 under conditions permissive to the formation of an antibody complex; and (b) detecting the presence of the complex formed in step (a), the presence of a complex formed indicating the presence of a primitive hemopoietic stem cell in the sample.

26. The method of claim 25, wherein the antibody is labeled with a detectable marker.

27. The method of claim 26, wherein the detectable marker is selected from the group consisting of a radioactive isotope, enzyme, magnetic bead, dye, fluorescent marker and biotin.

28. A method for generating a stem cell/progenitor cell from a primitive hematopoietic cell in a sample comprising contacting the sample with the protein of claim 17.

29. A method for identifying the presence in a sample of a compound that modulates hematopoietic stem cell activity comprising:
(a) contacting the hematopoietic stem cell with the sample;
(b) determining the hematopoietic stem cell activity;
(c) comparing the hematopoietic stem cell activity determined in step (b) with the activity determined in the absence of the compound an increase or decrease in hematopoietic stem cell activity indicating the presence in the sample of a compound that modulates hematopoietic stem cell activity.

30. The method of claim 29, wherein the activity is selected from the group consisting of gene expression, replication, differentiation, transplantation, and self regeneration.

31. A compound identified by the method of claim 29, previously unknown.

32. A method for identifying primitive hematopoietic stem cell-specific nucleic acids, comprising:
(a) creating a primitive hematopoietic stem cell cDNA library and a non-primitive stem cell immune cell cDNA library; and (b) subtracting the two libraries, thereby identifying primitive stem cell specific nucleic acids.

33. The method of claim 32, step (b) comprising:
(i) contacting the nucleic acids of the stem cell and non-stem cell libraries with each other under conditions permissive to hybridization, thereby forming hybrid complexes;
(ii) separating the hybrid complexes formed in step (b) from the nucleic acids which did not form complexes;
(iii) isolating the nucleic acids which did not form complexes, thereby identifying hematopoietic stem cell specific nucleic acids.

34. The method of claim 33, step (ii) further comprising amplification of the nucleic acids.

35. The method of claim 33, step (iii) further comprising ampliciation of the nucleic acids which did not form complexes.

36. The method of claim 33, further comprising displaying the amplified DNA on a chromatography gel.

37. The method of claim 32, step (b) comprising differential display of the two libraries, thereby identifying primitive stem cell specific nucleic acids.

38. The method of claim 32, step (b) comprising representation difference analysis of the two libraries, thereby identifying primitive stem cell specific nucleic acids.

39. The method of claim 32 further comprising cloning the stem cell specific nucleic acid.

40. The method of claim 32, wherein the stem cell is selected from the group consisting of AF024, 2012, and 2018.

41. A nucleic acid identified by the method of claim 32

42. A composition comprising the compound of claim 31 and a carrier.

43. A pharmaceutical composition comprising the compound of claim 31 and a pharmaceutically acceptable earner.

44. A primitive hematopoietic stem cell specifically expressing one selected from the group consisting of: SEQ.ID.No.: 1, SEQ.ID.No.: 2, SEQ.ID.No.: 3, SEQ.ID.No.:
4, SEQ.ID.No.: 5, SEQ.ID.No.: 6, SEQ.ID.No.: 7, SEQ.ID.No.: 8, SEQ.ID.No.: 9, SEQ.ID.No.: 10, SEQ.ID.No.: 11, SEQ.ID.No.: 12, SEQ.ID.No.: 13, SEQ.ID.No.:
14, SEQ.ID.No.: 15, SEQ.ID.No.: 16, SEQ.ID.No.: 17, SEQ.ID.No.: 18, SEQ.ID.No.:
19, SEQ.ID.No.: 20, SEQ.ID.No.: 21, SEQ.ID.No.: 22, SEQ.ID.No.: 23, SEQ.ID.No.:
24, SEQ.ID.No.: 25, SEQ.ID.No.: 26, SEQ.ID.No.: 27, SEQ.ID.No.: 28, SEQ.ID.No.:
29, SEQ.ID.No.: 30, SEQ.ID.No.: 31, SEQ.ID.No.: 32, SEQ.ID.No.: 33, SEQ.ID.No.:
34, SEQ.ID.No.: 35, SEQ.ID.No.: 3G, SEQ.ID.No.: 37, SEQ.ID.No.: 38, SEQ.ID.No.:
39, SEQ.ID.No.: 40, SEQ.ID.No.: 41, SEQ.ID.No.: 42, and SEQ.ID.No.: 43, SEQ.ID.No.: 45, SEQ.ID.No.: 47, SEQ.ID.No.: 49, SEQ.ID.No.: 51, SEQ.ID.No.:
53, SEQ.ID.No.: 55, SEQ.ID.No.: 57, SEQ.ID.No.: 59, SEQ.ID.No.: 61, SEQ.ID.No.:
63, SEQ.ID.No.: 65, SEQ.ID.No.: 67, SEQ.ID.No.: 72 or a functional portion thereof.

45. A primitive hematopoietic stem cell specifically expressing a nucleic acid identified by the method of claim 32.

46. A method for treating a condition in a subject comprising administering to the subject a therapeutically effective amount of the composition of claim 43.

47. The method of claim 46 wherein the condition is an immune system condition.

48. The method of claim 46 wherein the condition is leukemia.

49. A method of introducing an exogenous nucleic acid into a hematopoietic stem cell comprising contacting the stem cell with the composition of claim 42.

50. A method of ex vivo expansion of hematopoietic stem cells comprising contacting the cell with the composition of claim 42.