Bioactive fusion proteins comprising the p35 and p40 subunits of IL-12

Fusion proteins, such as a bio active IL-12 polypeptide, which comprise at least two polypeptide monomers (chains of amino acids) joined through a heterologous polypeptide linker and which are bioactive, as well as to their production.

BACKGROUND OF THE INVENTION 
Production of therapeutic proteins, such as those which are dimeric, is 
often difficult, inefficient and expensive. Production of a dimer may 
require separate expression of the two components, followed by joining of 
those components to form a functional dimer. Alternative methods of 
producing functional dimeric proteins would be useful. 
SUMMARY OF THE INVENTION 
The present invention relates to fusion proteins which comprise at least 
two polypeptide monomers (chains of amino acids) joined through a 
polypeptide linker and which are bioactive, as well as to their 
production. In one embodiment of the present invention, the bioactive 
fusion proteins of the present invention comprise two or more polypeptides 
which occur as subunits or monomers in a corresponding bioactive native 
dimeric protein and are linked through heterologous amino acid residues 
(amino acid residues which are not present between two subunits in the 
native protein). As it occurs in nature, IL-12 is a heterodimer made up of 
a 40 kDa subunit (p40) linked by a disulfide bond to a 35 kDa subunit 
(p35). Gillessen. S. et al., Eur. J. Immunology, 25:200-206 (1995); Ozmen 
et al., J. Exp. Med., 180:907-915 (1995); Heinsel et al., Inf. & Immun., 
62(10):4244-4249 (1994). For example, the fusion protein is a bioactive 
interleukin-12 (IL-12) fusion protein which comprises two subunits, 
designated p35 and p40, joined by a polypeptide linker. In further 
embodiments, the fusion protein comprises the subunits of other dimeric 
hematopoietic growth factors joined by a polypeptide linker, or the 
subunits of other dimeric cytokine proteins joined by a polypeptide 
linker. In another embodiment, the bioactive fusion protein comprises two 
subunits which are bioactive monomers (e.g., interleukin-2, GMCSF) in 
their native form and are joined through a polypeptide linker to produce a 
fusion protein which is chimeric or hybrid in nature in that it comprises 
at least two components or subunits which do not occur together in a 
native protein (e.g., an interleukin-2/GMCSF fusion protein). 
The present invention also relates to methods of producing the subject 
fusion proteins, constructs useful in their production and host cells 
containing the constructs from which the encoded fusion protein is 
expressed. The subject fusion proteins are expressed in an appropriate 
expression system, such as by a retrovirus vector which contains and 
expresses DNA encoding the subunits or monomers and the polypeptide linker 
as the desired fusion protein in an appropriate host cell, such as in 
mammalian cells. 
Fusion proteins of the present invention are useful for the same purposes 
(e.g., therapeutic or diagnostic uses) as the corresponding native 
protein. For example, IL-12 fusion protein can be used to enhance the 
lytic activity of NK/lymphokine--activated killer cells, act as a growth 
factor for activated human T and NK cells and stimulate production of 
IFN-.gamma. by resting peripheral blood mononuclear cells (PBMC). Because 
of its effects in enhancing cell-mediated immunity, IL-12 is potentially 
useful for the enhancement of antitumor immunity. The fusion proteins have 
certain advantages in that they can be made efficiently and reproducibly 
by the methods described herein.

DETAILED DESCRIPTION OF THE INVENTION 
Described herein are bioactive fusion proteins which comprise two subunits 
linked or joined by an intervening amino acid linker, a method of 
producing the bioactive fusion proteins, constructs useful for producing 
the fusion proteins and host cells containing the constructs, which can be 
expressed in the host cells. 
In one embodiment, the bioactive fusion proteins of the present invention 
comprise: 1) at least two polypeptide subunits or monomers which 
correspond to polypeptide subunits present in a native dimeric protein 
which has a specified bioactivity and 2) at least one polypeptide linker 
which joins the subunits in such a manner that the resulting fusion 
protein is bioactive. If the resulting fusion protein is dimeric (includes 
two subunits or monomers), the two components can be subunits which occur 
in the same native dimeric protein (e.g., two IL-12 subunits); subunits 
which occur in two different native dimeric proteins (e.g., one subunit 
from Il-12 and one subunit from IL-3) or monomers which are bioactive 
(e.g., IL-2, GMCSF). Multimeric fusion proteins, which comprise three or 
more subunits joined by polypeptide linkers, can comprise, for example, 
three or more of the subunits which occur in the same native dimeric 
protein (e.g., three or more IL-12 subunits), three or more subunits which 
occur in different native dimeric proteins (e.g., two IL-12 subunits and 
one IL-3 subunit), three or more bioactive monomers (e.g., three IL-2 
monomers, two IL-2 monomers and one GMCSF monomer) or a combination of 
subunits from native dimeric proteins and bioactive monomers (e.g., two 
IL-12 subunits and a GMCSF monomer). In each case, a polypeptide linker is 
present between two subunits (e.g., the order is 
subunit-linker-subunit-linker-subunit). As used herein, the terms subunit 
and monomer are used interchangeably to refer to the components of a 
dimeric or multimeric protein and the single component of a monomeric 
protein. The order of subunits in the fusion protein of the present 
invention can be p35-linker-p40 or p40-linker-p35. In either case, the 
polypeptide linker is positioned between the two subunits. A bioactive 
fusion protein of the present invention which includes subunits which 
occur in the same native dimeric protein "mimics" or is similar to what is 
referred to herein as a corresponding native dimeric protein in terms of 
its bioactivity, but differs from the corresponding native dimeric protein 
in that the fusion protein includes linker amino acid residues which do 
not occur in the corresponding native protein (heterologous amino acid 
residues) between each pair of polypeptide subunits. A corresponding 
native protein is one which includes the subunits present in the fusion 
protein and exhibits biological activity also exhibited by the fusion 
protein. For example, in the case of a bioactive IL-12 fusion protein, the 
two subunits, designated p35 and p40, of a mammalian native IL-12 
protein(e.g., human, mouse, rat, dog, cat, monkey, chimpanzee, pig IL-12 
protein) are joined through a polypeptide linker. Here, the corresponding 
native protein is the mammalian native IL-12 protein. Similarly, in the 
case of another bioactive fusion protein, such as IL-3, the corresponding 
native protein is IL-3. The amino acid residues of the subunits of the 
bioactive fusion protein can be the same as those of the subunits of the 
corresponding native protein or can be different, provided that the 
resulting fusion protein exhibits the desired bioactivity. For example, 
the subunit(s) can have a different amino acid sequence from that of the 
corresponding subunit of a native protein (the sequence of the native 
subunit can differ in that one or more amino acid residues has been 
deleted or replaced by a naturally-occurring or non-naturally-occurring 
amino acid residues or in that additional amino acid residues have been 
incorporated). The desired bioactivity is activity like that of the 
corresponding native protein (e.g., it produces a physiological response 
which also results from the activity of the corresponding native protein). 
The bioactivity of a fusion protein (e.g., the duration of its effect, 
extent of the resulting response) may be greater or lesser than that of 
the corresponding native protein. 
The polypeptide linker present in the fusion protein can be of any length 
and composition appropriate to join two subunits in such a manner that the 
resulting fusion protein has the desired biological activity and retains 
its integrity as a dimer or multimer. The appropriate length and 
composition of a linker can be determined empirically for the specific 
fusion protein to be produced. Generally, the polypeptide linker will be 
at least 10 amino acid residues. In one embodiment, the polypeptide linker 
is 11 to 16 amino acid residues and in specific embodiments is 11, 15 or 
16 amino acid residues. Specific linkers used in producing bioactive IL-12 
fusion proteins are represented in FIG. 2 and described in Example 4. In 
specific embodiments, the polypeptide linkers have the sequence (Gly.sub.4 
Ser).sub.3 SEQ ID NO:7; (Gly.sub.4 Ser).sub.3 Ser SEQ ID NO:6 or 
(Gly.sub.4 Ser).sub.2 Ser SEQ ID NO:5. These linkers can also be used to 
join subunits of other fusion proteins. Alternatively, other polypeptide 
linkers can be used to join two IL-12 subunits to produce a bioactive 
IL-12 fusion protein. 
The DNA encoding the bioactive fusion protein can be cDNA or genomic DNA 
and can be from a variety of animals, particularly mammals. For example, 
the DNA can be human, mouse, rat, dog, cat, monkey, chimpanzee, pig or 
ferret DNA. The DNA can encode a complete or entire subunit (e.g., a 
complete IL-12 p35 subunit and a complete IL-12 p40 subunit) or a fragment 
or portion of a subunit(s), provided that the encoded fusion protein has 
the desired biological activity when it is expressed. The nucleic acid 
sequences of DNA encoding mouse IL-12 p35 and p40 subunits are represented 
in FIGS. 4 and 5, respectively. The nucleic acid sequences of DNA encoding 
human IL-12 p35 and p40 subunits have been published. (See, e.g., Gubler 
et al. in Proceedings of the National Academy of Sciences, USA, 88:4143 
(1991)). All or a portion of IL-12 DNA can be used to produce the subject 
IL-12 fusion protein, provided that the encoded fusion protein is 
bioactive (has IL-12 activity). 
Any expression system appropriate for expressing a fusion protein of the 
present invention, such as a mammalian, bacterial, yeast or insect 
expression system, can be used. For example, as described herein, a viral 
(e.g., a retroviral) vector which expresses DNA (e.g., cDNA) encoding the 
desired fusion protein in a mammalian host cell has been used. As also 
described herein, retroviruses containing cDNA encoding the p35 and p40 
subunits of IL-12 and an intervening polypeptide linker (an IL-12 fusion 
protein) have been constructed and transfected into packaging cells (e.g., 
BOSC23 packaging cells). Target cells (e.g., CMS-5 fibrosarcoma cell line) 
were infected with virus-containing supernatants and cultured; media 
conditioned by infected cells was assayed for IL-12 activity using an 
interleukin-2 and concanavalin-A primed splenocyte proliferation bioassay. 
Packaging or producer cell lines other than BOSC23 cells can be used to 
produce infectious retroviruses containing the fusion protein-encodign 
DNA. In addition, target cells other than a fibrosarcoma cell line can be 
used to produce the fusion protein. IL-12 bioactivity was demonstrable in 
cells infected with the retroviruses, as described in Example 4. 
Specific retroviruses have been constructed for expression of an IL-12 
fusion protein (Example 1 and FIG. 1) and cells infected with the 
retroviruses have been shown to produce bioactive IL-12 fusion proteins. 
(See Example 4) The retroviruses used all include the SFG retroviral 
backbone whose sequence is shown in FIG. 3. The vectors designated 
pSFG.Il-12.p35 and pSFG.IL-12.p40 include, respectively, the cDNA for the 
IL-12 p35 subunit or the cDNA for the IL-12 p40 subunit. The vector 
designated pSFG.IL-12p35-IRES-p40 includes cDNA encoding the IL-12 p35 
subunit and cDNA encoding the IL-12 p40 subunit, separated by an internal 
ribosome entry site sequence. The vector designated pSFG.IL-12p40-IRES-p35 
includes the same components as plasmid pSFG.IL-12p35-IRES-p40 but the 
dimers are in the reverse order, as indicated. The vectors designated 
pSFG.IL-12.p35 - linker - p40 and pSFG.IL-12.p40 - linker - p35 include 
cDNAS encoding each IL-12 subunit linked by the (Gly.sub.4 Ser).sub.2 Ser 
and (Gly.sub.4 Ser).sub.3 Ser linker respectively. The vectors designated 
pSFG.IL-l2.p35 - linked - .DELTA.p40 and pSFG.IL-12.p40 - linker 
.DELTA.p35 include linked cDNAs in which sequences encoding a putative 22 
amino acid leader sequence were deleted from the second cDNA. As described 
in Example 4, IL-12 bioactivity was shown in conditioned medium from cells 
infected with the retroviruses. 
The present invention is illustrated by the following examples, which are 
not intended to be limiting in any way. 
EXAMPLE 1 
Construction of Plasmids 
The general structure of the plasmids used in these studies is shown 
schematically in FIG. 1. The confirmed sequences of the linkers in each of 
the four fusion proteins are given in FIG. 2. 
Source of Plasmids 
The plasmids containing cDNAs for the murine IL-12 p35 and p40 subunits 
(pBS.IL-12.p35 and pBS.IL-12.p40) were provided by Hoffmann-La Roche 
(Nutley, N.J.). The numbering of base pairs in this document corresponds 
to the maps of the inserts of these two plasmids (FIGS. 4 and 5). The 
plasmid containing the SFG retroviral backbone was provided by Dr. Dan Ory 
(Whitehead Institute, Cambridge, Mass.) as pSFG-TPA, a pUC plasmid 
containing the SFG retroviral backbone between the HindIII and EcoR1 sites 
with a tissue plasminogen activator cDNA between the unique Nco1 and BamH1 
sites in the SFG retrovirus. A nucleotide sequence map of the SFG 
retroviral backbone is shown in FIG. 3. 
Plasmid pSFG.IL-12.p35 
The IL-12p35 cDNA was provided in pBluescript with the sequences 
surrounding the translational initiation ATG optimized to ACCATGG 
according to the rules of Kozak. The IL-12p35 cDNA fragment was excised as 
a Nco1-EcoR1 fragment, the EcoR1 overhang having been filled using the 
Klenow fragment of E. coli DNA polymerase 1. This fragment was ligated 
using T4 DNA ligase into the Nco1-BamH1 sites of pSFG, the BamH1 overhang 
having been filled using the Klenow fragment of E. coli DNA polymerase 1. 
The resulting plasmid is designated pSFG.IL-12.p35. 
Plasmid pSFG.IL-12.p40 
The IL-12p40 cDNA was provided in pBluescript. The Nco1-BamH1 fragment 
containing the IL-12p40 cDNA was excised and ligated into the Nco1-BamH1 
sites of pSFG to make pSFG.IL-12.p40. 
General Strategy for Construction of SFG-based Vectors 
The general strategy for constructing the SFG-based retroviral vectors for 
IL-12 fusion protein expression is as follows: Two oligonucleotides 
encoding the sense and antisense strand of a (Gly.sub.4 Ser).sub.3 linker 
fragment and contiguous IL-12 cDNA sequences to be linked (with terminal 
sequences for the creation of cohesive ligatable overhangs) were 
synthesized using a "PCR-mate" 391 DNA synthesizer (Applied Biosystems, 
Foster City, Calif.). The sequence of the (Gly.sub.4 Ser).sub.3 linker was 
that of Huston et al. (Proc. Natl. Acad. Sci. USA, 85:5879-5883(1988)). 
For the two fusion proteins using complete IL-12 cDNAs, the 
oligonucleotides were designed to be cloned into a unique restriction 
enzyme site at the 3' end of the first cDNA, reconstructing the 3' end of 
the first cDNA and enabling a Nco1-Nco1 fragment encompassing the full 
cDNA and linker sequence to be cloned into the Nco1 site of the SFG 
plasmid containing the other cDNA. 
The cloning strategy was similar for the two fusion proteins with a 
deletion of 66 bp coding the first 22 amino acids of the second cDNA. 
Linker oligonucleotides were designed to be cloned into unique restriction 
enzyme sites that lay 3' of bp 66 of the translated bases of the second 
cDNA in the fusion protein construct. This enabled a fragment to be 
excised for cloning that reconstructed the 3' end of the first cDNA joined 
to the linker and contained the linker joined to codon 23 of the second 
cDNA. 
The sequence of the linker and contiguous cDNA regions in plasmids was 
determined using a "Sequenase" kit (Amersham, Cleveland, Ohio). 
Plasmid pSFG.IL-12.p35-linker-p40 
The oligonucleotides were: sense, 
5'-CCGCC.GGT.GGC.GGT.GGC.TCG.GGC.GGT.GGT.GGG.TCG.GGT.GGC.GG 
C.GGA.TCT.TCCATGGAGCT-3' (SEQ ID NO: 16); and antisense, 
5'-CCATGGA.AGA.TCC.GCC.GCC.ACC.CGA.CCC.ACC.ACC.GCC.CGA.GCC. 
ACC.GCC.ACC.GGCGGAGCT-3' (SEQ ID NO: 17). 
These two oligonucleotides were annealed, phosphorylated using T4 
polynucleotide kinase, and ligated into the Sac1 site of pBS.IL-12.p35 
which had been dephosphorylated using calf intestinal phosphatase. The 
Nco1-Nco1 fragment of the resulting plasmid containing the IL-12p35 cDNA 
and correctly orientated linker was excised and ligated into the 
dephosphorylated Nco1 site of pSFG.IL-12p40 to create 
pSFG.IL-12.p35-linker.p40 (the correct orientation of this ligated 
fragment was demonstrated by a Sac1 digest). 
This plasmid was sequenced using the following two primers: 
5'-CAGAGTGAAAATGAAGCT-3' (SEQ ID NO: 18) and 5'-GAAGCTCTGCATCCTGCT-3' (SEQ 
ID NO: 19), corresponding to bp 601-618 and 613-630 of the IL-12p35 cDNA. 
Sequencing demonstrated that a deletion had occurred during cloning 
resulting in a loss of 15 bp from the linker sequences, but maintaining an 
intact reading frame. The sequence of the linker in this plasmid is given 
in FIG. 2. 
Plasmid pSFG.IL-12.p40.linker.p35 
The oligonucleotides were: sense, 
5'-GGGTCCGATCC.GGT.GGC.GGT.GGC.TCG.GGC.GGT.GGT.GGG.TCG.GGT. 
GGC.GGC.GGA.TCT.TCCATG-3' (SEQ ID NO: 20); and antisense, 
5'-GATCCATGGA.AGA.TCC.GCC.GCC.ACC.CGA.CCC.ACC.ACC.GCC.CGA.G 
CC.ACC.GCC.ACC-GGATCGGACCCTGCA-3' (SEQ ID NO: 21). 
These two oligonucleotides were annealed and ligated into the Sse83871 and 
BamH1 sites of pBS.IL-12.p40. The Nco1-Nco1 fragment of the resulting 
plasmid containing the IL-12p40 cDNA and correctly orientated linker was 
excised and ligated into the dephosphorylated Nco1 site of pSFG.IL-12p35 
to create pSFG.IL-12.p40.linker.p35 (the correct orientation of this 
ligated fragment was demonstrated by a Xcm1 digest). 
This plasmid was sequenced using the following two primers: 
5'-CTATTACAATTCCTCATG-3' (SEQ ID NO: 22) and 5'-GAGGGCAAGGGTGGCCAA-3' (SEQ 
ID NO: 23), corresponding to bp 997-1014 of the IL-12 p40 cDNA and bp 
91-74 of the IL-12 p35 cDNA (an antisense primer). Sequencing confirmed 
that the sequence of the linker and contiguous IL-12 cDNA sequences were 
as expected. 
Subsequent restriction enzyme mapping of pSFG.IL-12.p40.linker.p35 after 
the transfection and expression studies were completed revealed that it 
probably contained a concatamer of Nco1-Nco1-fragments from the final 
cloning step. 
Plasmid pSFG.IL-12.p35.linker..DELTA.p40 
The oligonucleotides were: sense, 
5'-T.TGC.TGG.AGC.TCC.GCC.GGT.GGC.GGT.GGC.TCG.GGC.GGT.GGT.GG 
G.TCG.GGT.GGC.GGC.GGA.TCT.ATG.TGG-3' (SEQ ID NO: 24) and antisense, 
5'-CACAT.AGA.TCC.GCC.GCC.ACC.CGA.CCC.ACC.ACC.GCC.CGA.GCC.AC 
C.GCC.ACC.GGCGGAGCTCCAGCAAA-3' (SEQ ID NO: 25). 
These two oligonucleotides were annealed, phosphorylated using T4 
polynucleotide kinase, and ligated into pBS.IL-12.p40 from which the 30 bp 
5' Xcm1-Xcm1 fragment had been excised. The Sac1-Sac1 fragment from the 
resultant plasmid was excised and ligated into the Sac1 site of 
pBS.IL-12.p35 which had been dephosphorylated using calf intestinal 
phosphatase (the correct orientation of the ligated fragment was 
demonstrated by a Nco1-EcoR1 digest). The Nco1-EcoR1 fragment of the 
resultant vector was excised, the EcoR1 overhang having been filled using 
the Klenow fragment of E. coli DNA polymerase 1, and ligated into the Nco1 
and Klenow-filled BamH1 sites of pSFG to create 
pSFG.IL-12.p35.linker..DELTA.p40. 
This plasmid was sequenced using the following primers: 
5'-CAGAGTGAAAATGAAGCT-3'(SEQ ID NO: 18) and 5'-GAAGCTCTGCATCCTGCT-3' (SEQ 
ID NO: 19), corresponding to bp 601-618 and 613-630 of the IL-12p35 cDNA; 
and 5'-GTCATCTTCTTCAGGCGT-3' (SEQ ID NO: 34), an antisense primer 
corresponding to bp 217- 200 of the IL-12 p40 cDNA. Sequencing confirmed 
that the sequence of the linker and contiguous IL-12 cDNA sequences were 
as expected. 
Plasmid pSFG.IL-12.p40.linker..DELTA.p35 
The oligonucleotides were: sense, 
5'-CTG.GCC.TGC.AGG.GTC.CGA.TCC-GGT.GGC.GGT.GGC.TCG.GGC.GGT. 
GGT.GGG.TCG.GGT.GGC.GGC.GGA.TCT-AGG.GTC.ATT.CCA.GTC.T-3' (SEQ ID NO: 26) 
and antisense, 5'-CTGGAATGACCCT.AGA.TCC.GCC.GCC.ACC.CGA.CCC.ACC.ACC.GCC.CG 
A.GCC.ACC.GCC.ACC.GGATCGGACCCTGCAGGCCAGAGA-3' (SEQ ID NO: 27). 
These two oligonucleotides were annealed, phosphorylated using T4 
polynucleotide kinase, and ligated into the PflM1 site in pBS.IL-12.p35 
which had been dephosphorylated using calf intestinal phosphatase. (The 
orientation of this ligated fragment was confirmed by an Sse83871/EcoR1 
digest). The Sse83871-EcoR1 fragment from the resultant plasmid was 
excised, the EcoR1 overhang having been filled using the Klenow fragment 
of E. coli DNA polymerase 1, and ligated into the Sse83871 and 
Klenow-filled BamH1 sites of pSFG.IL-12.p40 to create 
pSFG.IL-12.p40.linker..DELTA.p35. 
This plasmid was sequenced using the primer 5'-GCAAAGGCGGGAATGTCT-3' (SEQ 
ID NO: 28), corresponding to bp 960-977 of the IL-12.p40 cDNA. The 
sequence of the second linker codon was difficult to read, but its 
sequence was determined by sequencing the cloned linker in the 
intermediate plasmid using the antisense primers 5'-AGGAATAATGTTTCAGTT-3' 
(SEQ ID NO: 29) and 5'-CAGCAGTGCAGGAATAAT-3' (SEQ ID NO: 30) corresponding 
to bp 224-207 and 233-216 of the IL-12 p35 cDNA respectively. Sequencing 
confirmed that the sequence of the linker and contiguous IL-12 cDNA 
sequences were as expected. 
Plasmids pSFG.IL-12.p35.IRES.p40 and pSFG.IL-12.p40.IRES.p35 
The encephalomyelocarditis virus (ECMV) internal ribosome entry site (IRES) 
fragment was provided by Dr. Michael Sadelain (Whitehead Institute, 
Cambridge, Mass.), and was as previously described (Ghattas et al., Mol. 
Cell. Biol., 11:5848-5859 (1991)). 
EXAMPLE 2 
Cells and Tissue Culture 
BOSC23 packaging cells (Pear et al., Proc. Natl. Acad. Sci. USA. 
90:8382-8396(1993)) were obtained from Dr. Dirk Lindemann (Whitehead 
Institute, Cambridge, Mass.). They were passaged in Dulbecco's modified 
Eagles medium (DMEM) supplemented with 10% calf serum, 50 U/ml penicillin 
and 50 .mu.g/ml streptomycin. 
CMS-5 tumour cells (DeLeo et al., J. Exp. Med., 146:720-734 (1977)) were 
obtained from Jason Salter (Whitehead Institute, Cambridge, Mass.). They 
were passaged in DMEM supplemented with 10% foetal calf serum, 50 U/ml 
penicillin and 50 .mu.g/ml streptomycin. The same medium was used for the 
collection of CMS-5 conditioned medium. 
C57BL/6 splenocytes for IL-12 assays were obtained by mincing a spleen 
through a sieve (Falcon 2350, Becton Dickinson, Franklin Lakes, N.J.) and 
collecting the cells in IL-12 medium (as detailed in Schoenhaut et al. (J. 
Immunol., 148:3433-3440 (1992)) supplemented with 2% foetal calf serum. 
EXAMPLE 3 
Generation of BOSC23-derived Producer Cells and Collection of Conditioned 
Media 
BOSC23 cells were plated at 2.times.10.sup.6 cells per 6 cm tissue culture 
dish and transfected by CaPO.sub.4 transfection with the various 
constructs as previously described (Pear et al., Proc. Natl. Acad. Sci. 
USA, 90:8382-8396 (1993)). Twenty-four hours after transfection, the 
medium was replaced with 5ml fresh medium. Virus-containing supernatants 
were collected 24 h later, filtered through a 0.45 .mu.m filter and 
polybrene added to a final concentration of 8 .mu.g/ml. 2.5 ml of 
virus-containing supernatant was used to infect CMS-5 cells immediately 
for 4 h (in preparation for this infection, CMS-5 cells had been plated at 
5.times.10.sup.4 cells/6 cm tissue culture dish the previous day) and the 
remaining 2.5 ml frozen at -70.degree. C. The following day, the frozen 
2.5 ml of virus-containing supernatant was thawed and used for a second 4 
h infection of the CMS-5 cells. To collect IL-12-containing conditioned 
medium, the medium was replaced the following day with 5 ml fresh medium 
which was harvested 24 h later. These conditioned media were filtered 
through a 0.2 .mu.m filter and frozen at -70.degree. C. for later assay 
for IL-12 bioactivity. 5 ml of fresh medium was added to the CMS-5 cells 
and a second set of conditioned media collected 24 h later which were also 
filtered and frozen for later assay. The infected CMS-5 cells were then 
lysed, and genomic DNA prepared for later analysis. 
EXAMPLE 4 
Bioassay for Murine Interleukin-12 
Levels of bioactive interleukin-12 were determined using a concanavalin-A 
and interleukin-2 primed splenocyte proliferation assay, as described in 
Schoenhaut et al. (J. Immunol.,148:3433-3440 (1992)). The concanavalin A 
was obtained commercially from Boehringer (Mannheim, Germany) and the 
recombinant human interleukin-2 commercially from Chiron Therapeutics 
(Emeryville, Calif.). To harvest cells for the measurement of .sup.3 
H!thymidine incorporation into cellular DNA, a Skatron (Sterling, Va.) 
cell harvester and filtermats (#7031) were used. To assay for inhibitory 
activity in conditioned media, the 50 .mu.l sample volume comprised 25 
.mu.l of 1000 pg/ml recombinant murine IL-12 and 25 .mu.l of the test 
sample. Samples of conditioned media were assayed in duplicate at several 
dilutions in the range 1:1 to 1:1000. A standard curve was constructed for 
each bioassay using recombinant murine IL-12 in the range 20-10,000 pg/ml. 
The recombinant murine IL-12 was obtained from Hoffmann--La Roche (Nutley, 
N.J.). To calculate the bioactive IL-12 concentration in test samples in 
pg/ml, the linear part of the standard curve was approximated using the 
curve-fit function of "KaleidaGraph 2.1.1" software and the resultant 
formula used for calculations. 
The following constructs (FIG. 1) were assessed for their ability to 
express a bioactive IL-12 fusion protein: 
A. pSFG.IL-12.p35.linker.p40 
B. pSFG.IL-12.p40.linker.p35 
C. pSFG.IL12-p35.linker..DELTA.p40 
D. pSFG.IL12-p40.linker..DELTA.p35 
The sequences for the linkers in each construct were as follows, as 
confirmed by sequencing (some adjacent confirmed IL-12 sequences are given 
for orientation): 
A. 5'-&gt;&gt;&gt;IL-12p35.AGC.TCC.GCC-GGT.GGT.GGT.GGG.TCG.GGT.GGC 
.GGC.GGA.TCT.TCC.ATG.GGT.CCT.CAG.&gt;&gt;&gt;IL-12p40-3' (SEQ ID NO: 1) 
B. 5'-&gt;&gt;&gt;IL-12p40.CCC.TGC.AGG.GTC.CGA.TCC-GGT.GGC.GGT.GGC 
.TCG.GGC.GGT.GGT.GGG.TCG.GGT.GGC.GGC.GGA.TCT.TCC.ATG.G 
GT.CAA.&gt;&gt;&gt;IL-12p35-3'0 (SEQ ID NO: 31) 
C. 5'-&gt;&gt;&gt;IL-12p35.5'-TAT.CTG.AGC.TCC.GCC-GGT.GGC.GGT.GGC. 
TCG.GGC.GGT.GGT.GGG.TCG.GGT.GGC.GGC.GGA.TCT.ATG.TGG.GA 
G.CTG.GAG.AAA.&gt;&gt;&gt;IL-12p40-3' (SEQ ID NO: 32) 
D. 5'-&gt;&gt;&gt;IL-12p40.TGT.GTT.CCC.TGC.AGG.GTC.CGA.TCC-GGT.GGC 
.GGT.GGC.TCG.GGC.GGT.GGT.GGG.TCG.GGT.GGC.GGC.GGA.TCT.A 
GG.GTC.ATT.CCA.GTC.TCT.GGA.CCT.GCC.&gt;&gt;&gt;IL-12p35-3' (SEQ ID NO: 33) 
No IL-12 bioactivity was detectable in media conditioned by 
mock-transfected CMS-5 cells, and CMS-5 cells infected with the SFG 
retrovirus alone, or by a related retrovirus (MFG) carrying the lac-z 
gene. However, media conditioned by these cells contained significant 
inhibitory activity at 1:2 and 1:10 dilutions, inhibiting as much as 95% 
of the bioactivity of 500 pg/ml of rmIL-12 (Table 1, and other data not 
shown). Despite this background of inhibitory activity in the conditioned 
media, bioactive II,-12 production proved to be still demonstrable. 
Constructs for the expression of single subunits of the IL-12 protein 
(pSFG.Il-12.p35 and pSFG.IL-12.p40) resulted in no detectable bioactivity 
on their own. 
However, cotransfection of BOSC23 cells with these constructs together 
resulted in bioactive IL-12 secretion by infected CMS-5 cells. Similarly, 
CMS-5 cells infected with the SFG.IL-12.p35 retrovirus and 24 hours later 
with the SFG.IL-12.p40 retrovirus also produced bioactive IL-12 (Table 1). 
The dicistronic constructs designed to express both IL-12 subunits using 
the IRES sequence resulted in similar levels of bioactive IL-12 production 
(this was despite an undetectable level of viral infection as determined 
by Southern hybridization analysis see below! (Table 1). The ability of 
IRES-containing retroviruses to result in bioactive IL-12 production has 
been confirmed by generating stable clonal retrovirus producing cell lines 
using both these constructs. 
All four IL-12 fusion protein constructs resulted in 20 significant 
bioactive IL-12 production by infected CMS-5 cells. Of particular note was 
the SFG.IL-12.p40 linker..DELTA.p35 construct, for which IL-12 bioactivity 
was demonstrable in undiluted conditioned medium (despite the background 
of substantial inhibitory activity) and for which a 1:1000 dilution of 
conditioned medium contained bioactivity equivalent to 301 pg/ml of 
rmIL-12 (Table 1). 
All four constructs resulted in titratable IL-12 bioactivity despite 
significant non-specific inhibitory activity in the conditioned media as 
well. 
TABLE 1 
______________________________________ 
Antagonist assay 
Agonist assay 
(% inhibition of 500 
(IL-12 bioactivity, pg/ml 
pg/ml IL-12 in assay) 
Dilution of CM in assay 
Dilution of CM in assay 
Construct 1:1 1:100 1:1000 
1:2 1:10 1:1000 
______________________________________ 
No DNA &lt;50 &lt;50 &lt;50 56 62 8.6 
SFG-empty &lt;50 &lt;50 &lt;50 12 47 -91 
MFG-lac-z &lt;50 &lt;50 &lt;50 66 56 64 
SFG.IL-12p35 
&lt;50 &lt;50 &lt;50 65 76 45 
SFG.IL-12p40 
&lt;50 &lt;50 &lt;50 94 84 38 
2X infection.sup.a 
199.7 234.2 137.2 1 1 -84 
2X transfection.sup.b 
244.7 118.8 &lt;50 12 -3 -2 
A 86.5 &lt;50 &lt;50 44 60 46 
B 253.8 &lt;50 &lt;50 41 12 -14 
C 189.2 57.0 &lt;50 43 42 47 
D 297.8 600.1 301.2 -48 -143 -93 
______________________________________ 
These data are from one of three separate assays. 
.sup.a Target cells infected sequentially with pSFG.IL 12.p35 and then 
pSFG.IL 12.p40 viruses (each containing only the respective cDNA between 
the Nco1 and BamH1 sites) 
.sup.b BOSC23 cells were transfected with a mixture of pSFG.IL 12p35 and 
pSFG.IL 12.p40 constructs 
These data indicate IL-12 agonist activity was present in media conditioned 
by cells infected with the four fusion protein retroviral constructs. It 
is presumed that this results from bioactivity of secreted respective 
fusion proteins. The fusion proteins can be demonstrated to be present 
using known methods, such as Western blotting or immunoprecipitation. 
EXAMPLE 5 
Southern Hybridization Analysis of Genomic DNA from Infected CMS-5 Cells 
Southern hybridization analysis of genomic DNA from the populations of 
infected CMS-5 cells was performed to demonstrate the presence of a 
hybridizing band consistent with infection of these cells by retroviruses 
of the expected structure, and to determine the efficiency of viral 
infection (by determination of retroviral copy number by genome). 
From these Nhe1 digests of genomic DNA, a hybridizing retrovirus-derived 
band of 985 bp plus the size of the insert cloned into the Nco1-BamH1 
sites of SFG was predicted (See FIG. 1). The size of the various cloned 
fragments were: IL-12.p35 cDNA, 0.6 kb; IL-12.p40 cDNA, 1.0 kb; IRES, 0.7 
kb; linker, 0.05 kb; the putative leader sequence deleted in two 
constructs was 0.066 bp. 
The BOSC23 cell supernatants resulted in viral copy numbers of between 0.1 
and 1.4 copies/genome (mostly 0.1-0.3 copies/genome) for all constructs 
except for the IRES-containing constructs, where no hybridizing band of 
the expected size (3.2 kb) was seen (Table 2). 
Of particular note are the comparative results for the IL-12 fusion 
proteins retrovirus constructs in these populations of infected cells. 
Although the pSFG.IL-12.p35.linker.p40 retrovirus was present at 1.4 
copies/genome, this corresponded with a relatively low level of bioactive 
IL-12 production (Table 2). However, the SFG.IL-12.p40 linker..DELTA.p35 
retrovirus resulted in a relatively high level of IL-12 bioactivity, 
although it was present at 0.2 copies/genome. 
TABLE 2 
______________________________________ 
Retrovirus Copy Number in CMS-5 Cells Infected by 
SFG.IL-12 Retroviruses 
SFG.IL-12 construct Retrovirus 
containing: copy number.sup.a 
______________________________________ 
Nil 0 
IL-12.p35 0.1 
IL-12.p40 0.3 
Sequential infection (p35/p40) 
0.3/0.3 
Co-transfection (p35/p40) 
0.1/0.1 
IL-12.p35-IRES-p40 &lt;&lt;0.1.sup.b 
IL-12.p40.IRES.p35 &lt;&lt;0.1.sup.b 
IL-12.p35.linker.p40 1.4.sup.b 
IL-12.p40.linker.p35 0.1.sup.b 
IL-12.p35.linker..DELTA.p40 
0.4.sup.b 
IL-12.p40.linker..DELTA.p35 
0.2.sup.b 
1 copy control 1.0.sup..sup.b 
0.1 copy control 0.1.sup..sup.b 
______________________________________ 
.sup.a Relative to a plasmid copy number control of 13.5 pg of pSFG.IL 
12.p35 linker.p40, calculated to be equimolar to 1 copy/genome for 10 
.mu.g genomic DNA. 
.sup.b Mean of results from one Southern blot probed first with a p35 and 
then with a p40 radiolabelled probe. Relative intensity of signals was 
quantitated using a Fuji BASII phosphoimager. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 34 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AGCTCCGCCGGTGGTGGTGGGTCGGGTGGCGGCGGATCTTCCATGGGTCCTCAG54 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTTCCATG60 
GGTCAA66 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AGCTCCGCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTATGTGG60 
GAGCTG66 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 66 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTAGGGTC60 
ATTCCA66 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GlyGlyGlyGlySerGlyGlyGlyGlySerSer 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerSer 
151015 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer 
151015 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6350 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AAGCTTTGCTCTTAGGAGTTTCCTAATACATCCCAAACTCAAATATATAAAGCATTTGAC60 
TTGTTCTATGCCCTAGGGGGCGGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATG120 
TTAATTCCATTTTAAATGCACAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAAT180 
GCTGCAATATTCCTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGGAAATTAC240 
TTAGAGTTTCTGTCATTAACGTTTCCTTCCTCAGTTGACAACATAAATGCGCTGCTGAGC300 
AAGCCAGTTTGCATCTGTCAGGATCAATTTCCCATTATGCCAGTCATATTAATTACTAGT360 
CAATTAGTTGATTTTTATTTTTGACATATACATGTGAATGAAAGACCCCACCTGTAGGTT420 
TGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAAT480 
AGAAAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGAT540 
ATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATA600 
TGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGA660 
TGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGG720 
GTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTC780 
TCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCAC840 
TCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC900 
TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGA960 
TTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCT1020 
GCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTG1080 
TCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAAC1140 
TAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAAC1200 
CCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAA1260 
TCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGT1320 
AGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGA1380 
CCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGAC1440 
TGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAG1500 
TTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGT1560 
CAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATG1620 
GCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTC1680 
ACCTGGCCCGCATGGACACCCAGACCAGGTGGGGTACATCGTGACCTGGGAAGCCTTGGC1740 
TTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCC1800 
TCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCT1860 
TTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATATGG1920 
GGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCC1980 
CTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCT2040 
GGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGG2100 
CGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACC2160 
TTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGAT2220 
ACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCCATG2280 
GCGCGGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGT2340 
TTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAAAATAAAA2400 
GATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAA2460 
GCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAA2520 
GTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGT2580 
GGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCC2640 
AAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCC2700 
CCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCC2760 
CAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTT2820 
CTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGG2880 
CGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGC2940 
AGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACT3000 
ACCCGTCAGCGGGGGTCTTTCACACATGCAGCATGTATCAAAATTAATTTGGTTTTTTTT3060 
CTTAAGTATTTACATTAAATGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAA3120 
ACATGGAGTATTCAGAATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGG3180 
CTTTCTACTTTTTCTTTTATTTTTTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGT3240 
TTGTTTGTTTGTTTGTTGGTTGGTTGGTTAATTTTTTTTTAAAGATCCTACACTATAGTT3300 
CAAGCTAGACTATTAGCTACTCTGTAACCCAGGGTGACCTTGAAGTCATGGGTAGCCTGC3360 
TGTTTTAGCCTTCCCACATCTAAGATTACAGGTATGAGCTATCATTTTTGGTATATTGAT3420 
TGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTGACTGTGAAAATGT3480 
GTGTATGGGTGTGTGTGAATGTGTGTATGTATGTGTGTGTGTGAGTGTGTGTGTGTGTGT3540 
GTGCATGTGTGTGTGTGTGACTGTGTCTATGTGTATGACTGTGTGTGTGTGTGTGTGTGT3600 
GTGTGTGTGTGTGTGTGTGTGTGTGTTGTGAAAAAATATTCTATGGTAGTGAGAGCCAAC3660 
GCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGTCTTTCACTTAGCTTGGAATTC3720 
ACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCG3780 
CCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCG3840 
CCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCT3900 
TACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGA3960 
TGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGC4020 
TTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTG4080 
TCAGAGGTTTTCACCGTCATCACCGAAACGCGCGATGACGAAAGGGCCTCGTGATACGCC4140 
TATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTC4200 
GGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATC4260 
CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGA4320 
GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTT4380 
TTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG4440 
TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG4500 
AACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTA4560 
TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG4620 
AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA4680 
GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG4740 
GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATC4800 
GTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTG4860 
TAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCC4920 
GGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG4980 
CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCG5040 
GTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGA5100 
CGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCAC5160 
TGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAA5220 
AACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCA5280 
AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAG5340 
GATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC5400 
CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA5460 
CTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCC5520 
ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAG5580 
TGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTAC5640 
CGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC5700 
GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTC5760 
CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA5820 
CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACC5880 
TCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG5940 
CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT6000 
TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATA6060 
CCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGC6120 
GCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACG6180 
ACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCA6240 
CTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTG6300 
TGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCC6350 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6350 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTCGAAACGAGAATCCTCAAAGGATTATGTAGGGTTTGAGTTTATATATTTCGTAAACTG60 
AACAAGATACGGGATCCCCCGCCCCCCTTCGATTCGGTCGAAAAAAATTGTAAATTTTAC120 
AATTAAGGTAAAATTTACGTGTCTACAAAAATAAAGTATTCCCAAAGTTACACGTACTTA180 
CGACGTTATAAGGACAATGGTTTCGATCATATTTATTTTTATCTATTTGCACCTTTAATG240 
AATCTCAAAGACAGTAATTGCAAAGGAAGGAGTCAACTGTTGTATTTACGCGACGACTCG300 
TTCGGTCAAACGTAGACAGTCCTAGTTAAAGGGTAATACGGTCAGTATAATTAATGATCA360 
GTTAATCAACTAAAAATAAAAACTGTATATGTACACTTACTTTCTGGGGTGGACATCCAA420 
ACCGTTCGATCGAATTCATTGCGGTAAAACGTTCCGTACCTTTTTATGTATTGACTCTTA480 
TCTTTTCAAGTCTAGTTCCAGTCCTTGTCTACCTTGTCGACTTATACCCGGTTTGTCCTA540 
TAGACACCATTCGTCAAGGACGGGGCCGAGTCCCGGTTCTTGTCTACCTTGTCGACTTAT600 
ACCCGGTTTGTCCTATAGACACCATTCGTCAAGGACGGGGCCGAGTCCCGGTTCTTGTCT660 
ACCAGGGGTCTACGCCAGGTCGGGAGTCGTCAAAGATCTCTTGGTAGTCTACAAAGGTCC720 
CACGGGGTTCCTGGACTTTACTGGGACACGGAATAAACTTGATTGGTTAGTCAAGCGAAG780 
AGCGAAGACAAGCGCGCGAATACGAGGGGCTCGAGTTATTTTCTCGGGTGTTGGGGAGTG840 
AGCCCCGCGGTCAGGAGGCTAACTGACTCAGCGGGCCCATGGGCACATAGGTTATTTGGG900 
AGAACGTCAACGTAGGCTGAACACCAGAGCGACAAGGAACCCTCCCAGAGGAGACTCACT960 
AACTGATGGGCAGTCGCCCCCAGAAAGTAAACCCCCGAGCAGGCCCTAGCCCTCTGGGGA1020 
CGGGTCCCTGGTGGCTGGGTGGTGGCCCTCCATTCGACCGGTCGTTGAATAGACACAGAC1080 
AGGCTAACAGATCACAGATACTGACTAAAATACGCGGACGCAGCCATGATCAATCGATTG1140 
ATCGAGACATAGACCGCCTGGGCACCACCTTGACTGCTCAAGCCTTGTGGGCCGGCGTTG1200 
GGACCCTCTGCAGGGTCCCTGAAGCCCCCGGCAAAAACACCGGGCTGGACTCAGGATTTT1260 
AGGGCTAGCAAATCCTGAGAAACCACGTGGGGGGAATCTCCTCCCTATACACCAAGACCA1320 
TCCTCTGCTCTTGGATTTTGTCAAGGGCGGAGGCAGACTTAAAAACGAAAGCCAAACCCT1380 
GGCTTCGGCGCGGCGCGCAGAACAGACGACGTCGTAGCAAGACACAACAGAGACAGACTG1440 
ACACAAAGACATAAACAGACTTTTATACCCGGGCCCGATCGGACAATGGTGAGGGAATTC1500 
AAACTGGAATCCAGTGACCTTTCTACAGCTCGCCTAGCGAGTGTTGGTCAGCCATCTACA1560 
GTTCTTCTCTGCAACCCAATGGAAGACGAGACGTCTTACCGGTTGGAAATTGCAGCCTAC1620 
CGGCGCTCTGCCGTGGAAATTGGCTCTGGAGTAGTGGGTCCAATTCTAGTTCCAGAAAAG1680 
TGGACCGGGCGTACCTGTGGGTCTGGTCCACCCCATGTAGCACTGGACCCTTCGGAACCG1740 
AAAACTGGGGGGAGGGACCCAGTTCGGGAAACATGTGGGATTCGGAGGCGGAGGAGAAGG1800 
AGGTAGGCGGGGCAGAGAGGGGGAACTTGGAGGAGCAAGCTGGGGCGGAGCTAGGAGGGA1860 
AATAGGTCGGGAGTGAGGAAGAGATCCGCGGGGGTATACCGGTATACTCTAGAATATACC1920 
CCGTGGGGGCGGGGAACATTTGAAGGGACTGGGACTGTACTGTTCTCAATGATTGTCGGG1980 
GAGAGAGGTTCGAGTGAATGTCCGAGAGATGAATCAGGTCGTGCTTCAGACCTCTGGAGA2040 
CCGCCGTCGGATGGTTCTTGTTGACCTGGCTGGCCACCATGGAGTGGGAATGGCTCAGCC2100 
GCTGTGTCACACCCAGGCGGCTGTGGTCTGATTCTTGGATCTTGGAGCGACCTTTCCTGG2160 
AATGTGTCAGGACGACTGGTGGGGGTGGCGGGAGTTTCATCTGCCGTAGCGTCGAACCTA2220 
TGTGCGGCGGGTGCACTTCCGACGGCTGGGGCCCCCACCTGGTAGGAGATCTGACGGTAC2280 
CGCGCCTAGGCCTAATCAGGTTAAACAATTTCTGTCCTATAGTCACCAGGTCCGAGATCA2340 
AAACTGAGTTGTTATAGTGGTCGACTTCGGATATCTCATGCTCGGTATCTATTTTATTTT2400 
CTAAAATAAATCAGAGGTCTTTTTCCCCCCTTACTTTCTGGGGTGGACATCCAAACCGTT2460 
CGATCGAATTCATTGCGGTAAAACGTTCCGTACCTTTTTATGTATTGACTCTTATCTCTT2520 
CAAGTCTAGTTCCAGTCCTTGTCTACCTTGTCGACTTATACCCGGTTTGTCCTATAGACA2580 
CCATTCGTCAAGGACGGGGCCGAGTCCCGGTTCTTGTCTACCTTGTCGACTTATACCCGG2640 
TTTGTCCTATAGACACCATTCGTCAAGGACGGGGCCGAGTCCCGGTTCTTGTCTACCAGG2700 
GGTCTACGCCAGGTCGGGAGTCGTCAAAGATCTCTTGGTAGTCTACAAAGGTCCCACGGG2760 
GTTCCTGGACTTTACTGGGACACGGAATAAACTTGATTGGTTAGTCAAGCGAAGAGCGAA2820 
GACAAGCGCGCGAAGACGAGGGGCTCGAGTTATTTTCTCGGGTGTTGGGGAGTGAGCCCC2880 
GCGGTCAGGAGGCTAACTGACTCAGCGGGCCCATGGGCACATAGGTTATTTGGGAGAACG2940 
TCAACGTAGGCTGAACACCAGAGCGACAAGGAACCCTCCCAGAGGAGACTCACTAACTGA3000 
TGGGCAGTCGCCCCCAGAAAGTGTGTACGTCGTACATAGTTTTAATTAAACCAAAAAAAA3060 
GAATTCATAAATGTAATTTACCGGTATCATGAATTTCAATGTAACCGAAGGAACTTTATT3120 
TGTACCTCATAAGTCTTACACAGTATTTATAAAGATTAAAATTCTATCATAGAGGTAACC3180 
GAAAGATGAAAAAGAAAATAAAAAAAAACAGGAGACAGAAGGTAAACAACAACAACAACA3240 
AACAAACAAACAAACAACCAACCAACCAATTAAAAAAAAATTTCTAGGATGTGATATCAA3300 
GTTCGATCTGATAATCGATGAGACATTGGGTCCCACTGGAACTTCAGTACCCATCGGACG3360 
ACAAAATCGGAAGGGTGTAGATTCTAATGTCCATACTCGATAGTAAAAACCATATAACTA3420 
ACTAACTAACTAACTACACACACACACACTAACACAAACACACACACTGACACTTTTACA3480 
CACATACCCACACACACTTACACACATACATACACACACACACTCACACACACACACACA3540 
CACGTACACACACACACACTGACACAGATACACATACTGACACACACACACACACACACA3600 
CACACACACACACACACACACACACAACACTTTTTTATAAGATACCATCACTCTCGGTTG3660 
CGAGGCCGAGTCCACAGTCCAACCAAAAACTCTGTCTCAGAAAGTGAATCGAACCTTAAG3720 
TGACCGGCAGCAAAATGTTGCAGCACTGACCCTTTTGGGACCGCAATGGGTTGAATTAGC3780 
GGAACGTCGTGTAGGGGGAAAGCGGTCGACCGCATTATCGCTTCTCCGGGCGTGGCTAGC3840 
GGGAAGGGTTGTCAACGCGTCGGACTTACCGCTTACCGCGGACTACGCCATAAAAGAGGA3900 
ATGCGTAGACACGCCATAAAGTGTGGCGTATACCACGTGAGAGTCATGTTAGACGAGACT3960 
ACGGCGTATCAATTCGGTCGGGGCTGTGGGCGGTTGTGGGCGACTGCGCGGGACTGCCCG4020 
AACAGACGAGGGCCGTAGGCGAATGTCTGTTCGACACTGGCAGAGGCCCTCGACGTACAC4080 
AGTCTCCAAAAGTGGCAGTAGTGGCTTTGCGCGCTACTGCTTTCCCGGAGCACTATGCGG4140 
ATAAAAATATCCAATTACAGTACTATTATTACCAAAGAATCTGCAGTCCACCGTGAAAAG4200 
CCCCTTTACACGCGCCTTGGGGATAAACAAATAAAAAGATTTATGTAAGTTTATACATAG4260 
GCGAGTACTCTGTTATTGGGACTATTTACGAAGTTATTATAACTTTTTCCTTCTCATACT4320 
CATAAGTTGTAAAGGCACAGCGGGAATAAGGGAAAAAACGCCGTAAAACGGAAGGACAAA4380 
AACGAGTGGGTCTTTGCGACCACTTTCATTTTCTACGACTTCTAGTCAACCCACGTGCTC4440 
ACCCAATGTAGCTTGACCTAGAGTTGTCGCCATTCTAGGAACTCTCAAAAGCGGGGCTTC4500 
TTGCAAAAGGTTACTACTCGTGAAAATTTCAAGACGATACACCGCGCCATAATAGGGCAT4560 
AACTGCGGCCCGTTCTCGTTGAGCCAGCGGCGTATGTGATAAGAGTCTTACTGAACCAAC4620 
TCATGAGTGGTCAGTGTCTTTTCGTAGAATGCCTACCGTACTGTCATTCTCTTAATACGT4680 
CACGACGGTATTGGTACTCACTATTGTGACGCCGGTTGAATGAAGACTGTTGCTAGCCTC4740 
CTGGCTTCCTCGATTGGCGAAAAAACGTGTTGTACCCCCTAGTACATTGAGCGGAACTAG4800 
CAACCCTTGGCCTCGACTTACTTCGGTATGGTTTGCTGCTCGCACTGTGGTGCTACGGAC4860 
ATCGTTACCGTTGTTGCAACGCGTTTGATAATTGACCGCTTGATGAATGAGATCGAAGGG4920 
CCGTTGTTAATTATCTGACCTACCTCCGCCTATTTCAACGTCCTGGTGAAGACGCGAGCC4980 
GGGAAGGCCGACCGACCAAATAACGACTATTTAGACCTCGGCCACTCGCACCCAGAGCGC5040 
CATAGTAACGTCGTGACCCCGGTCTACCATTCGGGAGGGCATAGCATCAATAGATGTGCT5100 
GCCCCTCAGTCCGTTGATACCTACTTGCTTTATCTGTCTAGCGACTCTATCCACGGAGTG5160 
ACTAATTCGTAACCATTGACAGTCTGGTTCAAATGAGTATATATGAAATCTAACTAAATT5220 
TTGAAGTAAAAATTAAATTTTCCTAGATCCACTTCTAGGAAAAACTATTAGAGTACTGGT5280 
TTTAGGGAATTGCACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTAGTTTC5340 
CTAGAAGAACTCTAGGAAAAAAAGACGCGCATTAGACGACGAACGTTTGTTTTTTTGGTG5400 
GCGATGGTCGCCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAGGCTTCCATT5460 
GACCGAAGTCGTCTCGCGTCTATGGTTTATGACAGGAAGATCACATCGGCATCAATCCGG5520 
TGGTGAAGTTCTTGAGACATCGTGGCGGATGTATGGAGCGAGACGATTAGGACAATGGTC5580 
ACCGACGACGGTCACCGCTATTCAGCACAGAATGGCCCAACCTGAGTTCTGCTATCAATG5640 
GCCTATTCCGCGTCGCCAGCCCGACTTGCCCCCCAAGCACGTGTGTCGGGTCGAACCTCG5700 
CTTGCTGGATGTGGCTTGACTCTATGGATGTCGCACTCGTAACTCTTTCGCGGTGCGAAG5760 
GGCTTCCCTCTTTCCGCCTGTCCATAGGCCATTCGCCGTCCCAGCCTTGTCCTCTCGCGT5820 
GCTCCCTCGAAGGTCCCCCTTTGCGGACCATAGAAATATCAGGACAGCCCAAAGCGGTGG5880 
AGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCCGCCTCGGATACCTTTTTGC5940 
GGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGAGTGTACAAGA6000 
AAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGGAAACTCACTCGACTAT6060 
GGCGAGCGGCGTCGGCTTGCTGGCTCGCGTCGCTCAGTCACTCGCTCCTTCGCCTTCTCG6120 
CGGGTTATGCGTTTGGCGGAGAGGGGCGCGCAACCGGCTAAGTAATTACGTCGACCGTGC6180 
TGTCCAAAGGGCTGACCTTTCGCCCGTCACTCGCGTTGCGTTAATTACACTCAATCGAGT6240 
GAGTAATCCGTGGGGTCCGAAATGTGAAATACGAAGGCCGAGCATACAACACACCTTAAC6300 
ACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCGG6350 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 713 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AAGCTTGGGCTGCAGGTCGATCGACTCTAGAGGATCGATCCCCACCATGGGTCAATCACG60 
CTACCTCCTCTTTTTGGCCACCCTTGCCCTCCTAAACCACCTCAGTTTGGCCAGGGTCAT120 
TCCAGTCTCTGGACCTGCCAGGTGTCTTAGCCAGTCCCGAAACCTGCTGAAGACCACAGA180 
TGACATGGTGAAGACGGCCAGAGAAAAACTGAAACATTATTCCTGCACTGCTGAAGACAT240 
CGATCATGAAGACATCACACGGGACCAAACCAGCACATTGAAGACCTGTTTACCACTGGA300 
ACTACACAAGAACGAGAGTTGCCTGGCTACTAGAGAGACTTCTTCCACAACAAGAGGGAG360 
CTGCCTGCCCCCACAGAAGACGTCTTTGATGATGACCCTGTGCCTTGGTAGCATCTATGA420 
GGACTTGAAGATGTACCAGACAGAGTTCCAGGCCATCAACGCAGCACTTCAGAATCACAA480 
CCATCAGCAGATCATTCTAGACAAGGGCATGCTGGTGGCCATCGATGAGCTGATGCAGTC540 
TCTGAATCATAATGGCGAGACTCTGCGCCAGAAACCTCCTGTGGGAGAAGCAGACCCTTA600 
CAGAGTGAAAATGAAGCTCTGCATCCTGCTTCACGCCTTCAGCACCCGCGTCGTGACCAT660 
CAACAGGGTGATGGGCTATCTGAGCTCCGCCTGAGAATTCATTGATCCACTAG713 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 713 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
TTCGAACCCGACGTCCAGCTAGCTGAGATCTCCTAGCTAGGGGTGGTACCCAGTTAGTGC60 
GATGGAGGAGAAAAACCGGTGGGAACGGGAGGATTTGGTGGAGTCAAACCGGTCCCAGTA120 
AGGTCAGAGACCTGGACGGTCCACAGAATCGGTCAGGGCTTTGGACGACTTCTGGTGTCT180 
ACTGTACCACTTCTGCCGGTCTCTTTTTGACTTTGTAATAAGGACGTGACGACTTCTGTA240 
GCTAGTACTTCTGTAGTGTGCCCTGGTTTGGTCGTGTAACTTCTGGACAAATGGTGACCT300 
TGATGTGTTCTTGCTCTCAACGGACCGATGATCTCTCTGAAGAAGGTGTTGTTCTCCCTC360 
GACGGACGGGGGTGTCTTCTGCAGAAACTACTACTGGGACACGGAACCATCGTAGATACT420 
CCTGAACTTCTACATGGTCTGTCTCAAGGTCCGGTAGTTGCGTCGTGAAGTCTTAGTGTT480 
GGTAGTCGTCTAGTAAGATCTGTTCCCGTACGACCACCGGTAGCTACTCGACTACGTCAG540 
AGACTTAGTATTACCGCTCTGAGACGCGGTCTTTGGAGGACACCCTCTTCGTCTGGGAAT600 
GTCTCACTTTTACTTCGAGACGTAGGACGAAGTGCGGAAGTCGTGGGCGCAGCACTGGTA660 
GTTGTCCCACTACCCGATAGACTCGAGGCGGACTCTTAAGTAACTAGGTGATC713 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 215 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
MetGlyGlnSerArgTyrLeuLeuPheLeuAlaThrLeuAlaLeuLeu 
151015 
AsnHisLeuSerLeuAlaArgValIleProValSerGlyProAlaArg 
202530 
CysLeuSerGlnSerArgAsnLeuLeuLysThrThrAspAspMetVal 
354045 
LysThrAlaArgGluLysLeuLysHisTyrSerCysThrAlaGluAsp 
505560 
IleAspHisGluAspIleThrArgAspGlnThrSerThrLeuLysThr 
65707580 
CysLeuProLeuGluLeuHisLysAsnGluSerCysLeuAlaThrArg 
859095 
GluThrSerSerThrThrArgGlySerCysLeuProProGlnLysThr 
100105110 
SerLeuMetMetThrLeuCysLeuGlySerIleTyrGluAspLeuLys 
115120125 
MetTyrGlnThrGluPheGlnAlaIleAsnAlaAlaLeuGlnAsnHis 
130135140 
AsnHisGlnGlnIleIleLeuAspLysGlyMetLeuValAlaIleAsp 
145150155160 
GluLeuMetGlnSerLeuAsnHisAsnGlyGluThrLeuArgGlnLys 
165170175 
ProProValGlyGluAlaAspProTyrArgValLysMetLysLeuCys 
180185190 
IleLeuLeuHisAlaPheSerThrArgValValThrIleAsnArgVal 
195200205 
MetGlyTyrLeuSerSerAla 
210215 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1061 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AAGCTTGGGCTGCAGGTCGATCGACTCTAGAGGATCGATCCCCACCATGGGTCCTCAGAA60 
GCTAACCATCTCCTGGTTTGCCATCGTTTTGCTGGTGTCTCCACTCATGGCCATGTGGGA120 
GCTGGAGAAAGACGTTTATGTTGTAGAGGTGGACTGGACTCCCGATGCCCCTGGAGAAAC180 
AGTGAACCTCACCTGTGACACGCCTGAAGAAGATGACATCACCTGGACCTCAGACCAGAG240 
ACATGGAGTCATAGGCTCTGGAAAGACCCTGACCATCACTGTCAAAGAGTTTCTAGATGC300 
TGGCCAGTACACCTGCCACAAAGGAGGCGAGACTCTGAGCCACTCACATCTGCTGCTCCA360 
CAAGAAGGAAAATGGAATTTGGTCCACTGAAATTTTAAAAAATTTCAAAAACAAGACTTT420 
CCTGAAGTGTGAAGCACCAAATTACTCCGGACGGTTCACGTGCTCATGGCTGGTGCAAAG480 
AAACATGGACTTGAAGTTCAACATCAAGAGCAGTAGCAGTTCCCCTGACTCTCGGGCAGT540 
GACATGTGGAATGGCGTCTCTGTCTGCAGAGAAGGTCACACTGGACCAAAGGGACTATGA600 
GAAGTATTCAGTGTCCTGCCAGGAGGATGTCACCTGCCCAACTGCCGAGGAGACCCTGCC660 
CATTGAACTGGCGTTGGAAGCACGGCAGCAGAATAAATATGAGAACTACAGCACCAGCTT720 
CTTCATCAGGGACATCATCAAACCAGACCCGCCCAAGAACTTGCAGATGAAGCCTTTGAA780 
GAACTCACAGGTGGAGGTCAGCTGGGAGTACCCTGACTCCTGGAGCACTCCCCATTCCTA840 
CTTCTCCCTCAAGTTCTTTGTTCGAATCCAGCGCAAGAAAGAAAAGATGAAGGAGACAGA900 
GGAGGGGTGTAACCAGAAAGGTGCGTTCCTCGTAGAGAAGACATCTACCGAAGTCCAATG960 
CAAAGGCGGGAATGTCTGCGTGCAAGCTCAGGATCGCTATTACAATTCCTCATGCAGCAA1020 
GTGGGCATGTGTTCCCTGCAGGGTCCGATCCTAGGAATTCC1061 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1060 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
TTCGAACCCGACGTCCAGCTAGCTGAGATCTCCTAGCTAGGGGTGGTACCCAGGAGTCTT60 
CGATTGGTAGAGGACCAAACGGTAGCAAAACGACCACAGAGGTGAGTACCGGTACACCCT120 
CGACCTCTTTCTGCAAATACAACATCTCCACCTGACCTGAGGGCTACGGGGACCTCTTTG180 
TCACTTGGAGTGGACACTGTGCGGACTTCTTCTACTGTAGTGGACCTGGAGTCTGGTCTC240 
TGTACCTCAGTATCCGAGACCTTTCTGGGACTGGTAGTGACAGTTTCTCAAAGATCTACG300 
ACCGGTCATGTGGACGGTGTTTCCTCCGCTCTGAGACTCGGTGAGTGTAGACGACGAGGT360 
GTTCTTCCTTTTACCTTAAACCAGGTGACTTTAAAATTTTTTAAAGTTTTTGTTCTGAAA420 
GGACTTCACACTTCGTGGTTTAATGAGGCCTGCCAAGTGCACGAGTACCGACCACGTTTC480 
TTTGTACCTGAACTTCAAGTTGTAGTTCTCGTCATCGTCAAGGGGACTGAGAGCCCGTCA540 
CTGTACACCTTACCGCAGAGACAGACGTCTCTTCCAGTGTGACCTGGTTTCCCTGATACT600 
CTTCATAAGTCACAGGACGGTCCTCCTACAGTGGACGGGTTGACGGCTCCTCTGGGACGG660 
GTAACTTGACCGCAACCTTCGTGCCGTCGTCTTATTTATACTCTTGATGTCGTGGTCGAA720 
GAAGTAGTCCCTGTAGTAGTTTGGTCTGGGCGGGTTCTTGAACGTCTACTTCGGAAACTT780 
CTTGAGTGTCCACCTCCAGTCGACCCTCATGGGACTGAGGACCTCGTGAGGGGTAAGGAT840 
GAAGAGGGAGTTCAAGAAACAAGCTTAGGTCGCGTTCTTTCTTTTCTACTTCCTCTGTCT900 
CCTCCCCACATTGGTCTTTCCACGCAAGGAGCATCTCTTCTGTAGATGGCTTCAGGTTAC960 
GTTTCCGCCCTTACAGACGCACGTTCGAGTCCTAGCGATAATGTTAAGGAGTACGTCGTT1020 
CACCCGTACACAAGGGACGTCCCAGGCTAGGATCTTAAGG1060 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 335 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
MetGlyProGlnLysLeuThrIleSerTrpPheAlaIleValLeuLeu 
151015 
ValSerProLeuMetAlaMetTrpGluLeuGluLysAspValTyrVal 
202530 
ValGluValAspTrpThrProAspAlaProGlyGluThrValAsnLeu 
354045 
ThrCysAspThrProGluGluAspAspIleThrTrpThrSerAspGln 
505560 
ArgHisGlyValIleGlySerGlyLysThrLeuThrIleThrValLys 
65707580 
GluPheLeuAspAlaGlyGlnTyrThrCysHisLysGlyGlyGluThr 
859095 
LeuSerHisSerHisLeuLeuLeuHisLysLysGluAsnGlyIleTrp 
100105110 
SerThrGluIleLeuLysAsnPheLysAsnLysThrPheLeuLysCys 
115120125 
GluAlaProAsnTyrSerGlyArgPheThrCysSerTrpLeuValGln 
130135140 
ArgAsnMetAspLeuLysPheAsnIleLysSerSerSerSerSerPro 
145150155160 
AspSerArgAlaValThrCysGlyMetAlaSerLeuSerAlaGluLys 
165170175 
ValThrLeuAspGlnArgAspTyrGluLysTyrSerValSerCysGln 
180185190 
GluAspValThrCysProThrAlaGluGluThrLeuProIleGluLeu 
195200205 
AlaLeuGluAlaArgGlnGlnAsnLysTyrGluAsnTyrSerThrSer 
210215220 
PhePheIleArgAspIleIleLysProAspProProLysAsnLeuGln 
225230235240 
MetLysProLeuLysAsnSerGlnValGluValSerTrpGluTyrPro 
245250255 
AspSerTrpSerThrProHisSerTyrPheSerLeuLysPhePheVal 
260265270 
ArgIleGlnArgLysLysGluLysMetLysGluThrGluGluGlyCys 
275280285 
AsnGlnLysGlyAlaPheLeuValGluLysThrSerThrGluValGln 
290295300 
CysLysGlyGlyAsnValCysValGlnAlaGlnAspArgTyrTyrAsn 
305310315320 
SerSerCysSerLysTrpAlaCysValProCysArgValArgSer 
325330335 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 61 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
CCGCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTTCCATGGAGC60 
T61 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 61 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CCATGGAAGATCCGCCGCCACCCGACCCACCACCGCCCGAGCCACCGCCACCGGCGGAGC60 
T61 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CAGAGTGAAAATGAAGCT18 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
GAAGCTCTGCATCCTGCT18 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 62 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GGGTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTTCCA60 
TG62 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GATCCATGGAAGATCCGCCGCCACCCGACCCACCACCGCCCGAGCCACCGCCACCGGATC60 
GGACCCTGCA70 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CTATTACAATTCCTCATG18 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GAGGGCAAGGGTGGCCAA18 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 67 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
TTGCTGGAGCTCCGCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATC60 
TATGTGG67 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 67 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CACATAGATCCGCCGCCACCCGACCCACCACCGCCCGAGCCACCGCCACCGGCGGAGCTC60 
CAGCAAA67 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 82 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CTGGCCTGCAGGGTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGC60 
GGATCTAGGGTCATTCCAGTCT82 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 82 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CTGGAATGACCCTAGATCCGCCGCCACCCGACCCACCACCGCCCGAGCCACCGCCACCGG60 
ATCGGACCCTGCAGGCCAGAGA82 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
GCAAAGGCGGGAATGTCT18 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
AGGAATAATGTTTCAGTT18 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CAGCAGTGCAGGAATAAT18 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 75 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
CCCTGCAGGGTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA60 
TCTTCCATGGGTCAA75 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 75 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CCCTGCAGGGTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGA60 
TCTTCCATGGGTCAA75 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 96 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
TGTGTTCCCTGCAGGGTCCGATCCGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGC60 
GGCGGATCTAGGGTCATTCCAGTCTCTGGACCTGCC96 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
GTCATCTTCTTCAGGCGT18 
__________________________________________________________________________