
GGGACCCCAATGACGAAGAGAGTATGAACAGCCCCTGCTGGCAAGTCAAGTGGCAACTCCGTCAG	(SEQ ID NO:6)

CTCGTTAGAAAGATGATTTTGAGAACCTCTGAGGAAACCATTTCTACAGTTCAAGAAAAGCAACA

AAATATTTCTCCCCTAGTGAGAGAAAGAGGTCCTCAGAGAGTAGCAGCTCACATAACTGGGACCA

GAGGAAGAAGCAACACATTGTCTTCTCCAAACTCCAAGAATGAAAAGGCTCTGGGCCGCAAAATA

AACTCCTGGGAATCATCAAGGAGTGGGCATTCATTCCTGAGCAACTTGCACTTGAGGAATGGTGA

ACTGGTCATCCATGAAAAAGGGTTTTACTACATCTATTCCCAAACATACTTTCGATTTCAGGAGG

A 60 bp oligonucleotide probe with the following sequence was employed in the screening: TGACGAAGAGAGTATGAACAGCCCCTGCTGGCAAGTCAAGTGGCAACTCCGTCAGCTCGT (SEQ ID NO:7)[0224]

Hybridization was conducted overnight at room temperature in buffer containing 20% formamide, 5× SSC, 10% dextran sulfate, 0.1% NaPiPO[0225]₄, 0.05M NaPO₄, 0.05 mg salmon sperm DNA, and 0.1% sodium dodecyl sulfate, followed by several washes at 42° C. in 5× SSC, and then in 2× SSC. Twelve positive clones were identified in the cDNA library, and the positive clones were rescreened by hybridization to a second 60 bp oligonucleotide probe (not overlapping the first probe) having the following sequence: GGTGAACTGGTCATCCATGAAAAAGGGTTTTACTACATCTATTCCCAAACATACTTTCGA (SEQ ID NO:8)

Hybridization was conducted as described above.[0226]

Four resulting positive clones were identified and amplified by polymerase chain reaction (PCR) using a primer based on the[0227]flanking 5′ vector sequence and adding an external ClaI restriction site and a primer based on the 3′ flanking vector sequence and adding an external HindIII restriction site. PCR products were gel purified and subcloned into pGEM-T (commercially available from Promega) by T-A ligation. Three independent clones from different PCRs were then subjected to dideoxy DNA sequencing. DNA sequence analysis of these clones demonstrated that they were essentially identical, with some length variation at their 5′ region.

The nucleotide sequence of the coding region of Apo-2 ligand is shown in FIG. 4. Sequencing of the downstream 3′ end region of one of the clones revealed a characteristic polyadenylation site (data not shown). The cDNA contained one long open reading frame with an initiation site assigned to the ATG codon at nucleotide positions 91-93. The surrounding sequence at this site is in reasonable agreement with the proposed consensus sequence for initiation sites [Kozak, supra]. The open reading frame ends at the termination codon TAA at nucleotide positions 934-936.[0228]

The predicted mature amino acid sequence of human Apo-2 ligand contains 281 amino acids, and has a calculated molecular weight of approximately 32.5 kDa and an isoelectric point of approximately 7.63. There is no apparent signal sequence at the N-terminus, although hydropathy analysis (data not shown) indicated the presence of a hydrophobic region between[0229]residues 15 and 40. The absence of a signal sequence and the presence of an internal hydrophobic region suggests that Apo-2 ligand is a type II transmembrane protein. The putative cytoplasmic, transmembrane and extracellular regions are 14, 26 and 241 amino acids long, respectively. The putative transmembrane region is underlined in FIG. 4. A potential N-linked glycosylation site is located atresidue 109 in the putative extracellular domain.

An alignment (using the Align™ computer program) of the amino acid sequence of the C-terminal region of Apo-2 ligand with other known members of the TNF cytokine family showed that, within the C-terminal region, Apo-2 ligand exhibits 23.2% identity to Apo-1 ligand. The alignment analysis showed a lesser degree of identity with other TNF family members: CD40L (20.8%), LT-α (20.2%), LT-β (19.6%), TNF-α (19.0%), CD30L and CD27L (15.5%), OX-40L (14.3%), and 4-1BBL (13.7%). In the TNF cytokine family, residues within regions which are predicted to form β strands, based on the crystal structures of TNF-α and LT-α [Eck et al.,[0230]J. Bio. Chem.,264:17595-17605 (1989); Eck et al.,J. Bio. Chem.,267:2119-2122 (1992)], tend to be more highly conserved with other TNF family members than are residues in the predicted connecting loops. It was found that Apo-2 ligand exhibits greater homology to other TNF family members in its putative β strand regions, as compared to homology in the predicted connecting loops. Also, the loop connecting putative β strands, B and B′, is markedly longer in Apo-2 ligand.

Example 5

Expression of Human Apo-2 Ligand (ECD)[0231]

A soluble Apo-2 ligand extracellular domain (“ECD”) fusion construct was prepared, in which another sequence was fused upstream of the C-terminal region of Apo-2 ligand.[0232]

A Met Gly His[0233]₁₀sequence (derived from the plasmid pET19B, Novagen), followed by a 12 amino acid enterokinase cleavage site Met Gly His His His His His His His His His His Ser Ser Gly His Ile Asp Asp Asp Asp Lys His Met (SEQ ID NO:9)

was fused upstream to codons 114-281 of Apo-2 ligand within a baculovirus expression plasmid (pVL1392, Pharmingen). Briefly, the Apo-2 ligand codon 114-281 region was amplified by PCR from the parent pRK5 Apo-2 ligand plasmid with primers complementary to the 5′ and 3′ regions which incorporate flanking NdeI and BamHI restriction sites respectively. The product was subcloned into pGEM-T (Promega) by T-A ligation, and the DNA sequence was confirmed. The insert was then excised by digestion with NdeI and BamHI and subcloned into a modified baculovirus expression vector pVL1392 (commercially available from Pharmingen) containing an amino terminal Met Gly His[0234]₁₀tag and enterokinase cleavage site.

Recombinant baculovirus was generated by co-transfecting the His[0235]₁₀-Apo-2 ECD plasmid and BaculoGold™ virus DNA (Pharmingen) intoSpodoptera frugiperda(“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28° C., the released viruses were harvested and used for further amplifications. Viral infection and protein expression was performed as described by O'Reilley et al., Baculovirus expression vectors: A laboratory Manual, Oxford:Oxford University Press (1994). The protein was purified by Ni²⁺-chelate affinity chromatography, as described in Example 6 below.

Example 6

Purification of Recombinant Human Apo-2 Ligand (ECD)[0236]

Extracts were prepared from recombinant virus-infected and mock-infected Sf9 cells (see Example 5) as described by Rupert et al.,[0237]Nature,362:175-179 (1993). Briefly, Sf9 cells were washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates were cleared by centrifugation, and the supernatant was diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTA agarose column (commercially available from Qiagen) was prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract was loaded onto the column at 0.5 mL per minute. The column was washed to baseline A₂₈₀with loading buffer, at which point fraction collection was started. Next, the column was washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH 6.0), which eluted nonspecifically bound protein. After reaching A280 baseline again, the column was developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions were collected and analyzed by SDS-PAGE and silver staining or western blot with Ni²-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His₁₀-Apo-2 ligand protein were pooled and dialyzed against loading buffer.

An identical procedure was repeated with mock-infected Sf9 cells as the starting material, and the same fractions were pooled, dialyzed, and used as control for the purified human Apo-2 ligand.[0238]

SDS-PAGE analysis of the purified protein revealed a predominant band of Mr 24 kDa, corresponding with the calculated molecular weight of 22.4 kDa for the His[0239]₁₀-Apo-2 ligand monomer (FIG. 5, lane 3); protein sequence microanalysis (data not shown) confirmed that the 24 kDa band represents the His₁₀-Apo-2 ligand polypeptide. Minor 48 kDa and 66 kDa bands were also observed, and probably represent soluble Apo-2 ligand homodimers and homotrimers. Chemical crosslinking of the purified His₁₀-Apo-2 ligand by incubation with sulfo-NHS (5 mM) (Pierce Chemical) and EDC (Pierce Chemical) at 25 mM and 50 mM (FIG. 5,

lanes

1 and 2, respectively), shifted the protein into the 66 kDa band primarily. These results suggest that the predominant form of Apo-2 ligand in solution is homotrimeric and that these trimers dissociate into dimers and monomers in the presence of SDS.

Example 7

Apoptotic Activity of Apo-2 Ligand on Human Lymphoid Cell Lines[0240]

Apoptotic activity of purified, soluble Apo-2 ligand (described in Example 6) was examined using several human lymphoid cell lines. In a first study, the effect of Apo-2 ligand on 9D cells (Genentech, Inc.), derived from Epstein-Barr virus (EBV)-transformed human peripheral blood B cells, was examined. The 9D cells (5×10[0241]⁴cells/well in RPMI 1640 medium plus 10% fetal calf serum) were incubated for 24 hours with either a media control, Apo-2 ligand (3 μg/ml, prepared as described in Example 6 above), or anti-Apo-1 monoclonal antibody, CH11 (1 μg/ml) [described by Yonehara et al., supra; commercially available from Medical and Biological Laboratories Co.]. The CH11 anti-Apo-1 antibody is an agonistic antibody which mimicks Fas/Apo-1 ligand activity.

After the incubation, the cells were collected onto cytospin glass slides, and photographed under an inverted light microscope. Both Apo-2 ligand and the anti-Apo-1 monoclonal antibody induced a similar apoptotic effect, characterized by cytoplasmic condensation and reduction in cell numbers. (see FIG. 6A).[0242]

The effects of the Apo-2 ligand on the 9D cells, as well as on Raji cells (human Burkitt's lymphoma B cell line, ATCC CCL 86) and Jurkat cells (human acute T cell leukemia cell line, ATCC TIB 152) were further analyzed by FACS. The FACS analysis was conducted, using established criteria for apoptotic cell death, namely, the relation of fluorescence staining of the cells with two markers: (a) propidium iodide (“PI”) dye, which stains apoptotic but not live cells, and (b) a fluorescent derivative of the protein, annexin V, which binds to the exposed phosphatidylserine found on the surface of apoptotic cells, but not on live cells [Darzynkiewicz et al.,[0243]Methods in Cell Biol.,41:15-38 (1994); Fadok et al.,J. Immunol.,148:2207-2214 (1992); Koopman et al.,Blood,84:1415-1420 (1994)].

The 9D cells (FIG. 6B), Raji cells (FIG. 6C), and Jurkat cells (FIG. 6D) were incubated (1×10[0244]⁶cells/well) for 24 hours with a media control (left panels), Apo-2 ligand (3 μg/ml, prepared as described in Example 6) (center panels), or anti-Apo-1 ligand antibody, CH11 (1 μg/ml) (right panels). The cells were then washed, stained with PI and with fluorescein thiocyanate (FITC)-conjugated annexin V (purchased from Brand Applications) and analyzed by flow cytometry. Cells negative for both PI and annexin V staining (quadrant 3) represent live cells; PI-negative, annexin V-positive staining cells (quadrant 4) represent early apoptotic cells; PI-positive, annexin V-positive staining cells (quadrant 2) represent primarily cells in late stages of apoptosis.

The Apo-2 ligand treated 9D cells exhibited elevated extracellular annexin V binding, as well as a marked increase in uptake of PI (FIG. 6B), indicating that Apo-2 ligand induced apoptosis in the cells. Comparable results were obtained with anti-Apo-1 antibody, CH11 (FIG. 6B). The Apo-2 ligand induced a similar response in the Raji and Jurkat cells, as did the anti-Apo-1 antibody. (see FIGS. 6C and 6D). The induction of apoptosis (measured as the % apoptotic cells) in these cell lines by Apo-2 ligand, as compared to the control and to the anti-Apo-1 antibody, is also shown in Table 1 below.[0245]

TABLE 1


		% apoptotic cells
Cell line	Control	Apo-2L	Anti-Apo-1Ab

Lymphoid


9D	22.5	92.4	90.8
Raji	35.9	73.4	83.7
Jurkat	5.9	77.0	18.1

The activation of internucleosomal DNA fragmentation by Apo-2 ligand was also analyzed. Jurkat cells (left lanes) and 9D cells (right lanes) were incubated (2×10[0246]⁶cells/well) for 6 hours with a media control or Apo-2 ligand (3 μg/ml, prepared as described in Example 6), The DNA was then extracted from the cells and labeled with³²P-ddATP using terminal transferase. The labeled DNA samples were subjected to electrophoresis on 2% agarose gels and later analyzed by autoradiography [Moore et al.,Cytotechnology,17:1-11 (1995)]. The Apo-2 ligand induced internucleosomal DNA fragmentation in both the Jurkat cells and 9D cells (FIG. 6E). Such DNA fragmentation is characteristic of apoptosis [Cohen,Advances in Immunol.,50:55-85 (1991)].

To examine the time-course of the Apo-2 ligand apoptotic activity, 9D cells were incubated in microtiter dishes (5×10[0247]⁴cells/well) with a media control or Apo-2 ligand (3 μg/ml, prepared as described in Example 6) for a period of time ranging from 0 hours to 50 hours. Following the incubation, the numbers of dead and live cells were determined by microscopic examination using a hemocytometer.

As shown in FIG. 7A, maximal levels of cell death were induced in 9D cells within 24 hours.[0248]

To determine dose-dependency of Apo-2 ligand-induced cell death, 9D cells were incubated (5×10[0249]⁴cells/well) for 24 hours with serial dilutions of a media control or Apo-2 ligand (prepared as described in Example 6). The numbers of dead and live cells following the incubation were determined as described above. The results are illustrated in FIG. 7B. Specific apoptosis was determined by subtracting the % apoptosis in the control from % apoptosis in Apo-2 ligand treated cells. Half-maximal activation of apoptosis occurred at approximately 0.1 μg/ml (approximately 1 nM), and maximal induction occurred at about 1 to about 3 μg/ml (approximately 10 to 30 nM).

Example 8

Inhibition Assay Using Fas/Apo-1 and TNF Receptors[0250]

An assay was conducted to determine if the Fas/Apo-1 receptor, as well as the[0251]type 1 andtype 2 TNF receptors (TNFR1 and TNFR2), are involved in mediating the apoptotic activity of Apo-2 ligand by testing if soluble forms of these receptors are capable of inhibiting the apoptotic activity of purified, soluble Apo-2 ligand (described in Example 6). 9D cells were incubated (5×10⁴cells/well) for 24 hours with a media control or Apo-2 ligand (0.3 μg/ml, prepared as described in Example 6) in the presence of buffer control, CD4-IgG control (25 μg/ml), soluble Apo-l-IgG (25 μg/ml), soluble TNFR1-IgG (25 μg/ml) or soluble TNFR2-IgG fusion protein (25 μg/ml). Soluble derivatives of the Fas/Apo-1, TNFR1 and TNFR2 receptors were produced as IgG fusion proteins as described in Ashkenazi et al.,Methods,8:104-115 (1995). CD4-IgG was produced as an IgG fusion protein as described in Byrn et al.,Nature,344:667-670 (1990) and used as a control.

As shown in FIG. 7C, none of the receptor-fusion molecules inhibited Apo-2 ligand apoptotic activity on the 9D cells. These results indicate that Apo-2 ligand apoptotic activity is independent of Fas/Apo-1 and of TNFR1 and TNFR2.[0252]

Example 9

Isolation of cDNA clones Encoding Human Apo-3[0253]

Human fetal heart and human fetal lung 1gt10 bacteriophage cDNA libraries (both purchased from Clontech) were screened by hybridization with synthetic oligonucleotide probes based on an EST (Genbank locus W71984), which showed some degree of homology to the intracellular domain (ICD) of human TNFR1 and CD95. W71984 is a 523 bp EST, which in its −1 reading frame has 27 identities to a 43 amino acid long sequence in the ICD of human TNFR1. The oligonucleotide probes used in the screening were 27 and 25 bp long, respectively, with the following sequences: GGCGCTCTGGTGGCCCTTGCAGAAGCC [SEQ ID NO:12] and TTCGGCCGAGAAGTTGAGAAATGTC [SEQ ID NO:13].[0254]

Hybridization was done with a 1:1 mixture of the two probes overnight at room temperature in buffer containing 20% formamide, 5× SSC, 10% dextran sulfate, 0.1% NaPiPO[0255]₄, 0.05 M NaPO₄, 0.05 mg salmon sperm DNA, and 0.1% sodium dodecyl sulfate (SDS), followed consecutively by one. wash at room temperature in 6× SSC, two washes at 37° C. in 1× SSC/0.1% SDS, two washes at 37° C. in 0.5× SSC/0.1% SDS, and two washes at 37° C. in 0.2× SSC/0.1% SDS. One positive clone from each of the fetal heart (FH20A.57) and fetal lung (FL8A.53) libraries were confirmed to be specific by PCR using the respective above hybridization probes as primers. Single phage plaques containing each of the positive clones were isolated by limiting dilution and the DNA was purified using a Wizard lambda prep DNA purification kit (Promega).

The cDNA inserts were excised from the lambda vector arms by digestion with EcoRI, gel-purified, and subcloned into pRK5 that was predigested with EcoRI. The clones were then sequenced in entirety.[0256]

Clone FH20A.57 (also referred to as[0257]Apo 3 clone FH20.57 deposited as ATCC 55820, as indicated below) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 89-91 and ending at the stop codon found at nucleotide positions 1340-1342 (FIG. 8; SEQ ID NO:11) [Kozak et al., supra]. The cDNA clone also contains a polyadenylation sequence at its 3′ end. The predicted polypeptide precursor is 417 amino acids long and has a calculated molecular weight of approximately 45 kDa and a PI of about 6.4. Hydropathy analysis (not shown) suggested the presence of a signal sequence (residues 1-24), followed by an extracellular domain (residues 25-198), a transmembrane domain (residues 199-224), and an intracellular domain (residues 225-417) (FIG. 8; SEQ ID NO:10). There are two potential N-linked glycosylation sites at amino acid positions 67 and 106.

The ECD contains 4 cysteine-rich repeats which resemble the corresponding regions of human TNFR1 (4 repeats), of human CD95 (3 repeats) (FIG. 9) and of the other known TNFR family members (not shown). The ICD contains a death domain sequence that resembles the death domains found in the ICD of TNFR1 and CD95 and in cytoplasmic death signalling proteins such as human FADD/MORT1, TRADD, RIP, and Drosophila Reaper (FIG. 10). Both globally and in individual regions, Apo-3 is related more closely to TNFR1 than to CD95; the respective amino acid identities are 29.3% and 22.8% overall, 28.2% and 24.7% in the ECD, 31.6% and 18.3% in the ICD, and 47.5% and 20% in the death domain.[0258]

The fetal lung cDNA clone, clone 5L8A.53, was identical to the fetal heart clone, with the following two exceptions: (1) it is 172 bp shorter at the 5′ region; and (2) it lacks the Ala residue at position 236, possibly due to differential mRNA splicing via two consecutive splice acceptor consensus sites (FIG. 10).[0259]

As mentioned in Example 1 above,[0260]amino acids 1 to 181 of the Apo-2LI clone 18.1 shown in FIG. 1 (SEQ ID NO:1) are identical to theamino acids 1 to 181 of the Apo-3 polypeptide, shown in FIG. 8 (SEQ ID NO:10).

Example 10

Expression of Apo-3[0261]

A pRK5 mammalian expression plasmid (described in Example 2) carrying clone FH20A.57 (referred to in Example 9) was transfected transiently into HEK293 cells (referred to in the Examples above) by calcium phosphate precipitation and into HeLa-S3 cells (ATCC No. CCL 2.2) by standard electroporation techniques.[0262]

Lysates of metabolically labeled transfected 293 cells were analyzed by immunoprecipitation with a mouse antiserum raised against an Apo-2LI-IgG fusion protein. Transfected cells (5×10[0263]⁵per lane) were labeled metabolically by addition of 50 μCi³⁵S-Met and³⁵S-Cys to the growth media 24 hours after transfection. After a 6 hour incubation, the cells were washed several times with PBS, lysed and subjected to immunoprecipitation by anti-Apo-3 antiserum as described in Marsters et al.,Proc. Natl. Acad. Sci.,92:5401-5405 (1995). The anti-Apo-3 antiserum was raised in mice against a fusion protein containing the Apo-2LI ECD (as described in Example 3).

A predominant radioactive band with a relative molecular weight of about 47 kDa was observed in the pRK5-Apo-3-transfected cells, but not in the cells transfected with pRK5 alone (control) (See FIG. 11,[0264]lanes 1, 2). Given the potential glycosylation sites of Apo-3, the observed size is consistent with the size of approximately 45 kDa predicted for the Apo-3 polypeptide precursor.

Example 11

Apoptotic Activity of Apo-3[0265]

The transiently transfected HEK293 and HeLa cells described in Example 10 were tested and analyzed for[0266]apoptotic activity 36 hours after transfection. Apoptosis was assessed morphologically or quantitated by FACS analysis of cells stained with fluoresceinisothiocyanate (FITC)-conjugated annexin V (Brand Applications) and propidium iodide (PI), as described in Example 7. The annexin V-positive/PI negative cells are in early stages of apoptosis and double-positive cells are in late apoptosis, while annexin V-negative/PI-positive cells are necrotic. Apoptosis was also assessed by DNA fragmentation testing.

Microscopic examination of the HEK 293 cells transfected with the pRK5-Apo-3 expression plasmid (see Example 10) showed a substantial loss of cell viability as compared to control cells transfected with pRK5 alone; many of the Apo-3 transfected cells exhibited a characteristic apoptotic morphology of membrane blebbing and loss of cell volume (FIGS. 12[0267]aandb), suggesting cell death by apoptosis [Cohen,Advances in Immunology,50:55-85 (1990)].

The FACS analysis also revealed that the Apo-3-transfected cells died by apoptosis, by virtue of the presence of exposed phosphatidylserine on their surface (FIGS. 12[0268]e-i). It was found that the transient transfection efficiency of the HEK 293 cells was 60-70%; therefore, to target FACS analysis to cells that had taken up the plasmid DNA, the 293 cells were co-transfected with a pRK5-CD4 expression vector (3 μg) as a marker and gated on CD4-positive cells (using phycoerythrin-conjugated anti-CD4 antibody) for analysis. For the co-transfection, the total amount of plasmid DNA was kept constant, but divided between different plasmids. The Apo-3-transfected cells showed a marked increase in PI and annexin V-FITC staining as compared to pRK5-transfected control cells indicating induction of apoptosis by Apo-3. (FIGS. 12eandf).

The effect of the dose of plasmid on apoptosis was also tested in the FACS assay. (FIG. 12[0269]i). Transfection of 293 cells with either Apo-3 or TNFR1 expression plasmids was associated with a dose-dependent increase in apoptosis; the effect of Apo-3 was more pronounced than that of TNFR1 (FIG. 12i). Similar results were obtained upon Apo-3 transfection of the HeLa cells (data not shown).

Apoptosis was also assayed by extraction of DNA from the cells, terminal transferase-mediated[0270]³²P-labelling of 3′ ends of DNA and 1.5% agarose gel electrophoresis as described by Moore et al.,Cytotechnology,17:1-11 (1995). Analysis of the cellular DNA revealed that the Apo-3-transfected cells showed a marked increase in DNA fragmentation as compared to controls (FIG. 12j,lanes 1, 2). The fragmented DNA migrated on agarose gels as a ladder of bands, indicating internucleosomal DNA cleavage, an indication of programmed cell death [Cohen, supra].

Example 12

Inhibition Assay Using CrmA[0271]

To investigate whether proteases such as ICE and CPP32/Yama play a role in apoptosis-induction by Apo-3, an assay was conducted to determine if CrmA inhibits Apo-3 function.[0272]

Co-transfection of HEK293 cells by a pRK5-CrmA expression plasmid (CrmA sequence reported in Ray et al., supra) and pRK5-Apo-3 did not affect the apparent levels of Apo-3 expressed by the cells (FIG. 11, lane 3) CrmA, however, blocked Apo-3 associated apoptosis as analyzed by morphological examination (FIGS. 12[0273]candd), FACS (FIGS. 12gandh) and DNA fragmentation (FIG. 12j,lanes 3,4) methods described in Example 11. A similar inhibitory effect of CrmA was observed in Apo-3-transfected HeLa cells (data not shown).

CrmA, a poxvirus-derived inhibitor of the death proteases ICE and CPP32/Yama, blocks death signalling by TNFR1 and CD95. Accordingly, the assay results suggest that Apo-3, TNFR1 and CD95 engage a common signalling pathway to activate apoptotic cell death. In particular, the results suggest that proteases such as ICE and CPP32/Yama may be required for Apo-3 induced apoptosis.[0274]

Example 13

Activation of NF-κB by Apo-3[0275]

An assay was conducted to determine whether Apo-3 activates NF-κB.[0276]

HEK 293 cells were harvested 36 hours after transfection (see Example 10) and nuclear extracts were prepared and 1 μg of nuclear protein was reacted with a[0277]³²P-labelled NF-κB-specific synthetic oligonucleotide probe ATCAGGGACTTTCCGCTGGGGACTTTCCG (SEQ ID NO:14) [see, also, MacKay et al.,J. Immunol.,153:5274-5284 (1994)], alone or together with a 50-fold excess of unlabelled probe, or with an irrelevant³²P-labelled synthetic oligonucleotide AGGATGGGAAGTGTGTGATATATCCTTGAT (SEQ ID NO:15). DNA binding was analyzed by an electrophoretic mobility shift assay as described by Hsu et al., supra; Marsters et al., supra, and MacKay et al., supra.

The results are shown in FIG. 13. The radioactive band at the bottom of the gel in all lanes is the free labelled probe, the two other radioactive bands seen in lanes 1-3 represent non-specific interaction, as does the band common to lanes 1-3 and lanes 4-6. The top radioactive band in lanes 4-6 represents the labelled NF-κB probe, whose migration is delayed by specific interaction with activated NF-κB protein in the nuclear extracts.[0278]

Apo-3 transfected cells showed a significant increase in NF-κB-specific DNA binding activity relative to pRK5-transfected controls. TNFR1-transfected cells showed NF-κB activation as well; this activation appeared to be enhanced as compared to the Apo-3-transfected cells. The data thus shows that Apo-3 is capable of regulating transcription of inflammatory response genes and in particular, may be linked to a NF-κB activation pathway.[0279]

Example 14

Northern Blot Analysis[0280]

Expression of Apo-3 mRNA in human tissues was examined by Northern blot analysis. Human RNA blots were hybridized to a 206 bp[0281]³²P-labelled DNA probe based on the 3′ untranslated region of Apo-3; the probe was generated by PCR with the 27 and 25 bp probes (described in Example 9) as PCR primers. Human fetal RNA blot MTN (Clontech) and human adult RNA blot MTN-II (Clontech) were incubated with the DNA probes. Blots were incubated with the probes in hybridization buffer (5× SSPE; 2× Denhardt's solution; 100 mg/mL denatured sheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hours at 42° C. The blots were washed several times in 2× SSC; 0.05% SDS for 1 hour at room temperature, followed by a 30 minute wash in 0.1× SSC; 0.1 SDS at 50° C. The blots were developed after overnight exposure.

As shown in FIG. 14, a predominant mRNA transcript of approximately 4 kb was detected in adult spleen, thymus, and peripheral blood lymphocytes, and less abundantly in small intestine, colon, fetal lung, and fetal kidney. Additional transcripts of approximately 7 and 9 kb were seen mainly in fetal brain, lung and kidney, and in adult spleen and ovary. These results suggest that Apo-3 mRNA is expressed in several types of tissues, including both lymphoid and non-lymphoid tissues.[0282]

Example 15

Chromosomal Localization of the Apo-3 Gene[0283]

Chromosomal localization of the Apo-3 gene was examined by fluorescence in situ hybridization (“FISH”) to normal human lymphocyte chromosomes.[0284]

Initial testing by direct hybridization with the Apo-2LI (clone 18.1) cDNA (see Example 1 and FIG. 1) as a probe gave a relatively poor signal to background ratio (data not shown) but suggested that the gene is located on chromosome 1p36. Further testing was conducted using the Apo-3 cDNA probe and FISH mapping [as described by Lichter et al.,[0285]Science,247:64-69 (1990)] of a human genomic p1-derived artificial chromosome (PAC) library (obtained from Dr. L. C. Tsui, University of Toronto, Toronto, Canada). The Apo-3 probes were biotinylated and detected with avidin-FITC. The normal human lymphocyte chromosomes were counterstained with PI and DAPI [Heng and Tsui,Chromosome,102:325-332 (1993)]. In addition to the “direct” FISH using the Apo-3 cDNA as a probe, the probe was used to identify clones in the genomic PAC library that contain the Apo-3 gene, and the PACs were used as confirmatory probes in FISH. The regional assignment of the genomic probe was determined by the analysis of 20 well-spread metaphases.

A positive PAC clone was mapped by FISH to the short arm of[0286]chromosome 1, at position 1p36.3. A second Apo-3-positive genomic PAC was mapped to the same position (data not shown). Positive hybridization signals at 1p36.3 were noted at >95% of the cells. Signals were seen in bothchromosome 1 homologues in >90% of the positive spreads.

Recent reports disclose that a genomic region which is deleted in certain human neuroblastomas maps within 1p36.2-1p36.3, indicating that a tumor suppressor gene may be present at this locus. Four additional TNFR gene family members, TNFR2, CD30, 4.1BB and OX40, reside in 1p36 [see Gruss and Dower, supra] but are outside the deleted region [White et al.,[0287]Proc. Natl. Acad. Sci.,92:5520-5524 (1995)].

Deposit of Material[0288]

The following materials have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., USA (ATCC):[0289]



Material	ATCC Dep. No.	Deposit Date

2935-pRK5-hApo-2L-	CRL-12014	Jan. 3, 1996
myc clone 2.1
Apo-2LI clone 18.1	97493	Mar. 27, 1996
Apo-3 clone FH20.57	55820	Sept. 5, 1996

This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).[0290]

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.[0291]

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.[0292]

1 Met Glu Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu 1 5 10 15 Leu Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser 20 25 30 Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu 35 40 45 Phe Cys Cys Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro 50 55 60 Cys Thr Glu Pro Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln 65 70 75 Asp Thr Phe Leu Ala Trp Glu Asn His His Asn Ser Glu Cys Ala 80 85 90 Arg Cys Gln Ala Cys Asp Glu Gln Ala Ser Gln Val Ala Leu Glu 95 100 105 Asn Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro Gly 110 115 120 Trp Phe Val Glu Cys Gln Val Ser Gln Cys Val Ser Ser Ser Pro 125 130 135 Phe Tyr Cys Gln Pro Cys Leu Asp Cys Gly Ala Leu His Arg His 140 145 150 Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr Asp Cys Gly Thr Cys 155 160 165 Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys Val Ser Cys Pro 170 175 180 Thr 181

2 CTGCTGGGGG CCCGGGCCAG NGGCGGCACT CGTAGCCCCA GGTGTGACTG 50 TGCCGGTGAC TTCCACAAGA AGATTGGTCT GTTTTGTTGC AGAGGCTGCC 100 CAGCGGGGCA ACTACCTGAA GGCCCCTTGC ACGGAGCCCT GCGCAACTCC 150 ACCTGCCTTG TGTGTCCCCA AGACACCTTC TTGGCCTGGG AGAACCACCA 200 TAATTCTGAA TGTGCCCGCT GCCAGGCCTG TGATGAGCAG GCCTCCCAGG 250 TGGCGCTGGA GAACTGTTCA GCAGTGGCCG ACACCCGCTG TGGCTGTAAG 300 CAGGGCTGGT TTGTGGAGTG CCAGGGTCAG CCAATGTGTC AGCAGTTTCA 350 CCCTTCTAAT GCCAACCATG CCTAGACTGC GGGGCCCTGC AACGCAACAC 400 ACGGCTAATN TGTTTCCCGC AGAGATNATT GTT 433

3 CCCGCTGCCA GGCCTGTGAT GAGCAGGC 28

4 CAGGGCCCCG CAGTCTAGGC ATGGTTGG 28

5 GAATTCCGGC GCGGAGGCCG AGAGAGAAGT CACTTGCCCT GGCTCTACCT 50 TGAAGTGGTT CTCAGGGTTG GGGCGAGAGT CGGGGTGGGG ACCGAGATGC 100 AGCTCTATCC TGTGCCCCTG GTCGCAGCAG GCAGCCCAGC GCTTCGCGTG 150 TTCTACTTGG CCTGTCCGCT GCCGCCTAAT GAGCTCAGGT CTAGGCCGAG 200 CAGAGGGGGC ACCTGGTCGG ACTCGGTTGG GCTCGGGCGG CCCCGCCTCC 250 CCCCGCCCGC CAGGCGGGCC CTTCTCGACG GCGCGGGGCG GGCCCTGCGG 300 GCGCGGGGCT GAAGGCGGAA CCACGACGGG CAGAGAGCAC GGAGCCGGGA 350 AGCCCCTGGG CGCCCGTCGG AGGGCTATGG AGCAGCGGCC GCGGGGCTGC 400 GCGGCGGTGG CGGCGGCGCT CCTCCTGGTG CTGCTGGGGG CCCGGGCCCA 450 GGGCGGCACT CGTAGCCCCA GGTGTGACTG TGCCGGTGAC TTCCACAAGA 500 AGATTGGTCT GTTTTGTTGC AGAGGCTGCC CAGCGGGGCA CTACCTGAAG 550 GCCCCTTGCA CGGAGCCCTG CGGCAACTCC ACCTGCCTTG TGTGTCCCCA 600 AGACACCTTC TTGGCCTGGG AGAACCACCA TAATTCTGAA TGTGCCCGCT 650 GCCAGGCCTG TGATGAGCAG GCCTCCCAGG TGGCGCTGGA GAACTGTTCA 700 GCAGTGGCCG ACACCCGCTG TGGCTGTAAG CCAGGCTGGT TTGTGGAGTG 750 CCAGGTCAGC CAATGTGTCA GCAGTTCACC CTTCTACTGC CAACCATGCC 800 TAGACTGCGG GGCCCTGCAC CGCCACACAC GGCTACTCTG TTCCCGCAGA 850 GATACTGACT GTGGGACCTG CCTGCCTGGC TTCTATGAAC ATGGCGATGG 900 CTGCGTGTCC TGCCCCACGT AATTCCTAGC TGTCGTGGGA TGGAGGGAAG 950 GGCGGCTGGG AGCAGAGCAG GGGCCTGGGG TGGGGCAGGT GCTGCTGGTT 1000 CAGGAATAGG AAGAGGGGAT AGGGAGGAGG GAGCCTTGGC CCTGTGATGG 1050 GTGGGCCCCA CTTCAGGCAA ACTTAGATGG CAAAAGAGCA ATCTGGATCC 1100 GCCTTAGCCA GATACATAAG GGTATTTGCC TTCACTTTCA GCCAGCATTC 1150 CCCCCAGCGA TCCTAGCCAG ATATTACAGA TGATTTGTCA CTTACACAGA 1200 GAGTCACATT GATATAGCTT TAAAACTTGG GCTGAAGGAG GTTGAGGCTG 1250 CAGTGAGCTA TGATCGTGCC ACTGCACTTC AGCCTGGGCA ACAGAGCGAG 1300 ACCTATTAAA TAAATAAATA AATATTAAAT CTATTAAATA TTAAATATTA 1350 AATCTATTAA ATAAATAAAT ACAAAGGGCT GAGAGTCAGG ACTGTGCTGC 1400 TAGTTCTCTA GGGGATCTTG GGCAAGTGCA GAGAATTC 1438

6 GGGACCCCAA TGACGAAGAG AGTATGAACA GCCCCTGCTG GCAAGTCAAG 50 TGGCAACTCC GTCAGCTCGT TAGAAAGATG ATTTTGAGAA CCTCTGAGGA 100 AACCATTTCT ACAGTTCAAG AAAAGCAACA AAATATTTCT CCCCTAGTGA 150 GAGAAAGAGG TCCTCAGAGA GTAGCAGCTC ACATAACTGG GACCAGAGGA 200 AGAAGCAACA CATTGTCTTC TCCAAACTCC AAGAATGAAA AGGCTCTGGG 250 CCGCAAAATA AACTCCTGGG AATCATCAAG GAGTGGGCAT TCATTCCTGA 300 GCAACTTGCA CTTGAGGAAT GGTGAACTGG TCATCCATGA AAAAGGGTTT 350 TACTACATCT ATTCCCAAAC ATACTTTCGA TTTCAGGAGG 390

7 TGACGAAGAG AGTATGAACA GCCCCTGCTG GCAAGTCAAG TGGCAACTCC 50 GTCAGCTCGT 60

8 GGTGAACTGG TCATCCATGA AAAAGGGTTT TACTACATCT ATTCCCAAAC 50 ATACTTTCGA 60

24 amino acids

Amino Acid

Linear

9 Met Gly His His His His His His His His His His Ser Ser Gly 1 5 10 15 His Ile Asp Asp Asp Asp Lys His Met 20 24

417 amino acids

Amino Acid

Linear

10 Met Glu Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu 1 5 10 15 Leu Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser 20 25 30 Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu 35 40 45 Phe Cys Cys Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro 50 55 60 Cys Thr Glu Pro Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln 65 70 75 Asp Thr Phe Leu Ala Trp Glu Asn His His Asn Ser Glu Cys Ala 80 85 90 Arg Cys Gln Ala Cys Asp Glu Gln Ala Ser Gln Val Ala Leu Glu 95 100 105 Asn Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro Gly 110 115 120 Trp Phe Val Glu Cys Gln Val Ser Gln Cys Val Ser Ser Ser Pro 125 130 135 Phe Tyr Cys Gln Pro Cys Leu Asp Cys Gly Ala Leu His Arg His 140 145 150 Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr Asp Cys Gly Thr Cys 155 160 165 Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys Val Ser Cys Pro 170 175 180 Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys Ala Ala Val Cys 185 190 195 Gly Trp Arg Gln Met Phe Trp Val Gln Val Leu Leu Ala Gly Leu 200 205 210 Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr Tyr Arg 215 220 225 His Cys Trp Pro His Lys Pro Leu Val Thr Ala Asp Glu Ala Gly 230 235 240 Met Glu Ala Leu Thr Pro Pro Pro Ala Thr His Leu Ser Pro Leu 245 250 255 Asp Ser Ala His Thr Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys 260 265 270 Ile Cys Thr Val Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr 275 280 285 Pro Glu Thr Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp 290 295 300 Asp Gln Leu Pro Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr 305 310 315 Leu Ser Pro Glu Ser Pro Ala Gly Ser Pro Ala Met Met Leu Gln 320 325 330 Pro Gly Pro Gln Leu Tyr Asp Val Met Asp Ala Val Pro Ala Arg 335 340 345 Arg Trp Lys Glu Phe Val Arg Thr Leu Gly Leu Arg Glu Ala Glu 350 355 360 Ile Glu Ala Val Glu Val Glu Ile Gly Arg Phe Arg Asp Gln Gln 365 370 375 Tyr Glu Met Leu Lys Arg Trp Arg Gln Gln Gln Pro Ala Gly Leu 380 385 390 Gly Ala Val Tyr Ala Ala Leu Glu Arg Met Gly Leu Asp Gly Cys 395 400 405 Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly Pro 410 415 417

11 CGGGCCCTGC GGGCGCGGGG CTGAAGGCGG AACCACGACG GGCAGAGAGC 50 ACGGAGCCGG GAAGCCCCTG GGCGCCCGTC GGAGGGCT ATG GAG 94 Met Glu 1 CAG CGG CCG CGG GGC TGC GCG GCG GTG GCG GCG GCG CTC 133 Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu 5 10 15 CTC CTG GTG CTG CTG GGG GCC CGG GCC CAG GGC GGC ACT 172 Leu Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr 20 25 CGT AGC CCC AGG TGT GAC TGT GCC GGT GAC TTC CAC AAG 211 Arg Ser Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys 30 35 40 AAG ATT GGT CTG TTT TGT TGC AGA GGC TGC CCA GCG GGG 250 Lys Ile Gly Leu Phe Cys Cys Arg Gly Cys Pro Ala Gly 45 50 CAC TAC CTG AAG GCC CCT TGC ACG GAG CCC TGC GGC AAC 289 His Tyr Leu Lys Ala Pro Cys Thr Glu Pro Cys Gly Asn 55 60 65 TCC ACC TGC CTT GTG TGT CCC CAA GAC ACC TTC TTG GCC 328 Ser Thr Cys Leu Val Cys Pro Gln Asp Thr Phe Leu Ala 70 75 80 TGG GAG AAC CAC CAT AAT TCT GAA TGT GCC CGC TGC CAG 367 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys Gln 85 90 GCC TGT GAT GAG CAG GCC TCC CAG GTG GCG CTG GAG AAC 406 Ala Cys Asp Glu Gln Ala Ser Gln Val Ala Leu Glu Asn 95 100 105 TGT TCA GCA GTG GCC GAC ACC CGC TGT GGC TGT AAG CCA 445 Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro 110 115 GGC TGG TTT GTG GAG TGC CAG GTC AGC CAA TGT GTC AGC 484 Gly Trp Phe Val Glu Cys Gln Val Ser Gln Cys Val Ser 120 125 130 AGT TCA CCC TTC TAC TGC CAA CCA TGC CTA GAC TGC GGG 523 Ser Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys Gly 135 140 145 GCC CTG CAC CGC CAC ACA CGG CTA CTC TGT TCC CGC AGA 562 Ala Leu His Arg His Thr Arg Leu Leu Cys Ser Arg Arg 150 155 GAT ACT GAC TGT GGG ACC TGC CTG CCT GGC TTC TAT GAA 601 Asp Thr Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu 160 165 170 CAT GGC GAT GGC TGC GTG TCC TGC CCC ACG AGC ACC CTG 640 His Gly Asp Gly Cys Val Ser Cys Pro Thr Ser Thr Leu 175 180 GGG AGC TGT CCA GAG CGC TGT GCC GCT GTC TGT GGC TGG 679 Gly Ser Cys Pro Glu Arg Cys Ala Ala Val Cys Gly Trp 185 190 195 AGG CAG ATG TTC TGG GTC CAG GTG CTC CTG GCT GGC CTT 718 Arg Gln Met Phe Trp Val Gln Val Leu Leu Ala Gly Leu 200 205 210 GTG GTC CCC CTC CTG CTT GGG GCC ACC CTG ACC TAC ACA 757 Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr 215 220 TAC CGC CAC TGC TGG CCT CAC AAG CCC CTG GTT ACT GCA 796 Tyr Arg His Cys Trp Pro His Lys Pro Leu Val Thr Ala 225 230 235 GAT GAA GCT GGG ATG GAG GCT CTG ACC CCA CCA CCG GCC 835 Asp Glu Ala Gly Met Glu Ala Leu Thr Pro Pro Pro Ala 240 245 ACC CAT CTG TCA CCC TTG GAC AGC GCC CAC ACC CTT CTA 874 Thr His Leu Ser Pro Leu Asp Ser Ala His Thr Leu Leu 250 255 260 GCA CCT CCT GAC AGC AGT GAG AAG ATC TGC ACC GTC CAG 913 Ala Pro Pro Asp Ser Ser Glu Lys Ile Cys Thr Val Gln 265 270 275 TTG GTG GGT AAC AGC TGG ACC CCT GGC TAC CCC GAG ACC 952 Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr Pro Glu Thr 280 285 CAG GAG GCG CTC TGC CCG CAG GTG ACA TGG TCC TGG GAC 991 Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp Asp 290 295 300 CAG TTG CCC AGC AGA GCT CTT GGC CCC GCT GCT GCG CCC 1030 Gln Leu Pro Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro 305 310 ACA CTC TCG CCA GAG TCC CCA GCC GGC TCG CCA GCC ATG 1069 Thr Leu Ser Pro Glu Ser Pro Ala Gly Ser Pro Ala Met 315 320 325 ATG CTG CAG CCG GGC CCG CAG CTC TAC GAC GTG ATG GAC 1108 Met Leu Gln Pro Gly Pro Gln Leu Tyr Asp Val Met Asp 330 335 340 GCG GTC CCA GCG CGG CGC TGG AAG GAG TTC GTG CGC ACG 1147 Ala Val Pro Ala Arg Arg Trp Lys Glu Phe Val Arg Thr 345 350 CTG GGG CTG CGC GAG GCA GAG ATC GAA GCC GTG GAG GTG 1186 Leu Gly Leu Arg Glu Ala Glu Ile Glu Ala Val Glu Val 355 360 365 GAG ATC GGC CGC TTC CGA GAC CAG CAG TAC GAG ATG CTC 1225 Glu Ile Gly Arg Phe Arg Asp Gln Gln Tyr Glu Met Leu 370 375 AAG CGC TGG CGC CAG CAG CAG CCC GCG GGC CTC GGA GCC 1264 Lys Arg Trp Arg Gln Gln Gln Pro Ala Gly Leu Gly Ala 380 385 390 GTT TAC GCG GCC CTG GAG CGC ATG GGG CTG GAC GGC TGC 1303 Val Tyr Ala Ala Leu Glu Arg Met Gly Leu Asp Gly Cys 395 400 405 GTG GAA GAC TTG CGC AGC CGC CTG CAG CGC GGC CCG T 1340 Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly Pro 410 415 417 GACACGGCGC CCACTTGCCA CCTAGGCGCT CTGGTGGCCC TTGCAGAAGC 1390 CCTAAGTACG GTTACTTATG CGTGTAGACA TTTTATGTCA CTTATTAAGC 1440 CGCTGGCACG GCCCTGCGTA GCAGCACCAG CCGGCCCCAC CCCTGCTCGC 1490 CCCTATCGCT CCAGCCAAGG CGAAGAAGCA CGAACGAATG TCGAGAGGGG 1540 GTGAAGACAT TTCTCAACTT CTCGGCCGGA GTTTGGCTGA GATCGCGGTA 1590 TTAAATCTGT GAAAGAAAAC AAAAAAAAAA AAAAAAAAAA AAAA 1634

12 GGCGCTCTGG TGGCCCTTGC AGAAGCC 27

13 TTCGGCCGAG AAGTTGAGAA ATGTC 25

14 ATCAGGGACT TTCCGCTGGG GACTTTCCG 29

15 AGGATGGGAA GTGTGTGATA TATCCTTGAT 30