Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-02-10
2003-05-27
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C424S093200, C435S320100, C435S419000, C536S023500
Reexamination Certificate
active
06570069
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
FIELD OF THE INVENTION
The present invention is directed to polynucleotides encoding inhibitors of apoptosis. The polynucleotides can be used to control apoptosis in target cells.
BACKGROUND OF THE INVENTION
Apoptosis denotes a type of programmed cell death in which the cell nucleus shrinks, the genetic material (DNA) progressively degrades, and the cell collapses (see, e.g. Kawabe, et al.
Nature
349:245-248 (1991). In many organisms, apoptosis plays an important physiological role in development, tissue homeostasis, eradication of virus-infected cells, and other events (Wyllie, A. H., Kerr, J. F. R. & Currie, A. R. (1980)
Int. Rev. Cytol.
68, 251-306)). Caspases are a family of intracellular proteases responsible for execution of the apoptotic program (Salvesen, G. S. & Dixit, V. M. (1997)
Cell
91, 443-446). They are initially synthesized as inactive zymogens that are activated by proteolytic processing, generating the requisite large- and small-subunits which comprise the active caspase enzyme. The functional conservation of caspases in inducing apoptosis within various insect, plant, and animal species makes them appropriate targets for influencing the apoptosis process.
Some viruses harbor genes which encode caspase inhibitory proteins, thereby suppressing host defense mechanisms which would otherwise eliminate virus-infected cells by apoptosis. Examples of viral caspase inhibitors include the baculoviral p35 protein (Clem, R. J. et al., (1991)
Science
254, 1388-1390 and the crmA protein of the Poxviridae-family cowpox virus (Ray, C., et al., (1992)
Cell
69, 597-604). IAP family proteins were first discovered in baculoviruses (Birnbaum, M., et al., (1994)
Journal of Virology
68:2521-2525; Crook, N. E., et al., (1993)
J. Virol.
67: 2168-2174). Genetic complementation analysis revealed that the Inhibitor of Apoptosis Protein (“IAP”) genes of the CpGV and OpMNPV baculoviruses can rescue p35-deficient viruses, maintaining host cell survival so that viral replication successfully occurs (Birnbaum, M., (1994), supra, Crook, N. E., et al., (1993) supra). Baculoviral IAPs contain two tandem copies of a Baculovirus Inhibitory Repeat (BIR) domain followed by a C-terminal RING domain. Mutagenesis studies suggest a requirement for both the BIR and RING domains for their anti-apoptotic function in insect cells. Since these initial discoveries, cellular IAP homologs have been found in many animal species, including Drosophila, mammals, and humans (reviewed in (Miller, L. (1999)
Trends in Cell Biology
9:323-328; Deveraux, Q. & Reed, J. C. (1998)
Genes Dev.
13:239-252)). All cellular IAPs contain one to three copies of a baculoviral inhibitory repeat (BIR) domain and most also contain a RING domain located near their C-termini. A mechanism for IAP-family proteins was shown when it was reported that several human IAPs, including XIAP, cIAP1, cIAP2, can directly bind and inhibit certain caspases, including caspases-3, -7, and -9 (Deveraux, Q., et al., (1999)
EMBO J., in press.; Deveraux, Q. & Reed, J. C. (
1998) supra; Deveraux, Q., et al. (1997)
Nature
388:300-303; Roy, N., et al., (1997)
EMBO J.
16, 6914-6925). Subsequent deletional analysis indicated that the second BIR domain (BIR2) of XIAP is sufficient for inhibiting mammalian caspases-3 and -7 (Roy, N., et al., (1997), supra; Takahashi, R. et al., (1998)
J. Biol. Chem.
273, 7787-7790). However, recently it was shown that a fragment of XIAP encompassing the third BIR domain (BIR3) and RING domain specifically inhibits mammalian caspase-9 (Takahashi, R. et al., (1998), supra). Thus, among mammalian IAPs, different regions of these proteins appear to mediate inhibitory interactions with specific caspases.
Though other types of mechanisms have not been excluded, it has been suggested that Drosophila and baculovirus IAPs also may inhibit some caspases. It has been shown that Drosophila IAP1 (DIAP1) is able to inhibit drICE and DCP-1 in insect cells and in yeast (Kaiser, W. et al., (1998)
FEBS Lett.
440, 243-248; Hawkins, C., et al., (1999)
Proc. Natl. Acad. Sci. USA
96, 2885-2890). It has also been shown that CpIAP and OpIAP require both BIR and RING domains to inhibit activation of Sf-caspase-1 during baculovirus-induced apoptosis in Sf-21 cells. (Seshagiri, S. & Miller, L. K. (1997)
Proc. Natl. Acad. Sci. USA
94, 13606-13611.)
Interestingly, a group of apoptosis-inducing genes which encode IAP-binding proteins has been identified in Drosophila, including reaper, hid, and grim (White, E. & Cipriani, R. (1989)
Proc. Natl. Acad. Sci. USA
86, 9886-9890). The Reaper, Hid, and Grim proteins contain a homologous 14 amino acid N-terminal domain which is both necessary and sufficient for binding DIAP 1 and for inducing apoptosis (Vucic, D., et al., (1997)
Proc. Natl. Acad. Sci. USA
94, 10183-10188; Vucic, D., et al., (1998)
Mol. Cell. Biol.
18, 3300-3309). Though initially controversial (reviewed in Deveraux, Q. & Reed, J. C. (1998)
Genes Dev.
13, 239-252; Miller, L. (1999)
Trends in Cell Biology
9, 323-328), recent data suggest that Reaper, Hid, and Grim induce apoptosis by inhibiting IAPs thus interfering with IAP-mediated suppression of caspases (Wang, S., et al., (1999)
Cell
98, 453-463).
SUMMARY OF THE INVENTION
The invention provides a cDNA for an Inhibitor of Apoptosis Protein (“IAP”) from
Spodoptera frugiperda
(fall armyworm) (SEQ ID NO:1), as well as nucleic acids that are 85% or more identical to that cDNA. Further, the invention provides polypeptides that are at least 90% identical to a polypeptide encoded by SEQ ID NO:1 (the polypeptide is SEQ ID NO:3). In preferred embodiments, the polypeptides are at least 95% identical to SEQ ID NO:3.
The invention further provides host cells comprising recombinant expression cassettes comprising a promoter operably linked to a polynucleotide at least 85% identical to SEQ ID NO:1, at least 95% identical to SEQ ID NO:1, or that comprises SEQ ID NO:1. The promoter can be inducible or can be constitutive. The host cell can be an insect cell, a plant cell, a mammalian cell. The invention provides recombinant expression cassettes comprising polynucleotides that are 85% or more identical to, 95% or more identical to, or that comprises SEQ ID NO:1. The recombinant expression cassette will typically comprise a promoter, which promoter can be inducible or can be constitutive.
The invention further provides recombinant baculoviruses which have been engineered to contain a nucleic acid which is 85% or more identical to SEQ ID NO:1, which is 95% or more identical to SEQ ID NO:1, or which comprises SEQ ID NO:1. The invention further provides transgenic plants which contain a nucleic acid which is 85% or more identical to SEQ ID NO:1, which is 95% or more identical to SEQ ID NO:1, or which comprises SEQ ID NO:1.
The invention further provides in vitro methods of assaying for compounds capable of specifically binding to an IAP, wherein the method comprises combining an IAP with a test compound and assaying whether the test compound specifically binds to the IAP, where the IAP has a sequence at least 90% or more identical to SEQ ID NO:3. In preferred embodiments, the LAP has a sequence at least 95% identical to SEQ ID NO:3. The IAP can be immobilized on a solid support or can be in an aqueous solution. In some embodiments, the test compound can be bound to a solid support and contacted with the IAP.
The invention further provides in vitro methods of assaying for modulators of IAP activity wherein the method comprises combining an IAP with a test compound and assaying whether the test compound can increase or decrease IAP binding specifically to a capsase polypeptide. The IAP can be immobilized on a solid support or can be in an aqueous solution. In some embodiments, the test compound can be bound to a solid support and contacted with the IAP.
The invention further provides an in vitro method of assaying for the presence of IAP cDNA comprising hybridizing said cDNA to a nucleic acid 85% or more identical to SEQ ID
Hammock Bruce D.
Huang Qihong
Maeda Hiroko
Maeda Susumu
Collins Cynthia
Maeda Hiroko
McElwain Elizabeth F.
Regents of the University of California
Townsend and Townsend / and Crew LLP
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