ZA200100084B - Poly(ADP-ribose)polymerase gene. - Google Patents

Description

0050/49790 ® 20010084 yo .

Novel poly(ADP-ribose) polymerase genes

The present invention relates to novel poly(ADP-ribose) polymerase (PARP) genes and to the proteins derived therefrom; antibodies with specificity for the novel proteins; pharmaceutical and gene therapy compositions which comprise products according to the invention; methods for the analytical determination of the proteins and nucleic acids according to the invention; methods for identifying effectors or binding partners of the proteins according to the invention; methods for determining the activity of such effectors and use thereof for the diagnosis or therapy of pathological states.

In 1966, Chambon and co-workers discovered a 116 kD enzyme which was characterized in detail in subsequent years and is now called

PARP (EC 2.4.2.30) (poly(adenosine-5'-diphosphoribose) polymerase), PARS (poly(adenosine-5'-diphosphoribose) synthase) or ADPRT (adenosine-5'-diphosphoribose transferase). In the plant kingdom (Arabidopsis thaliana) a 72 kD (637 amino acids) PARP was found in 1995 (Lepiniec, L. et al., FEBS Lett. 1995: 364(2): 103-8). It was unclear whether this shorter form of PARP is a plant-specific peculiarity or an artefact (splice variant or the like). The 116 kD PARP enzyme has to date been unique in its activity, which is described below, in the animal kingdom and in humans. It is referred to as PARPl below to avoid ambiguity.

The primary physiological function of PARP 1 appears to be its involvement in a complex repair mechanism which cells have developed to repair DNA strand breaks. The primary cellular response to a DNA strand break appears moreover to consist of

PARPl-catalyzed synthesis of poly(ADP-ribose) from NAD* (cf.

De Murcia, G. et al. (1994) TIBS, 19, 172).

PARP] has a modular molecular structure. Three main functional elements have been identified to date: an N-terminal 46 kD DNA binding domain; a central 22 kD automodification domain to which poly(ADP-ribose) becomes attached, with the PARP1 enzyme activity decreasing with increasing elongation; and a C-terminal 54 kD NAD* 40 binding domain. A leucine zipper region has been found within the automodification domain, indicating possible protein-protein interactions, only in the PARP from Drosophila. All PARPs known to date are presumably active as homodimers. 45 The high degree of organization of the molecule is reflected in the strong conservation of the amino acid sequence. Thus, 62% conservation of the amino acid sequence has been found for PARP1

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0050/49790 o : from humans, mice, cattle and chickens. There are greater structural differences from the PARP from Drosophila. The individual domains themselves in turn have clusters of increased conservation. Thus, the DNA binding region contains two so-called zinc fingers as subdomains (comprising motifs of the type

CX;CX2g8/30HX2C), which are involved in the Zn2+-dependent recognition of DNA single strand breaks or single-stranded DNA overhangs (e.g. at the chromosome ends, the telomeres). The

C-terminal catalytic domain comprises a block of about 50 amino acids (residues 859-908), which is approximately 100% conserved among vertebrates (PARP “signature”). This block binds the natural substrate NAD* and thus governs the synthesis of poly(ADP-ribose) (cf. de Murcia, loc.cit.). The GX3GKG motif in particular is characteristic of PARPs in this block.

The beneficial function described above contrasts with a pathological one in numerous diseases (stroke, myocardial infarct, sepsis etc.). PARP is involved in cell death resulting from ischemia of the brain (Choi, D.W., (1997) Nature Medicine, 3, 10, 1073), of the myocardium (Zingarelli, B., et al (1997),

Cardiovascular Research, 36, 205) and of the eye (Lam, T.T. (1997), Res. Comm. in Molecular Pathology and Pharmacology, 95, 3, 241). PARP activation induced by inflammatory mediators has also been observed in septic shock (Szabo, C., et al. (1997),

Journal of Clinical Investigation, 100, 3, 723). In these cases, activation of PARP is accompanied by extensive consumption of

NAD*. Since four moles of ATP are consumed for the biosynthesis of one mole of NADY, the cellular energy supply decreases drasticallly. The consequence is cell death.

PARP1 inhibitors described in the abovementioned specialist literature are nicotinamide and 3-aminobenzamide. 3,4-Dihydro-5-[4-(1l-piperidinyl)butoxy]~-1(2H)-isoquinolone is disclosed by Takahashi, K., et al (1997), Journal of Cerebral

Blood Flow and Metabolism 17, 1137. Further inhibitors are described, for example, in Banasik, M., et al. (1992) J. Biol.

Chem., 267, 3, 1569 and Griffin, R.J., et al. (1995), Anti-Cancer

Drug Design, 10, 507. 40 High molecular weight binding partners described for human PARP1 include the base excision repair (BER) protein XRCCl (X-ray repair cross-complementing 1) which binds via a zinc finger motif and a BRCT (BRCAl C terminus) module (amino acids 372-524) (Masson, M., et al., (1998) Molecular and Cellular Biology, 18,6, 45 3563).

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Kk Because of the diverse physiological and pathological functions of PARP, a need exists to provide novel PARP homologs. The reason for this is that the provision of homologous PARPs would be particularly important for developing ® 5 novel targets for drugs, and novel drugs, in order to improve diagnosis and/or therapy of pathological states in which PARP,

PARP homologs or substances derived therefrom are involved.

The present invention provides PARP : homologs, preferably from human or non-human mammals, having an amino acid sequence which has ..a) a functional NAD+ binding domain i.e. a PARP “gignature” sequence having the characteristic GX3GKG motif; and b) especially in the N-terminal sequence region, i.e. in the region of the first 200, such as, for example, in the region of the first 100, N-terminal amino acids, no PARP zinc finger sequence motifs of the general formula

CX,CX EX,C in which m is an integral value from 28 or 30, and the X radicals are, independently of one another, any amino acid; and the functional equivalents thereof. N

Since the PARP molecules according to the invention represent in particular functional homologs, they naturally also have a poly (ADP-ribose)-synthesizing activity. The NAD binding domain essentially corresponds to this activity and is localized to the

C terminus.

Thus an essential characteristic of the PARPs according to the invention is the presence of a functional NAD* binding domain (PARP signature) which is located in the C-terminal region of the amino acid sequence (i.e. approximately in the region of the last 400, such as, for example, the last 350 or 300, C-terminal amino acids), in combination with an N-terminal sequence having no zinc finger motifs. Since the zinc finger motifs in known PARPs presumably contribute to recognition of the DNA breakages, it is to be assumed that the proteins according to the invention do not 40 interact with DNA or do so in another way. It has been demonstrated by appropriate biochemical tests that the PARP2 according to the invention can be activated by ‘activated DNA’ (i.e. DNA after limited DNaselI digestion). It can be concluded from this further that the PARP? according to the invention has 45 DNA binding properties. However, the mechanism of the DNA binding and enzyme activation differs between the PARPs according to the invention and PARP1. Its DNA binding and enzyme activation is, as

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0050/49790 ] ¢ 4 ) mentioned, mediated by a characteristic zinc finger motif. No such motifs are present in the PARPs according to the invention.

Presumably these properties are mediated by positively charged amino acids in the N-terminal region of the PARPs according to the invention. Since the ‘activated DNA’ (i.e. for example DNA after limited treatment with DNaseI) has a large number of defects (single strand breaks, single strand gaps, single- stranded overhangs, double strand breaks etc.), it is possible that although PARP1 and the PARPs according to the invention are activated by the same ‘activated DNA‘, it is by a different subpopulation of defects (e.g. single strand gaps instead of single strand breaks).

The functional NAD* binding domain (i.e. catalytic domain) binds the substrate for the synthesis of poly(ADP-ribose). Consistent with known PARPs, the sequence motif GX1X2X3GKG, in which G is glycine, K is lysine, and X!, X2 and X3 are, independently of one another, any amino acid, is present in particular. However, as shown, surprisingly, by comparison of the amino acid sequences of the NAD* binding domains of PARP molecules according to the invention with previously disclosed human PARP1l, the sequences according to the invention differ markedly from the known sequence for the NAD* binding domain.

A group of PARP molecules which is preferred according to the invention preferably has the following general sequence motif in the catalytic domain in common:

PXn(S/T)GX3GKGIYFA (SEQ ID NO: 11), in particular (S/T)XGLR(I/V)XPX,(S/T)GX3GKGIYFA (SEQ ID NO: 12), preferably

LLWHG(S/T)X7IL(S/T)XGLR(I/V)XPX,(S/T)GX3GKGIYFAX1SKSAXY (SEQ ID NO: 13) in which (S/T) describes the alternative occupation of this sequence position by S or T, (I/V) describes the alternative occupation of this sequence position by I or V, and n is an integral value from 1 to 5, and the X radicals are, independently : of one another, any amino acid. The last motif is also referred 40 to as the "PARP signature” motif.

The automodification domain is preferably likewise present in the

PARPs according to the invention. It can be located in the region from about 100 to 200 amino acids in front of the N-terminal end 45 of the NAD* binding domain.

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0050/49790 3 PARP homologs according to the invention may additionally comprise, N-terminally of the NAD* binding domain (i.e. about 30 to about B0 amino acids closer to the N terminus), a leucine zipper-like sequence motif of the general formula (L/V)XgLXgLX¢L (SEQ ID NO: 14) in which (L/V) represents the alternative occupation of this sequence position by L or V, and the X radicals are, independently of one another, any amino acid. The leucine zipper motifs observed according to the invention differ distinctly in position from those described for PARP from Drosophila. Leucine zippers may lead to homodimers (two PARP molecules) or heterodimers (one PARP molecule with a binding partner differing therefrom).

The PARP homologs according to the invention preferably additionally comprise, N-terminally of the abovementioned leucine zipper-like sequence motifs, i.e. about 10 to 250 amino acid residues closer to the N terminus, at least another one of the following part-sequence motifs:

LXgNX,YX,QLLX (D/E) XpWGRVG, (motif 1; SEQ ID NO: 15)

AX3FXKX4KTXNXWX5FX3PXK, (motif 2; SEQ ID NO: 16)

QXL(I/L)X2IXgMX;oPLGKLX3QIX¢L, (motif 3; SEQ ID NO: 17)

FYTXIPHXFGX3PP, (motif 4; SEQ ID NO: 18) and

KX3LX,LXDIEXAX,L (motif 5; SEQ ID NO: 19), in which (D/E) describes the alternative occupation of this sequence position by D or E, (I/L) describes the alternative occupation of this sequence position by I or L, b is the integral value 10 or 11, and the X radicals are, independently of one another, any amino acid. It is most preferred for these motifs 1 to 5 all to be present in the stated sequence, with motif 1 being closest to the N terminus.

The abovementioned PARP signature motif is followed in the proteins according to the invention by at least another one of the following motifs: 40 GX3LXEVALG (motif 6; SEQ ID NO: 20)

GX SX 4GX3PX,LXGX,V (motif 7; SEQ ID NO: 21) and

E(Y/F)X;YX30X4YLL (motif 8; SEQ ID NO: 22) in which (Y/F) describes the alternative occupation of this 45 sequence position by Y or F, a is equal to 7 to 9 and X is in each case any amino acid. It is most preferred for the three

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3 C-terminal motifs all to be present and in the stated sequence, with motif 8 being closest to the C terminus.

A preferred PARP structure according to the invention may be @ 5 described schematically as follows:

Motifs 1 to 5/PARP signature/motifs 6 to 8 or motifs 1 to 5/leucine zipper/PARP signature/motifs 6 to 8 it being possible for further amino acid residues, such as, for example, up to 40, to be arranged between the individual motifs “and for further amino acid residues, such as, for example, up to 80, to be arranged at the N terminus and/or at the C terminus.

PARP homologs which are particularly preferred according to the invention are the proteins human PARP2, human PARP3, mouse PARP3 and the functional equivalents thereof. The protein referred to as human PARP2 comprises 570 amino acids (cf. SEQ ID NO:2). The protein referred to as human PARP3 possibly exists in two forms.

Type 1 comprises 533 amino acids (SEQ ID NO:4) and type 2 comprises 540 amino acids (SEQ ID NO:6). The forms may arise through different initiation of translation. The protein referred to as mouse PARP3 exists in two forms which differ from one another by a deletion of 5 amino acids (15 bp). Type 1 comprises: 533 amino acids (SEQ ID NO: 8) and type 2 comprises 528 amino acids (SEQ ID NO:10). The PARP homologs according to the invention differ in their sequences distinctly from the PARP protein known from Arabidopsis thaliana (loc. cit.). Thus, PARP2 and PARP3 for example do not have the peptide sequence AAVLDOWIPD which is characteristic of plant PARP and corresponds to amino acid residues 143 to 152 of the Arabidopsis protein.

The invention further relates to the binding partners for the

PARP homologs according to the invention. These binding partners are preferably selected from a) antibodies and fragments such as, for example, Fv, Fab,

F(ab)’, , thereof b) protein-like compounds which interact, for example via the above leucin€ zipper region or another sequence section, with 40 PARP, and c) low molecular weight effectors which modulate a biological

PARP function such as, for example, the catalytic PARP activity, i.e. NAD*-consuming ADP ribosylation, or the binding to an activator protein or to DNA. 45

The invention further relates to nucleic acids comprising

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0050/49790 / oo a) a nucleotide sequence coding for at least one PARP homolog according to the invention, or the complementary nucleotide sequence thereof; b) a nucleotide sequence which hybridizes with a sequence as specified in a), preferably under stringent conditions; or c) nucleotide sequences which are derived from the nucleotide sequences defined in a) and b) through the degeneracy of the genetic code.

Nucleic acids which are suitable according to the invention comprise in particular at least one of the part-sequences which code for the abovementioned amino acid sequence motifs.

Nucleic acids which are preferred according to the invention comprise nucleotide sequences as shown in SEQ ID NO: 1 and 3, and, in particular, part-sequences thereof which are characteristic of PARP homologs according to the invention, such as, for example, nucleotide sequences comprising a) nucleotides +3 to +1715 shown in SEQ ID NO:1; b) nucleotides +242 to +1843 shown in SEQ ID NO:3; c) nucleotides +221 to +1843 shown in SEQ ID NO:5; d) nucleotides +112 to +1710 shown in SEQ ID NO:7; or e) nucleotides +1 to +1584 shown in SEQ ID NO:9 or part-sequences of a), b), ¢), d) and e) which code for the abovementioned characteristic amino acid sequence motifs of the

PARP homologs according to the invention.

The invention further relates to expression cassettes which comprise at least one of the above-described nucleotide sequences according to the invention under the genetic control of regulatory nucleotide sequences. These can be used to prepare recombinant vectors according to the invention, such as, for example, viral vectors or plasmids, which comprise at least one expression cassette according to the invention.

Recombinant microorganisms according to the invention are transformed with at least one of the abovementioned vectors. 40 .

The invention also relates to transgenic mammals transfected with a vector according to the invention.

The invention further relates to an in vitro detection method, 45 which can be carried out homogeneously or heterogeneously, for

PARP inhibitors, which comprises

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0050/49790 i a) incubating an unsupported or supported polyADP-ribosylatable target with a reaction mixture comprising al) a PARP homolog according to the invention; a2) a PARP activator; and a3) a PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected; b) carrying out the polyADP ribosylation reaction; and c) determining the polyADP ribosylation of the target qualitatively or quantitatively.

The detection method is preferably carried out by preincubating the PARP homolog with the PARP activator and the PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected, for example for about 1-30 minutes, before carrying out the polyADP ribosylation reaction.

After activation by DNA with single strand breaks (referred to as “activated DNA” according to the invention), PARP polyADP ribosylates a large number of nuclear proteins in the presence of

NAD. These proteins include, on the one hand, PARP itself, but also histones etc.

The polyADP-ribosylatable target preferably used in the detection method is a histone protein in its native form or a polyADP-ribosylatable equivalent derived therefrom. A histone preparation supplied by Sigma (SIGMA, catalogue No. H-7755; histone type II-AS from calf thymus, Luck, J. M., et al.,

J. Biol. Chem., 233, 1407 (1958), Satake K., et al., J. Biol.

Chem, 235, 2801 (1960)) was used by way of example. It is possible in principle to use all types of proteins or parts thereof amenable to polyADP-ribosylation by PARP. These are preferably nuclear proteins, e.g. histones, DNA polymerase, telomerase or PARP itself. Synthetic peptides derived from the corresponding proteins can also act as target.

In the ELISA according to the invention it is possible to use amounts of histones in the range from about 0.1 pug/well to about 100 pg/well, preferably about 1 pg/well to about 10 pg/well. The amounts of the PARP enzyme are in a range from about 40 0.2 pmol/well to about 2 nmol/well, preferably from about 2 pmol/well to about 200 pmol/well, the reaction mixture comprising in each case 100 pug/well. Reductions to smaller wells and correspondingly smaller reaction volumes are possible. 45 In the HTRF assay according to the invention, identical amounts of PARP are employed, and the amount of histone or modified histones is in the range from about 2 ng/well to about 25 pg/well,

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0050/49790 oo preferably about 25 ng/well to about 2.5 pg/well, the reaction mixture comprising in each case 50 pl/well. Reductions to smaller wells and correspondingly smaller reaction volumes are possible.

The PARP activator used according to the invention is preferably activated DNA.

Various types of damaged DNA can function as activator. DNA damage can be produced by digestion with DNses or other

DNA-modifying enzymes (e.g. restriction endonucleases), by irradiation or other physical methods or chemical treatment of the DNA. It is further possible to simulate the DNA damage situation in a targeted manner using synthetic oligonucleotides.

In the assays indicated by way of example, activated DNA from calf thymus was employed (Sigma, product No. D4522; CAS: 91080-16-9, prepared by the method of Aposhian and Kornberg using calf thymus DNA (SIGMA D-1501) and deoxyribonuclease type I (D-4263). Aposhian H. V. and Kornberg A., J. Biol. Chem., 237, 519 (1962)). The activated DNA was used in a concentration range from 0.1 to 1000 pg/ml, preferably from 1 to 100 ug/ml, in the reaction step.

The polyADP ribosylation reaction is started in the method according to the invention by adding NAD*. The NAD concentrations were in a range from about 0.1 pM to about 10 mM, preferably in a range from about 10 pM to about 1 mM.

In the variant of the above method which can be carried out heterogeneously, the polyADP ribosylation of the supported target is determined using anti-poly (ADP-ribose) antibodies. To do this, the reaction mixture is separated from the supported target, washed and incubated with the antibody. This antibody can itself be labeled. Alternatively, bound anti-poly(ADP-ribose) antibody is detected using a labeled secondary antibody or a corresponding labeled antibody fragment. Suitable labels are, for example, radiolabeling, chromophore- or fluorophore-labeling, biotinylation, chemiluminescence labeling, labeling with paramagnetic material or, in particular, enzyme labels, e.g. with horseradish peroxidase. Appropriate detection techniques are 40 generally known to the skilled worker.

In the variant of the above process which can be carried out homogeneously, the unsupported target is labeled with an acceptor fluorophore. The target preferably used in this case is 45 biotinylated histone, the acceptor fluorophore being coupled via avidin or streptavidin to the biotin groups of the histone.

Particularly suitable as acceptor fluorophore are

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0050/49790 ; d 10 ) phycobiliproteins (e.g. phycocyanins, phycoerythrins), e.g.

R-phycocyanin (R-PC), allophycocyanin (APC), R-phycoerythrin (R-PE), C-phycocyanin (C-PC), B-phycoerythrin (B-PE) or their combinations with one another or with fluorescent dyes such as

Cy5, Cy7 or Texas Red (Tandem system) (Thammapalerd, N. et al.,

Southeast Asian Journal of Tropical Medicine & Public Health, 27(2): 297-303 (1996); Kronick, M. N. et al., Clinical Chemistry, 29(9), 1582-1586 (1986); Hicks, J. M., Human Pathology, 15(2), 112-116 (1984)). The dye XL665 used in the examples is a crosslinked allophycocyanin (Glazer, A. N., Rev. Microbiol., 36, 173-198 (1982); Kronick, M. N., J. Imm. Meth., 92, 1-13 (1986);

MacColl, R. et al., Phycobiliproteins, CRC Press, Inc., Boca

Raton, Florida (1987); MacColl, R. et al., Arch. Biochem.

Biophys., 208(1), 42-48 (1981)).

It is additionally preferred in the homogeneous method to determine the polyADP ribosylation of the unsupported target using anti-poly(ADP-ribose) antibody which is labeled with a donor fluorophore which is able to transfer energy to the acceptor fluorophore when donor and acceptor are close in space owing to binding of the labeled antibody to the polyADP-ribosylated histone. A a [sic] europium cryptate is preferably used as donor fluorophore for the anti-poly (ADP-ribose) antibody.

Besides the europium cryptate used, other compounds are also possible as potential donor molecules. This may entail, on the one hand, modification of the cryptate cage. Replacement of the europium by other rare earth metals such as terbium is also conceivable. It is crucial that the fluorescence has a long duration to guarantee the time delay (Lopez, E. et al., Clin.

Chem. 39/2, 196-201 (1993); US Patent 5,534,622).

The detection methods described above are based on the principle that there is a correlation between the PARP activity and the amount of ADP-ribose polymers formed on the histones. The assay described herein makes it possible to quantify the ADP-ribose polymers using specific antibodies in the form of an ELISA and an

HTRF (homogenous time-resolved fluorescence) assay. Specific 40 embodiments of these two assays are described in detail in the following examples.

The developed HTRF (homogeneous time-resolved fluorescence) assay system measures the formation of poly(ADP-ribose) on histones 45 using specific antibodies. In contrast to the ELISA, this assay is carried out in homogeneous phase without separation and washing steps. This makes a higher sample throughput and a

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0050/49790 ) smaller susceptibility to errors possible. HTRF is based on the fluorescence resonance energy transfer (FRET) between two fluorophores. In a FRET assay, an excited donor fluorophore can transfer its energy to an acceptor fluorophore when the two are close to one another in space. In HTRF technology, the donor fluorophore is a europium cryptate [(Eu)K] and the acceptor is

XL665, a stabilized allophycocyanin. The europium cryptate is based on studies by Jean Marie Lehn (Strasbourg) (Lopez, E. et al., Clin. Chem. 39/2, 196-201 (1993); US Patent 5,534,622).

In a homogeneous assay, all the components are also present during the measurement. Whereas this has advantages for carrying out the assay (rapidity, complexity), it is necessary to preclude interference by assay components (inherent fluorescence, quenching by dyes etc.). HTRF precludes such interference by time-delayed measurement at two wavelengths (665 nm, 620 nm). The

HTRF has a very long decay time and time-delayed measurement is therefore possible. There is no longer any interference from short-lived background fluorescence (e.g. from assay components or inhibitors of the substance bank). In addition, measurement is always carried out at two wavelengths in order to compensate for quench effects of colored substances. HTRF assays can be carried out, for example, in 96- or 384-well microtiter plate format and are evaluated using a discovery HTRF microplate analyzer (Canberra Packard).

Also provided according to the invention are the following in vitro screening methods for binding partners for PARP, in particular for a PARP homolog according to the invention.

A first variant is carried out by al) immobilizing at least one PARP homolog on a support; bl) contacting the immobilized PARP homolog with an analyte in which at least one binding partner is suspected; and cl) determining, where appropriate after an incubation period, analyte constituents bound to the immobilized PARP homolog.

A second variant entails a2) immobilizing on a support an analyte which comprises at least 40 one possible binding partner for the PARP homolog; b2) contacting the immobilized analyte with at least one PARP homolog for which a binding partner is sought; and c3) examining the immobilized analyte, where appropriate after an incubation period, for binding of the PARP homolog. 45

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0050/49790 4 12 ’ The invention also relates to a method for the qualitative or quantitative determination of a nucleic acid encoding a PARP homolog, which comprises a) incubating a biological sample with a defined amount of an exogenous nucleic acid according to the invention (e.g. with a length of about 20 to 500 bases or longer), hybridizing, preferably under stringent conditions, determining the hybridizing nucleic acids and, where appropriate, comparing with a standard; or b) incubating a biological sample with a defined amount of oligonucleotide primer pairs with specificity for a PARP homolog-encoding nucleic acid, amplifying the nucleic acid, determining the amplification product and, where appropriate, comparing with a standard.

The invention further relates to a method for the qualitative or quantitative determination of a PARP homolog according to the invention, which comprises a) incubating a biological sample with at least one binding partner specific for a PARP homolog, b) detecting the binding partner/PARP complex and, where appropriate, c) comparing the result with a standard.

The binding partner in this case is preferably an anti-PARP antibody or a binding fragment thereof, which carries a detectable label where appropriate.

The determination methods according to the invention for PARP, in particular for PARP homologs and for the coding nucleic acid sequences thereof, are suitable and advantageous for diagnosing sepsis- or ischemia-related tissue damage, in particular strokes, myocardial infarcts, diabetes or septic shock.

The invention further comprises a method for determining the efficacy of PARP effectors, which comprises a) incubating a PARP homolog according to the invention with an analyte which comprises an effector of a physiological or 40 pathological PARP activity; removing the effector again where appropriate; and b) determining the activity of the PARP homolog, where appropriate after adding substrates or cosubstrates. 45 The invention further relates to gene therapy compositions which comprise in a vehicle acceptable for gene therapy a nucleic acid construct which

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0050/49790 @® 13 ) a) comprises an antisense nucleic acid against a coding nucleic acid according to the invention; or b) a ribozyme against a noncoding nucleic acid according to the invention; or ¢) codes for a specific PARP inhibitor.

The invention further relates to pharmaceutical compositions comprising, in a pharmaceutically acceptable vehicle, at least one PARP protein according to the invention, at least one PARP binding partner according to the invention or at least one coding nucleotide sequence according to the invention.

Finally, the invention relates to the use of binding partners of a PARP homolog for the diagnosis or therapy of pathological states in the development and/or progress of which at least one

PARP protein, in particular a PARP homolog according to the invention, or a polypeptide derived therefrom, is involved. The binding partner used can be, for example, a low molecular weight binding partner whose molecular weight can be, for example, less than about 2000 dalton or less than about 1000 dalton.

The invention additionally relates to the use of PARP binding partners for the diagnosis or therapy of pathological states mediated by an energy deficit. An energy deficit for the purpose of the present invention is, in particular, a cellular energy deficit which is to be observed in the unwell patient systemically or in individual body regions, organs or organ regions, or tissues or tissue regions. This is characterized by an NAD and/or ATP depletion going upwardly or downwardly beyond the physiological range of variation of the NAD and/or ATP level and mediated preferably by a protein with PARP activity, in particular a PARP homolog according to the invention, or a polypeptide derived therefrom. “Disorders mediated by energy deficits” furthermore comprise for the purpose of the invention those in which tissue damage is attributable to cell death as a result of necrosis or apoptosis.

The methods according to the invention are suitable for the treatment and prevention of tissue damage resulting from cell 40 damage due to apoptosis or necrosis; damage to nervous tissue due to ischemias and/or reperfusion; neurological disorders; neurodegenerative disorders; vascular stroke; for the treatment and prevention of cardiovascular disorders; for the treatment of other disorders or conditions such as, for example, age-related 45 macular degeneration, AIDS or other immunodeficiency disorders; arthritis; atherosclerosis; cachexia; cancer; degenerative disorders of the skeletal muscles; diabetes; cranial trauma;

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L) inflammatory disorders of the gastrointestinal tract such as, for example, Crohn’s disease; muscular dystrophy; osteoarthritis; osteoporosis; chronic and/or acute pain; kidney failure; retinal ischemia; septic shock (such as, for example, endotoxin shock); ® 5 aging of the skin or aging in general; general manifestations of aging. The methods according to the invention can additionally be employed for prolonging the lifespan and the proliferative capacity of body cells and for sensitizing tumor cells in irradiation therapy.

The invention particularly relates to the use of a PARP binding .- partner as defined above for the diagnosis or therapy (acute or prophylactic) of pathological states mediated by energy deficits and selected from neurodegenerative disorders, or tissue damage caused by sepsis or ischemia, in particular of neurotoxic disturbances, strokes, myocardial arrest, damage during or after infarct lysis (e.g. with TPa, reteplase or mechanically with laser or Rotablator) and of microinfarcts during and after heart valve replacement, aneurysm resections and heart transplants, trauma to the head and spinal cord, infarcts of the kidney (acute kidney failure, acute renal insufficiency or damage during and after kidney transplant), infarcts of the liver (liver failure, damage during or after a liver transplant), damage to skeletal muscles, peripheral neuropathies, AIDS dementia, septic’ shock, diabetes, neurodegenerative disorders occurring after ischemia, trauma (craniocerebral trauma), massive bleeding, subarachnoid hemorrhages and stroke, and neurodegenerative disorders such as Alzheimer’s disease, multi-infarct dementia,

Huntington'’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, epilepsy, especially of generalized epileptic seizures such as petit mal and tonoclonic seizures and partial epileptic seizures, such as temporal lobe, and complex partial seizures, kidney failure, also in the chemotherapy of tumors and prevention of metastasis and for the treatment of inflammations and rheumatic disorders, e.g. of rheumatoid arthritis; further for the treatment of revascularization of critically narrowed coronary arteries and critically narrowed peripheral arteries, e.g. leg arteries. 40 “Ischemia” for the purpose of the invention comprises a localized undersupply of oxygen to a tissue caused by blockage of arterial blood flow. Global ischemia occurs when the blood flow to the whole brain is interrupted for a limited period. This may be caused, for example, by cardiac arrest. Focal ischemia occurs 45 when a part of the brain is cut off from its normal blood supply.

Focal ischemia may be caused by thromboembolic occlusion of a blood vessel, by cerebral trauma, edema or cerebral tumor. Even

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0050/49790 ® 15 temporary ischemias may lead to extensive neuronal damage.

Although damage to “nervous tissue” may appear days or weeks after the start of the ischemia, some permanent damage (for example necrotic cell death) occurs in the first few minutes after interruption of the blood supply. This damage is caused, for example, by glutamate neurotoxicity and the consequences of secondary reperfusion, such as, for example, release of free radicals (for example oxygen free radicals, NO free radicals).

Ischemias may likewise occur in other organs and tissues such as, for example, in the heart (myocardial infarction and other cardiovascular disorders caused by occlusion of coronary arteries) or in the eye (ischemia of the retina).

The invention additionally relates to the use of an effective therapeutic amount of a PARP binding partner for influencing neuronal activity. “Neuronal activity” may consist for the purpose of the invention of stimulation of damaged neurons, promotion of neuronal regeneration or treatment of pathological states of neurons. “Neuronal damage” comprises for the purpose of the invention every type of damage to “nerve tissue” and every physical or mental impairment or death resulting from this damage. The cause of the damage may be, for example, of a metabolic, toxic, chemical or thermal nature and includes by way of example ischemias, hypoxias, trauma, cerebrovascular damage, operations, pressure, hemorrhages, irradiation, vasospasms, neurodegenerative disorders, infections, epilepsy, perception disorders, disorders of glutamate metabolism and the secondary effects caused thereby. “Nervous tissue” comprises for the purpose of the invention the various components which form the nervous system selected from, inter alia, neurons, glia cells, astrocytes, Schwann cells, the vascular system inside and for supply, the CNS, brain, brain stem, spinal cord, peripheral nervous system etc. “Neuroprotective” for the purpose of the invention comprises reducing, stopping, slowing down or improving neuronal damage and protecting, reviving and regenerating nervous tissue which has 40 been exposed to neuronal damage. “Prevention of neurodegenerative disorders” includes the possibility of preventing, slowing down and improving neurodegenerative disorders in people for whom such a disorder 45 has been diagnosed or who belong to corresponding risk groups for these neurodegenerative disorders. Likewise meant is the

M/40043 }

AMENDLD SHILA

} 0050749790

L 16 ’ treatment of people already suffering from symptoms of these disorders. ® “Treatment” for the purpose of the invention comprises (i) preventing a disorder, a disturbance or a state in people with a predisposition thereto; (ii) preventing a disorder, a disturbance or a state by slowing down its advance; : (iii) improving a disorder, a disturbance or a state.

Examples of “neurological disorders” which can be treated by the methods according to the invention are neuralgias (tigeminal, glossopharyngeal), myasthenia gravis, muscular dystrophies, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, peripheral neuropathies caused by poisoning (for example lead poisoning), Guillain-Barré syndrome, Huntington's disease, :

Alzheimer’s disease, Parkinson’s disease or Plexus disorders. The methods according to the invention are preferably suitable for treating neurological disorders selected from peripheral neuropathies caused by physical injury or disease; cranial trauma * such as, for example, traumatic brain injury; physical damage to the spinal cord; stroke associated with brain damage such as vascular stroke in conjunction with hypoxia and brain damage, and cerebral reperfusion damage; demyelinizing disorders (myelopathies, Alzheimer’s disease, Parkison disease,

Huntington’s disease, amyotrophic lateral sclerosis).

The methods according to the invention can also be used for treating cardiovascular disorders. “Cardiovascular disorders” for the purpose of the invention comprise those which cause ischemias or are caused by ischemias or ischemia/reperfusion of the heart.

Examples are coronary vascular disorders (for example atherosclerosis), angina pectoris, myocardial infarction, cardiovascular damage due to cardiac arrest or bypass operation.

The methods according to the invention can be used for treating 40 cancer or for sensitizing cancer cells in irradiation therapy.

The term “cancer” is to be understood in its widest sense.

Modulators of the proteins according to the invention can be used as “anticancer therapeutics”. The methods can, for example, be used for treating types of cancer or tumor cells such as 45 ACTH-producing tumors, acute lymphatic or lymphoblastic leukemia; acute or chronic lymphocytic leukemia; acute non-lymphocytic leukemia; bladder cancer, brain tumors; breast cancer; cervical

M/40043 :

0050/49790 o 17 carcinoma; chronic myelocytic leukemia; bowel cancer; T-zone lymphoma; endometriosis; esophageal cancer; gall bladder cancer;

Ewing's sarcoma; cancer of the head and neck; cancer of the tongue; Hodgkin’s lymphoma; Kaposi's sarcoma; renal cancer; hepatic cancer; lung cancer; mesothelioma; multiple myeloma; neroblastoma; non-Hodgkin lymphoma; osteosarcoma; ovarian carcinoma; gliablastoma; carcinoma of the breast; cervical carcinoma; prostate cancer; pancreatic cancer; penis cancer; retinoblastoma; skin cancer; stomach cancer, thyroid cancer; uterine carcinoma; vaginal carcinoma; Wilm’s tumor; or trophoblastoma. “Radiosensitizers” or “irradiation sensitizers” for the purpose of the invention relate to molecules which increase the sensitivity of the cells in the body to irradiation with electromagnetic radiation (for example x-rays) or expedite this irradiation treatment. Irradiation sensitizers increase the sensitivity of cancer cells to the toxic effects of the electromagnetic radiation. The literature discloses inter alia mitomycin C, 5-bromodeoxyuridine and metronidazole. It is possible to use radiation with wavelengths in the range from 10-20 to 10 meters, preferably gamma radiation (10-20 to 10-13 m), x-rays (10-11 to 10-2 m), ultraviolet radiation (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1 mm) and microwave radiation (1 mm to 30 cm).

Disorders which can be treated by such a therapy are, in particular, neoplastic disorders, benign or malignant tumors and cancer. The treatment of other disorders using electromagnetic radiation is likewise possible.

The present invention will now be described in more detail with reference to the appended figures. These show:

In Figure 1 a sequence alignment of human PARP (human PARP1l) and two PARPs preferred according to the invention (human PARP2, human PARP3, murine PARP3). Sequence agreements between human

PARP1 and human PARP2, human PARP3 or murine PARP3 are depicted within frames. The majority sequence is indicated over the 40 alignment. The zinc finger motifs of human PARP1 are located in the sequence sections corresponding to amino acid residues 21 to 56 and 125 to 162;

In Figure 2 Northern blots with various human tissues to 45 illustrate the tissue distribution of PARP2 and PARP3 molecules according to the invention. Lane 1: brain; lane 2: heart; lane 3: skeletal muscle; lane 4: large bowel; lane 5: thymus; lane 6:

M/40043 :

0050/49790 ® 18 ’ spleen; lane 7: kidney; lane 8: liver; lane 9: small bowel; lane 10: placenta; lane 11: lung; lane 12: peripheral blood leukocytes; the respective position of the size standard (kb) is indicated.

In Figure 3 a Northern blot with further various human tissues to illustrate the tissue distribution of the PARP3 molecule according to the invention. Lane 1: heart; lane 2: brain; lane 3: placenta; lane 4: lung; lane 5: liver; lane 6: skeletal muscle; lane 7: kidney; lane 8: pancreas; the respective position of the size standard (kb) is indicated.

In Figure 4 a Western blot with various human tissues to illustrate the tissue distribution of the PARP3 molecule according to the invention at the protein level. Lane 1: heart; lane 2: lung; lane 3: liver; lane 4: spleen; lane 5: kidney; lane 6: large bowel; lane 7: muscle; lane 8: brain; the respective position of the size standard (kD) is indicated.

In Figure 5 a Western blot with various human tissues to illustrate the tissue distribution of the PARP3 molecule according to the invention. Lane 1: frontal cortex; lane 2: posterior cortex; lane 3: cerebellum; lane 4: hippocampus; lane 5: olfactory bulb; lane 6: striatum; lane 7: thalamus; lane 8: midbrain; lane 9: entorhinal cortex; lane 10: pons; lane 11: medulla; lane 12: spinal cord.

In Figure 6 a diagrammatic representation of the PARP assay (ELISA)

In Figure 7 a diagrammatic representation of the PARP assay (HTRF)

Further preferred embodiments of the invention are described in the following sections.

PARP homologs and functional equivalents

Unless stated otherwise, for the purposes of the present 40 description amino acid sequences are indicated starting with the

N terminus. If the one-letter code is used for amino acids, then

G is glycine, A is alanine, V is valine, L is leucine, I is isoleucine, S is serine, T is threonine, D is aspartic acid, N is asparagine, E is glutamic acid, Q is glutamine, W is tryptophan, 45 H is histidine, R is arginine, P is proline, K is lysine, Y is tyrosine, F is phenylalanine, C is cysteine and M is methionine.

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] 0050/49790 ® 19

The present invention is not confined to the PARP homologs specifically described above. On the contrary, those homologs which are functional equivalents thereof are also embraced.

Functional equivalents comprise both natural, such as, for example, species-specific or organ-specific, and artificially produced variants of the proteins specifically described herein.

Functional equivalents according to the invention differ by addition, substitution, inversion, insertion and/or deletion of one or more amino acid residues of human PARP2 (SEQ ID NO:2), human PARP3 (SEQ ID NO: 4 and 6) and mouse PARP3 (SEQ ID:8 and 10), there being at least retention of the NAD-binding function of the protein mediated by a functional catalytic C-terminal domain. Likewise, the poly(ADP-ribose)-producing catalytic activity should preferably be retained. Functional equivalents also comprise where appropriate those variants in which the region similar to the leucine zipper is essentially retained.

It is moreover possible, for example, starting from the sequence for human PARP2 or human PARP3 to replace certain amino acids by those with similar physicochemical properties (bulk, basicity, hydrophobicity, etc.). It is possible, for example, for arginine residues to be replaced by lysine residues, valine residues by isoleucine residues or aspartic acid residues by glutamic acid residues. However, it is also possible for one or more amino acids to be exchanged in sequence, added or deleted, or several of these measures can be combined together. The proteins which have been modified in this way from the human PARP2 or human

PARP3 sequence have at least 60%, preferably at least 75%, very particularly preferably at least 85%, homology with the starting sequence, calculated using the algorithm of Pearson and Lipman,

Proc. Natl. Acad. Sci (USA) 85(8), 1988, 2444-2448,

The following homologies have been determined at the amino acid level and DNA level between human PARP1l, 2 and 3 (FastA program,

Pearson and Lipman, loc. cit.):

Amino acid homologies: 40 Percent identity Percent identity in

PARP signature

EE CE EY

Se FT PTE

EE FE FR

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0050/49790 ® 20

Co Numbers in parentheses indicate the number of overlapping amino acids.

DNA Homologies:

Percent identity Percent identity in the ORF in

PARP signature eevee [so.ens as) [ress en)

EY PTT FS lessor/onres [e0.22s 269) [es.aen 22) se

Numbers in parentheses indicate the number of overlapping nucleotides.

The polypeptides according to the invention can be classified as homologous poly(ADP-ribose) polymerases on the basis of the great similarity in the region of the catalytic domain.

It is also essential to the invention that the novel PARP homologs do not have conventional zinc finger motifs. This means that these enzymes are not necessarily involved in DNA repair or are so in a way which differs from PARP1, but are still able to carry out their pathological mechanism (NAD* consumption and thus energy consumption due to ATP consumption). The strong protein expression, particularly of PARP3, observable in the Western blot suggests a significant role in the NAD consumption. This is particularly important for drug development. Potential novel inhibitors of the polymerases according to the invention can thus inhibit the pathological functions without having adverse effects on the desired physiological properties. This was impossible with inhibitors against the PARPs known to date since there was always also inhibition of the DNA repair function. The potentially mutagenic effect of known PARP inhibitors is thus easy to understand. It is also conceivable to design PARP inhibitors so that they efficiently inhibit all PARP homologs with high 40 affinity. In this case, a potentiated effect is conceivable where appropriate.

The PARP homolog which is preferred according to the invention and is shown in SEQ ID NO:2 (human PARP2) can advantageously be 45 isolated from human brain, heart, skeletal muscle, kidney and

M/40043 )

0050/49790 ® 21 oo liver. The expression of human PARP2 in other tissues or organs is distinctly weaker.

The PARP homolog which is preferred according to the invention and is shown in SEQ ID NO: 4 and 6 (human PARP3) can advantageously be isolated from human brain (in this case preferably from the hippocampus), heart, skeletal muscle, liver or kidney. The expression of human PARP3 in other tissues or organs, such as muscle or liver, is distinctly weaker.

The skilled worker familiar with protein isolation will make use of the combination of preparative methodologies which is most suitable in each case for isolating natural PARPs according to the invention from tissues or recombinantly prepared PARPs according to the invention from cell cultures. Suitable standard preparative methods are described, for example, in Cooper, T.G.,

Biochemische Arbeitsmethoden, published by Walter de Gruyter,

Berlin, New York or in Scopes, R. Protein Purification, Springer

Verlag, New York, Heidelberg, Berlin.

The invention additionally relates to PARP2 and PARP3 homologs which, although they can be isolated from other eukaryotic species, i.e. invertebrates or vertebrates, especially other mammals such as, for example, mice, rats, cats, dogs, pigs, sheep, cattle, horses or monkeys, or from other organs such as, for example the myocardium, have the essential structural and functional properties predetermined by the PARPs according to the invention.

In particular, the human PARP2 which can be isolated from human brain, and its functional equivalents, are preferred agents for developing inhibitors of neurodegenerative disorders such as stroke. This is because it can be assumed that drug development based on PARP2 as indicator makes it possible to develop inhibitors which are optimized for use in the human brain.

However, it cannot be ruled out that inhibitors developed on the basis of PARP2 can also be employed for treating PARP-mediated pathological states in other organs too (cf. tissue distribution of the proteins according to the invention). 40

PARPZ2 and presumably PARP3 are also, similar to PARP1l, activated by damaged DNA, although by a presumably different mechanism.

Significance in DNA repair is conceivable. Blockade of the PARPs according to the invention would also be beneficial in 45 indications such as cancer (e.g. in the radiosensitization of tumor patients).

M/40043

0050/49790 ® 22 oo Another essential biological property of PARPs according to the invention and their functional equivalents is to be seen in their ability to bind an interacting partner. Human PARP2 and 3 differ from previously disclosed PARPs from higher eukaryotes such as, in particular, mammals by having potential so-called leucine zipper motifs. This is a typical motif for protein-protein interactions. It is possible that these motifs permit modulation of PARP activity by an interacting partner. This additional structural element thus also provides a possible starting point for development of PARP effectors such as, for example, inhibitors.

The invention thus further relates to proteins which interact with PARP2 and/or 3, preferably those which bring about their activation or inactivation.

The invention further relates to proteins which still have the abovementioned ligand-binding activity and which can be prepared starting from the specifically disclosed amino acid sequences by targeted modifications.

It is possible, starting from the peptide sequence of the proteins according to the invention, to generate synthetic peptides which are employed, singly or in combination, as antigens for producing polyclonal or monoclonal antibodies. It is also possible to employ the PARP protein or fragments thereof for generating antibodies. The invention thus also relates to peptide fragments of PARP proteins according to the invention which comprise characteristic part-sequences, in particular those oligo- or polypeptides which comprise at least one of the abovementioned sequence motifs. Fragments of this type can be obtained, for example, by proteolytic digestion of PARP proteins or by chemical synthesis of peptides.

Novel PARP2 and PARP3 binding partners

Active and preferably selective inhibitors against the proteins according to the invention were developed using the specific assay systems described above for binding partners for PARP2 and 40 PARP3. These inhibitors are where appropriate also active against

PARP1.

Inhibitors provided according to the invention have a strong inhibitory activity on PARP2. The Kj values may in this case be 45 less than about 100 nM, such as less than about 700 nM, less than about 200 nM and less than about 30 nM, e.g. about 1 to 20 nM.

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] 0050/49790

C

Inhibitors provided according to the invention may additionally have a surprising selectivity for PARP2. The Kj; (PARP1):K; (PARP2) ratio for such inhibitors according to the invention is, for example, greater than 3 or greater than 5, such as, for example, greater than 10, or greater than 20.

An example which should be mentioned is 4-(N-(4-hydroxyphenyl)aminomethyl)-(2H)~dihydrophthalazin-1l-one.

This and analogous compounds are prepared in accordance with the instructions in Puodzhyunas et al., Pharm. Chem. J., 1973, 7, 566 et seq., or Mazkanowa et al., Zh. Obshch. Khim., 1958, 28, 2798 et seq., or Mohamed et al., Ind. J. Chem. B. 1994, 33, 769 et seq., to which express reference is hereby made.

The abovementioned compound has a K; for PARP2 of 113 nM and acts about 8-fold more selectively on PARP2 than on PARPI.

Nucleic acids coding for PARP homologs:

Unless stated otherwise, nucleotide sequences are indicated in the present description from the 5’ to the 3’ direction.

The invention further relates to nucleic acid sequences which code for the abovementioned proteins, in particular for those having the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8 and 10, but without being restricted thereto. Nucleic acid sequences which can be used according to the invention also comprise allelic variants which, as described above for the amino acid sequences, are obtainable by deletion, inversion, insertion, addition and/or substitution of nucleotides, preferably of nucleotides shown in SEQ ID NO: 1, 3, 7 and 9, but with essential retention of the biological properties and the biological activity of the corresponding gene product. Nucleotide sequences which can be used are obtained, for example, by nucleotide substitutions which cause silent (without alteration of the amino acid sequence) or conservative amino acid changes (exchange of amino acids of the same size, charge, polarity or solubility).

Nucleic acid sequences according to the invention also embrace 40 functional equivalents of the genes, such as eukaryotic homologs for example from invertebrates such as Caenorhabditis or

Drosophila, or vertebrates, preferably from the mammals described above. Preferred genes are those from vertebrates which code for a gene product which has the properties essential to the 45 invention as described above.

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0050/49790 ® 24

The nucleic acids according to the invention can be obtained in a conventional way by various routes:

For example, a genomic or a cDNA library can be screened for DNA which codes for a PARP molecule or a part thereof. For example, a cDNA library obtained from human brain, heart or kidney can be screened with a suitable probe such as, for example, a labeled single-stranded DNA fragment which corresponds to a part-sequence of suitable length selected from SEQ ID NO: 1 or 3, or sequence complementary thereto. For this purpose, it is possible, for example, for the DNA fragments of the library which have been transferred into a suitable cloning vector to be, after transformation into a bacterium, plated out on agar plates. The clones can then be transferred to nitrocellulose filters and, after denaturation of the DNA, hybridized with the labeled probe.

Positive clones are then isolated and characterized.

The DNA coding for PARP homologs according to the invention or partial fragments can also be synthesized chemically starting from the sequence information contained in the present application. For example, it is possible for this purpose for oligonucleotides with a length of about 100 bases to be synthesized and sequentially ligated in a manner known per se by, for example, providing suitable terminal restriction cleavage sites.

The nucleotide sequences according to the invention can also be prepared with the aid of the polymerase chain reaction (PCR). For this, a target DNA such as, for example, DNA from a suitable full-length clone is hybridized with a pair of synthetic oligonucleotide primers which have a length of about 15 bases and which bind to opposite ends of the target DNA. The sequence section lying between them is then filled in with DNA polymerase.

Repetition of this cycle many times allows the target DNA to be amplified (cf. White et al. (1989), Trends Genet. 5, 185).

The nucleic acid sequences according to the invention are also to be understood to include truncated sequences, single-stranded DNA or RNA of the coding and noncoding, complementary DNA sequences, 40 mRNA sequences and cDNAs derived therefrom.

The invention further embraces nucleotide sequences hybridizing with the above sequences under stringent conditions. Stringent hybridization conditions for the purpose of the present invention 45 exist when the hybridizing sequences have a homology of about 70 to 100%, such as, for example about 80 to 100% or 90 to 100% (preferably in an amino acid section of at least about 40, such

M/40043

0050/49790 ® 25 as, for example, about 50, 100, 150, 200, 400 or 500 amino acids).

Stringent conditions for the screening of DNA, in particular cDNA banks, exist, for example, when the hybridization mixture is washed with 0.1X SSC buffer (20X SSC buffer = 3M NaCl, 0.3M sodium citrate, pH 7.0) and 0.1% SDS at a temperature of about 600°C.

Northern blot analyses are analyses are washed under stringent conditions with 0.1X SSC, 0,1% SDS at a temperature of about 65°C, for example.

Nucleic acid derivatives and expression constructs:

The nucleic acid sequences are also to be understood to include derivatives such as, for example, promoter variants or alternative splicing variants. The promoters operatively linked in front of the nucleotide sequences according to the invention may moreover be modified by nucleotide addition(s) or substitution(s), inversion(s), insertion(s) and/or deletion(s), but without impairing the functionality or activity of the promoters. The promoters can also have their activity increased by modifying their sequence, or be completely replaced by more effective promoters even from heterologous organisms. The promoter variants described above are used to prepare expression cassettes according to the invention.

Specific examples of human PARP2 splicing variants which may be mentioned are:

Variant human PARP2a: Deletion of base pairs 766 to 904 (cf.

SEQ ID NO:1). This leads to a frame shift with a new stop codon ("TAA” corresonding to nucleotides 922 to 924 in SEQ ID NO:l).

Variant human PARP2b: Insertion of 5'- gta tgc cag gaa ggt cat ggg cca gca aaa ggg tct ctg -3’ after nucleotide 204 (SEQ ID NO:1). This extends the amino acid sequence by the insertion: GMPGRSWASKRVS 40 Nucleic acid derivatives also mean variants whose nucleotide sequences in the region from -1 to -1000 in front of the start codon have been modified so that gene expression and/or protein expression is increased. 45 Besides the nucleotide sequence described above, the nucleic acid constructs which can be used according to the invention comprise in functional, operative linkage one or more other regulatory

M/40043 .

0050/49790 ° 2 ’ sequences, such as promoters, amplification signals, enhancers, polyadenylation sequences, origins of replication, reporter genes, selectable marker genes and the like. This linkage may, depending on the desired use, lead to an increase or decrease in gene expression.

In addition to the novel regulatory sequences, it is possible for the natural regulatory sequence still to be present in front of the actual structural genes. This natural regulation can, where appropriate, be switched off by genetic modification, and the expression of the genes increased or decreased. However, the gene construct may also have a simpler structure, that is to say no additional regulatory signals are inserted in front of the structural genes, and the natural promoter with its regulation is not deleted. Instead, the natural regulatory sequence is mutated in such a way that regulation no longer takes place, and gene expression is enhanced or diminished. It is also possible to insert additional advantageous regulatory elements at the 3’ end of the nucleic acid sequences. The nucleic acid sequences can be present in one or more copies in the gene construct.

Advantageous regulatory sequences for the expression method according to the invention are, for example, present in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, 17, T5, T3, gal, trc, ara, SP6, 1-PR or the 1-PL promoter, which are advantageously used in Gram-negative bacteria. Other advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SPO2, in the yeast promoters

ADCl1l, MFa, AC, P-60, CYC1l, GAPDH or in the plant promoters

CaMv/35s, SSuU, OCS, 1lib4, usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.

It is possible in principle to use all natural promoters with their regulatory sequences. It is also possible and advantageous to use synthetic promoters.

Said regulatory sequences are intended to make specific expression of the nucleic acid sequences and protein expression possible. This may mean, for example, depending on the host 40 organism that the gene is expressed or overexpressed only after induction, or that it is immediately expressed and/or overexpressed.

The regulatory sequences or factors may moreover preferably have 45 a positive influence on, and thus increase or decrease, the expression. Thus, enhancement of the regulatory elements may advantageously take place at the level of transcription by using

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0050/49790 ® 27

Co strong transcription signals such as promoters and/or enhancers.

However, it is also possible to enhance translation by, for example, improving the stability of the mRNA.

Enhancers mean, for example, DNA segences which bring about increased expression via an improved interaction between RNA polymerase and DNA.

The recombinant nucleic acid construct or gene construct is, for expression in a suitable host organism, advantageously inserted into a host-specific vector which makes optimal expression of the genes in the host possible. Vectors are well known to the skilled worker and are to be found, for example, in “Cloning Vectors” (Pouwels P. H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985). Apart from plasmids, vectors also mean all other vectors known to the skilled worker, such as, for example, phages, viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors may undergo autonomous replication in the host organism or chromosomal replication.

Expression of the constructs:

The recombinant constructs according to the invention described above are advantageously introduced into a suitable host system and are expressed. Cloning and transfection methods familiar to the skilled worker are preferably used in order to bring about expression of said nucleic acids in the particular expression system. Suitable systems are described, for example, in Current

Protocols in Molecular Biology, F. Ausubel et al., ed., Wiley

Interscience, New York 1997.

Suitable host organisms are in principle all organisms which make it possible to express the nucleic acids according to the invention, their allelic variants, their functional equivalents or derivatives or the recombinant nucleic acid construct. Host organisms mean, for example, bacteria, fungi, yeasts, plant or animal cells. Preferred organisms are bacteria such as those of the genera Escherichia, such as, for example, Escherichia coli, 40 Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic cells from animals or plants, for example Sf9 or CHO cells.

The gene product can also, if required, be expressed in 45 transgenic organisms such as transgenic animals such as, in particular, mice, sheep, or transgenic plants. The transgenic organisms may also be so-called knock-out animals or plants in

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) 0050/49790 ® 28 which the corresponding endogenous gene has been switched off, such as, for example, by mutation or partial or complete deletion.

The combination of the host organisms and the vectors appropriate for the organisms, such as plasmids, viruses or phages, such as, for example, plasmids with the RNA polymerase/promoter system, phages A, p or other temperate phages or transposons and/or other advantageous regulatory sequences forms an expression system. The term expression systems preferably means, for example, a combination of mammalian cells such as CHO cells, and vectors, such as pcDNA3neo vector, which are suitable for mammalian cells.

As described above, the gene product can also be expressed advantageously in transgenic animals, e.g. mice, sheep, or transgenic plants. It is likewise possible to program cell-free translation systems with the RNA derived from the nucleic acid.

The gene product can also be expressed in the form of therapeutically or diagnostically suitable fragments. To isolate the recombinant protein it is possible and advantageous to use vector systems or oligonucleotides which extend the cDNA by certain nucleotide sequences and thus code for modified polypeptides which serve to simplify purification. Suitable modifications of this type are, for example, so-called tags which act as anchors, such as, for example, the modification known as the hexa-histidine anchor, or epitopes which can be recognized as antigens by antibodies (described, for example, in Harlow, E. and

Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring

Harbor (N.Y.) Press). These anchors can be used to attach the proteins to a solid support such as, for example, a polymer matrix, which can, for example, be packed into a chromatography column, or to a microtiter plate or to another support.

These anchors can also at the same time be used to recognize the proteins. It is also possible to use for recognition of the proteins conventional markers such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination 40 with the anchors for derivatizing the proteins.

Production of antibodies:

Anti-PARP2 antibodies are produced in a manner familiar to the 45 skilled worker. Antibodies mean both polyclonal, monoclonal, human or humanized antibodies or fragments thereof, single chain antibodies or else synthetic antibodies, likewise antibody

M/40043 :

) 0050/49790 AMENDED SHEET k 29 i fragments such as Fv, Fab and F(ab)’; Suitable production methods are described, for example, in Campbell, A.M., Monoclonal ® Antibody Technology, (1987) Elsevier Verlag, Amsterdam, New York,

Oxford and in Breitling, F. and Diibel, S., Rekombinante

Antikdrper (1997), Spektrum Akademischer Verlag, Heidelberg.

Further use of the coding sequence:

The present cDNA additionally provides the basis for cloning the genomic sequence of the novel PARP gene. This also includes the relevant regulatory or promoter sequences, which are available, - for example, by sequencing the region located 5° upstream of the

CDNA according to the invention or which are to be found in the introns of the genes. The cDNA sequence information is also the basis for producing antisense molecules or ribozymes with the aid of known methods (cf. Jones, J.T. and Sallenger, B.A. (1997) Nat.

Biotechnol. 15, 902; Nellen, W. and Lichtenstein, C. (1993) TIBS, 18, 419). The genomic DNA can likewise be used to produce the gene constructs described above. .

Another possibility of using the nucleotide sequence or parts thereof is to generate transgenic animals. Transgenic overexpression or genetic knock-out of the sequence information in suitable animal models may provide further valuable information about the (patho)physiology of the novel genes.

Therapeutic applications: .

In situations where there is a prevailing deficiency of a protein according to the invention it is possible to employ several methods for replacement. On the one hand, the protein, natural or recombinant, can be administered directly or by gene therapy in the form of its coding nucleic acid (DNA or RNA). It is possible to use any suitable vectors for this, for example both viral and non-viral vehicles. Suitable methods are described, for example, by Strauss and Barranger in Concepts in Gene Therapy (1997),

Walter de Gruyter, publisher. Another alternative is provided by stimulation of the endogenous gene by suitable agents. 40 It is also possible to block the turnover or the inactivation of

PARPs according to the invention, for example by proteases.

Finally, inhibitors or agonists of PARPs according to the invention can be employed. 45 In situations where a PARP is present in excess or is overactivated, various types of inhibitors can be employed. This inhibition can be achieved both by antisense molecules,

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) 0050/49790 ® a 30 ribozymes, oligonucleotides or antibodies, and by low molecular weight compounds.

The active substances according to the invention, i.e. PARP proteins, nucleic acids and PARP binding partners, such as, for example, antibodies or modulators, can be administered either as single therapeutic active substances or as mixtures with other therapeutic active substances. They can be administered as such, but in general will be administered in the form of pharmaceutical compositions, i.e. as mixtures of the active substance(s) with at least one suitable pharmaceutical carrier or diluent. The active substances or compositions can be administered in any way suitable for the particular therapeutic purpose, for example, orally or parenterally.

The nature of the pharmaceutical composition and of the pharmaceutical carrier or diluent depends on the required mode of administration. Oral compositions can, for example, be in the form of tablets or capsules and may contain conventional excipients such as binders (for example syrup, acacia, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone), bulking agents (for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine), lubricants (for example magnesium stearate, talc, polyethylene glycol or silicon dioxide), disintegrating agents (for example starch) or wetting agents (for example sodium lauryl sulfate). Liquid oral products can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs or sprays etc. or can be in the form of dry powders for reconstitution with water or another suitable carrier. Liquid products of these types may contain conventional additives, for example suspending agents, flavorings, diluents or emulsifiers.

Solutions or suspensions with conventional pharmaceutical carriers can be employed for parenteral administration.

Parenteral administration of active substances according to the invention advantageously takes place using a liquid pharmaceutical composition which can be administered parenterally, in particular intravenously. This contains, preferably in a pharmaceutically acceptable carrier which is suitable for this purpose, an effective amount of at least one 40 active substance, preferably in dissolved form. Examples of pharmaceutical carriers suitable for this purpose are, in particular, aqueous solutions such as, for example, physiological saline, phosphate-buffered saline, Ringer's solution, lactated

Ringer’s solution and the like. The composition may additionally 45 contain other additives such as antioxidants, chelating agents or antimicrobial agents.

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8 The particular choice of the dosage of the active substances according to the invention and the particular dosage regimen are subject to the decision of the treating physician. The latter will select, depending on the chosen route of administration, on 9 5 the activity of the particular medicine, on the nature and severity of the disorder to be treated, on the wellbeing of the patient and his response to the therapy, a suitable dose and an appropriate dosage regimen. Thus, for example, the pharmacologically active substances according to the invention can be administered to a mammal (human and animal) in doses of about 0.5 mg to about 100 mg per kg of body weight per day. They can be administered in a single dose or in several doses.

Nontherapeutic applications:

The nucleic acids according to the invention, such as, for example, cDNA, the genomic DNA, the promoter, and the . polypeptide, and partial fragments thereof, can also be used in recombinant or nonrecombinant form for developing various test systems.

For example, it is possible to establish a test system which is suitable for measuring the activity of the promoter or of the protein in the presence of a test substance. The methods of . measurement in this case are preferably simple ones, e.g. colorimetric, luminometric, fluorimetric, immunological or radioactive, and allow preferably a large number of test substances to be measured rapidly. Tests of this type are suitable and advantageous for so-called high-throughput screening. These test systems allow test substances to be assessed for their binding to or their agonism, antagonism or inhibition of proteins according to the invention.

Determination of the amount, activity and distribution of the proteins according to the invention or their underlying mRNA in the human body can be used for the diagnosis, for the determination of the predisposition and for the monitoring of certain diseases.. Likewise, the sequence of the cDNA and of the genomic sequence may provide information about genetic causes 40 of and predispositions to certain diseases. It is possible to use for this purpose both DNA/RNA probes and antibodies of a wide variety of types. The nucleotide sequences according to the invention or parts thereof can further be used in the form of suitable probes for detecting point mutations, deletions or 45 insertions.

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0050/49790

R 4 32 ) The proteins according to the invention can further be used to identify and isolate their natural ligands or interacting partners. The proteins according to the invention can additionally be used to identify and isolate artificial or synthetic ligands. For this purpose, the recombinantly prepared or purified natural protein can be derivatized in such a way that it has modifications which permit linkage to support materials.

Proteins bound in this way can be incubated with various analytes, such as, for example, protein extracts or peptide libraries or other sources of ligands. Specifically bound peptides, proteins or low molecular weight, non-proteinogenous substances can be isolated and characterized in this way.

Non-proteinogenous substances mean, for example, low molecular weight chemical substances which may originate, for example, from classical drug synthesis or from so-called substance libraries which have been synthesized combinatorially.

The protein extracts used are derived, for example, from homogenates of plants or parts of plants, microorganisms, human or animal tissues or organs.

Ligands or interacting partners can also be identified by methods like the yeast two-hybrid system (Fields, S. and Song, O. (1989)

Nature, 340, 245). The expression banks which can be employed in this case may be derived, for example, from human tissues such as, for example, brain, heart, kidney etc.

The nucleic acid sequences according to the invention and the proteins encoded by them can be employed for developing reagents, agonists and antagonists or inhibitors for the diagnosis and therapy of chronic and acute diseases associated with the expression or activation of one of the protein sequences according to the invention, such as, for example, with increased or decreased expression thereof. The reagents, agonists, antagonists or inhibitors developed can subsequently be used to produce pharmaceutical preparations for the treatment or diagnosis of disorders. Examples of possible diseases in this connection are those of the brain, of the peripheral nervous system, of the cardiovascular system or of the eye, of septic 40 shock, of rheumatoid arthritis, diabetes, acute kidney failure, or of cancer.

The relevance of the proteins according to the invention for said indications was verified using specific inhibitors in relevant 45 animal models.

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The invention is now illustrated in detail with reference to the following examples.

Example 1: Isolation of the PARP2 and PARP3 cDNA

The present cDNA sequences were found for the first time on sequence analysis of cDNA clones of a cDNA library from human brain (Human Brain 5’Stretch Plus cDNA Library, # HL3002a,

Clontech). The mouse PARP3 clones were isolated from a “lambda-triplex mouse brain cDNA library” (Clontech order No. :

ML5004t). The sequences of these clones are described in SEQ ID

NO:1, 3, 7 and 9.

Example 2: Expression of PARP2 and PARP3 in human tissues

The expression of human PARP2 and human PARP3 was investigated in twelve different human tissues by Northern blot analysis. A Human

Multiple Tissue Northern Blot (MTN™) supplied by Clontech (#7760-1 and #7780-1) was hybridized for this purpose with an RNA probe. The probe was produced by in vitro transcription of the corresponding cDNA of human PARP2 and human PARP3 in the presence of digoxigenin-labeled nucleotides in accordance with the manufacturer’s method (BOEHRINGER MANNHEIM DIG Easy Hyb order No. 1603 558, DIG Easy Hyb method for RNA:RNA hybridization). The protocol was modified to carry out the prehybridization: 2x1lh with addition of herring sperm DNA (10 mg/ml of hybridization solution). Hybridization then took place overnight with addition of herring sperm DNA (10 mg/ml of hybridization solution). The bands were detected using the CDP-Star protocol (BOEHRINGER

MANNHEIM CDP-Star™ order No. 1685 627).

After stringent washing, the transcript of PARP2 was mainly detected in human brain, heart, skeletal muscle, kidney and liver. The transcript size of about 1.9 kb corresponds to the length of the cDNA determined (1.85kb) (cf. Figure 2(A)).

In other tissues or organs, human PARP2 expression is considerably weaker. 40 After stringent washing, the expression of the transcript of

PARP3 was mainly detected in the heart, brain, kidney, skeletal muscle and liver. Expression in other tissues (placenta, lung, pancreas) is distinctly weaker (cf. Figure 2(B)). There are at least 2 transcripts for human PARP3, which can presumably be 45 explained by different polyadenylation sites or alternative splicing. Their size (about 2.2 kb and 2.5 kb respectively) corresponds to the length of the cDNA determined (2.3kb).

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Washing was carried out with 0.2 SSC/0.2% SDS at room temperature for 2 x 15 minutes and then with 0.1 x SSC/0.1% SDS at 65°C for 2 x 15 minutes (prepared from 20X SSC: 3M NaCl, 0.3M sodium citrate, pH 7.0).

Example 3: Production of antibodies

Specific antibodies against the proteins according to the invention were produced. These were used inter alia for analyzing the tissue distribution at the protein level of PARP2 and PARP3 by immunoblot (Western blot) analysis. Examples of the production of such antibodies are indicated below.

The following peptides were prepared by synthesis in the manner familiar to the skilled worker for the antibody production. In some cases, a cysteine residue was attached to the N or C terminals of the sequences in order to facilitate coupling to

KLH (keyhole limpet hemocyanin).

PARP-2: NH;-MAARRRRSTGGGRARALNES-CO,H (amino acids 1-20;

SEQ ID NO:23)

NH,-KTELQSPEHPLDQHYRNLHC-CO,H (amino acids 335-353;

SEQ ID NO:24)

PARP-3: NH;-CKGRQAGREEDPFRSTAEALK-CO,H (amino acids 25-44;

SEQ ID NO: 25)

NH,-CKQQIARGFEALEALEEALK-CO,H (amino acids 230-248;

SEQ ID NO: 26)

The production of an anti-PARP3 antibody is described as a representative example.

For human PARP3, polyclonal antibodies were raised in rabbits using a synthetic peptide having the peptide sequence

H;N-KQQIARGFEALEALEEALK~CO2H; SEQ ID NO: 27) (amino acids 230-248 of the human PARP3 protein sequence). The corresponding mouse sequence differs in this region only by one amino acid (HoN-KQQIARGFEALEALEEAMK-CO,H; SEQ ID NO: 28). A cysteine was also attached to the N terminus in order to make it possible for the protein to couple to KLH. 40

Rabbits were immunized a total of five times, at intervals of 7-14 days, with the KLH-peptide conjugate. The antiserum obtained was affinity-purified using the antigen. The specific IgG fraction was isolated from the serum using the respective 45 peptides which, for this purpose, were initially immobilized on an affinity column in the manner familiar to the skilled worker.

The respective antiserum was loaded onto this affinity column,

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. 0050/49790 . 3s ) and nonspecifically sorbed proteins were eluted with buffer. The specifically bound IgG fraction was eluted with 0.2 M glycine/HCl buffer pH 2.2. The pH was immediately increased using a 1M

TRIS/HC1 buffer pH 7.5. The eluate containing the IgG fraction was mixed 1:1 (volume) with saturated ammonium sulfate solution and incubated at +4°C for 30 min to complete the precipitation.

The resulting precipitate was centrifuged at 10,000 g and, after removal of the supernatant, dissolved in the minimum amount of

PBS/TBS. The resulting solution was then dialyzed against PBS/TBS in the ratio 1:100 (volume). The antibodies were adjusted to a concentration of about 100 pg of IgG/ml. The PARP3 antibodies purified in this way had high specificity for PARP3. Whereas mouse PARP3 was recognized well, there was no observable cross-reaction with PARP1 or PARP2.

Example 4: Analysis of the tissue distribution by immunoblot (Western blot)

The tissue distribution at the protein level was also investigated for PARP2 and PARP3 by immunoblot (Western blot) analysis.

Preparation of the mouse tissues for protein gels:

Tissues or cells were homogenized using a Potter or Ultra-Turrax.

For this, 0.5 g of tissue (or cells) was incubated in 5 ml of buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 6 mM MgCl;), one tablet of protease inhibitor cocktail (Boehringer Mannheim, order No.: 1836153) and benzonase (purity grade I, MERCK) at 37°C for 30 min.

Tissue samples from mice were produced for heart, lung, liver, spleen, kidney, intestine, muscle, brain and for human embryonic kidney cells (HEK293, human embryonal kidney).

Protein gels:

The NuPAGE system supplied by NOVEX was used according to the instructions for protein gels. Polyacrylamide gels (NuPAGE 4-12%

BisTris, NOVEX NP 0321), running buffer (MES-Running Buffer,

NOVEX NP 0002), antioxidant (NOVEX NP 0005), protein size 40 standard (Multi Mark Multi Colored Standard, NOVEX LC 5725), sample buffer (NuPAGE LDS Sample Buffer (4X), NOVEX NP 0007) were used. The gels were run for 45 minutes at a voltage of 200 V.

Western blot: 45

Western blots were carried out using the NOVEX system in accordance with instructions. A nitrocellulose membrane

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Co (Nitrocellulose Pore size 45 um, NOVEX LC 2001) was used. The transfer took 1 hour at a current of 200 mA. The transfer buffer consisted of 50 ml of transfer buffer concentrate (NOVEX NP 0006), 1 ml of antioxidant (NOVEX NP 0002), 100 ml of analytical grade methanol and 849 ml of double-distilled water.

Besides the blots produced in this way, also used were premade blots, for example from Chemicon (mouse brain blot, Chemicon, catalog No.: NS 106 with the tissues 1. frontal cortex, 2. posterior cortex, 3. cerebellum, 4. hippocampus, 5. olfactory bulb, 6. striatum, 7. thalamus, 8. mid brain, 9. entorhinal cortex, 10. pons, 11. medulla, 12. spinal cord).

Antibody reaction with PARP3:

The Western blots were blocked in TBST (TBS + 0.3 % Tween 20) with 5% dry milk powder for at least 2 hours (TBS: 100 mM Tris pH 7.5, 200 mM NaCl). The antibody reaction with the primary antibody (dilution 1:1000) took place in TBST with 5% dry milk powder (see above) at room temperature for at least 2 hours or at © 49°C overnight, with gentle agitation (vertical rotator). This was followed by washing three times in TBST for 5 minutes. Incubation with the secondary antibody (anti-rabbit IgG, peroxidase-coupled,

SIGMA A-6154, dilution 1:2000) took place in TBST with 5% dry milk powder for 1 hour. This was followed by washing three times for 5 minutes each time as above. The subsequent detection was based on chemiluminescence using the SUPER BLAZE kit (Pierce,

Signal BLAZE Chemiluminescent Substrate 34095) as stated by the manufacturer. The ”“Lumi-Film” (Chemiluminescent Detection Film,

Boehringer order No: 1666916) was used. The films were developed for about 2 min (X-ray developer concentrate, ADEFO-Chemie GmbH), hydrated, fixed for about 4 min (Acidofix 85 g/l /AGFA), hydrated and then dried.

Example 5: Preparation of the enzymes

For comparison, human PARP1l was expressed recombinantly in the baculovirus system in the manner familiar to the skilled worker and partially purified as described (Shah et al., Analytical 40 Biochemistry 1995, 227, 1-13). Bovine PARPl in a purity of 30-50% (c= 0.22 mg/ml, spec. activity 170 nmol of ADP-ribose/min/mg of total protein at 25°C) was purchased from BIOMOL (order No.

SE-165). Human and mouse PARP2 and PARP3 were expressed recombinantly in the baculovirus system (Bac-to-Bac system, BRL 45 LifeScience). For this purpose, the appropriate cDNAs were cloned to the pFASTBAC-1 vector. Preparation of recombinant baculovirus

DNA by recombination in E. coli was followed by transfection of

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- AMENDED SHEET insect cells (Sf9 or High-Five) with the appropriate recombinant baculovirus DNAs. Expression of the corresponding proteins was verified by Western blot analysis. Virus strains were amplified in the manner familiar to the skilled worker. Larger amounts of ® 5 recombinant proteins were produced by infecting 500 ml of insect cell culture (2 x 106 cells/ml, infected with viruses in an MOI (multiplicity of infection; ratio of viruses to cells) of 5-10 and incubated for 3 to 4 days). The insect cells were then pelleted by centrifugation, and the proteins were purified from the pellet. . The purification took place by classical methods of protein purification familiar to the skilled worker, detecting the enzymes with appropriate specific antibodies. In some cases, the proteins were also affinity-purified on a 3-aminobenzamide affinity column as described (Burtscher et al., Anal Biochem 1986, 152:285-290). The purity was >90%.

Example 6: Assay systems for determining the activity of

PARP2 and PARP3 and the inhibitory action of effectors on PARP],

PARP2 and PARP3. a) Production of antibodies against poly(ADP-ribose)

It is possible to use poly(ADP-ribose) as antigen for generating anti-poly (ADP-ribose) antibodies. The production of anti-poly (ADP-ribose) antibodies is described in the literature (Ranai Y et al. (1974) Biochem Biophys Res Comm 59:1, 300-306;

Kawamaitsu H et al. (1984) Biochemistry 23, 3771-3777; Kanai Y et al. (1978) Immunology 34, 501-508). : The following were used, inter alia: anti-poly (ADP-ribose) antibodies (polyclonal antiserum, rabbits), BIOMOL; order

No. SA-276, anti-poly(ADP-ribose) antibodies (monoclonal, mouse; clone 10H; hybridoma supernatant, affinity-purified).

The antisera or monoclonal antibodies obtained from hybridoma supernatant were purified by protein A affinity chromatography in the manner familiar to the skilled worker. 40 b) ELISA

Materials: 45 ELISA color reagent: TMB mix, SIGMA T-8540

A 96-well microtiter plate (FALCON Micro-Test IIIT Flexible Assay

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Plate, # 3912) was coated with histones (SIGMA, H-7755). Histones were for this purpose dissolved in carbonate buffer (0.05M

Na HCO3; pH 9.4) in a concentration of 50 pg/ml. The individual wells of the microtiter plate were each incubated with 150 ul of this histone solution at room temperature for at least 2 hours or at 4°C overnight. The wells are then blocked by adding 150 pl of a 1% strength BSA solution (SIGMA, A-7888) in carbonate buffer at room temperature for 2 hours. This is followed by three washing steps with washing buffer (0.05% TweenlO in 1x PBS; PBS (Phosphate buffered saline; Gibco, order No. 10010): 0.21g/1

KH,PO4, 9g/1 NaCl, 0.726g/1 Na,HPO4 - 7H0, pH 7.4). Washing steps were all carried out in a microtiter plate washer (”Columbus” microtiter plate washer, SLT-Labinstruments, Austria).

Required for the enzyme reaction were an enzyme reaction solution and a substrate solution, in each case as a premix. The absolute amount of these solutions depended on the intended number of assay wells.

Composition of the enzyme reaction solution per well: - 4 pl of PARP reaction buffer (1M Tris-HCl pH 8.0, 100mM MgCl, 10mM DTT) - 20ng of PARP1 (human or bovine) or 8ng PARP2 (human or mouse) - 4 ul of activated DNA (1 mg/ml; SIGMA, D-4522) - H,0 ad 40 pl

Composition of the substrate solution per well: - 5 ul of PARP reaction buffer (10x) ~ 0.8 ul of NAD solution (10mM, SIGMA N-1511) - 44 pl HR0

Inhibitors were dissolved in 1x PARP reaction buffer. DMSO, which was occasionally used to dissolve inhibitors in higher concentrations, was no problem up to a final concentration of 2%.

For the enzyme reaction, 40 pul of the enzyme reaction solution were introduced into each well and incubated with 10 ul of inhibitor solution for 10 minutes. The enzyme reaction was then started by adding 50 pl of substrate solution per well. The reaction was carried out at room temperature for 30 minutes and 40 then stopped by washing three times with washing buffer.

The primary antibodies employed were specific anti-poly (ADP-ribose) antibodies in a dilution of 1:5000.

Dilution took place in antibody buffer (1% BSA in PBS; 0.05% 45 Tween20). The incubation time for the primary antibodies was one hour at room temperature. After subsequently washing three times with washing buffer, incubation was carried out with the

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- 0050/49790 e 39 ) secondary antibody (anti-mouse IgG, Fab fragments, peroxidase-coupled, Boehringer Mannheim, order No. 1500.686; anti-rabbit IgG, peroxidase-coupled, SIGMA, order No. A-6154) in a dilution of 1:10,000 in antibody buffer at room temperature for one hour. Washing three times with washing buffer was followed by the color reaction using 100 ul of color reagent (TMB mix, SIGMA) per well at room temperature for about 15 min. The color reaction was stopped by adding 100 ul of 2M H;SO4. This was followed by immediate measurement in an ELISA plate reader (EAR340AT “Easy

Reader”, SLT-Labinstruments, Austria) (450nm versus 620nm). The measurement principle is depicted diagrammatically in Figure 6.

Various concentrations were used to construct a dose-effect plot to determine the Kj value of an inhibitor. Values are obtained in triplicate for a particular inhibitor concentration. Arithmetic means are determined using Microsoft® Excel. The ICsq is determined using the Microcal© Origin Software (Vers. 5.0) (”"Sigmoidal Fit”). Conversion of the ICsy value is calculated in this way into Kj values took place by using “calibration inhibitors”. The “calibration inhibitors” were also measured in each analysis. The Kj values of the “calibration inhibitors” were determined in the same assay system by analysis of the Dixon diagram in the manner familiar to the skilled worker. b) HTRF (homogenous time-resolved fluorescence) assay

In the HTRF PARP assay according to the invention, histones, as target proteins for modification by PARP, are labeled indirectly with an XL665 fluorophore. The anti-poly (ADP-ribose) antibody is directly labeled with a europium cryptate (anti-PAR cryptate). If the XL665 fluorophore is in the direct vicinity in space, which is ensured by binding to the poly(ADP-ribose) on the histone, then energy transfer is possible. The emission at 665 nm is thus directly proportional to the amount of bound antibody, which in turn is equivalent to the amount of poly(ADP-ribose). The measured signal thus corresponds to the PARP activity. The measurement principle is depicted diagrammatically in Figure 7.

The materials used are identical to those used in the ELISA (see above) unless expressly indicated. 40

Histones were dissolved in a concentration of 3 mg/ml in Hepes buffer (50mM, pH=7.5). Biotinylation took place with sulfo-NHS-LC-biotin (Pierce, #21335T). A molar ratio of 4 biotin molecules per histone was used. The incubation time was 45 90 minutes (RT). The biotinylated histones were then purified on a G25 SF HR10/10 column (Pharmacia, 17-0591-01) in Hepes buffer (50mM, pH=7.0) in order to remove excess biotinylation reagent.

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A 40

The anti-poly (ADP-ribose) antibody was labeled with europium cryptate using bifunctional coupling reagents (Lopez, E. et al.,

Clin. Chem. 39(2), 196-201 (1993); US Patent 5,534,622).

Purification took place on a G25SF HR10/30 column. A molar ratio of 3.1 cryptates per antibody was achieved. The yield was 25%.

The conjugates were stored at -80°C in the presence of 0.1% BSA in phosphate buffer (0.1M, pH=7).

For the enzyme reaction, the following were pipetted into each well: - 10 pl of PARP solution in PARP HTRF reaction buffer (50mM

Tris-HCl pH 8.0, 10mM MgCl2 [sic], 1mM DTT) with 20ng of PARP1 (human or bovine) or 8ng of PARP2 (human or mouse) - 10 pl of activated DNA in PARP HTRF reaction buffer (50ug/ml) - 10 pl of biotinylated histones in PARP HTRF reaction buffer (1.25pM) - 10 pl of inhibitor in PARP HTRF reaction buffer

These reagents were incubated for 2 minutes before the reaction was started by adding - 10 pl of NAD solution in PARP HTRF reaction buffer (41 uM/ml).

The reaction time was 30 minutes at room temperature.

The reaction was then stopped by adding - 10 pl of PARP inhibitor (25 uM, K;=10nM) in “Revelation” buffer (100mM Tris-HCl pH 7.2, 0.2M KF, 0.05% BSA).

The following were then added: - 10 pl of EDTA solution (SIGMA, E-7889, 0.5M in H,0) - 100 pl of Sa-XL665 (Packard Instruments) in ”"Revelation” buffer (15-31.25nM) - 50 ul of anti-PAR cryptate in "Revelation” buffer (1.6-3.3nM).

Measurement was then possible after 30 minutes (up to 4 hours).

The measurement took place in a “discovery HTRF microplate analyzer” (Canberra Packard Instruments). The K; values were calculated as described for the ELISA.

Example 7: Test systems for determining the therapeutic efficacy 40 of PARP inhibitors

Novel PARP inhibitors can have their therapeutic efficacy checked in relevant pharmacological models. Examples of some suitable models are listed in Table 1. 45

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AMENDED SHEET

!

Neurodegenerative NMDA excitotoxicity [See below for disorders (stroke, in mice or rats description ® 5 |Parkinson’s, etc.)

Stroke Permanent MCAO Tokime, T. et al., ("middle cerebral J. Cereb. Blood Flow arterial occlusion”) |Metab., 18(9):

Guegan, C., Brain

Research. Molecular ’ Brain Research, 55(1): 133-40, 1998.

Transient, focal Eliasson MJL et al.,

MCAO in rats or mice |Nat Med 1997, 3:1089-1095. ' Endres, M et al., J : Cereb Blood Flow

Metab 1997, 17:1143-1151.

Takahashi K et al.,

J Cereb Blood Flow

Metab 1997, 17:1137-1142.

Parkinson’s disease |MPTP (l-methyl- Cosi C, et al. , 4-phenyl-1,2,3,6-~ Brain Res. , 1998 : tetrahydropyridine) 809(1):58-67. toxicity in mice/ Cosi C, et al., rats Brain Res., 1996 729(2):264-9.

Myocardial arrest Coronary vessel Richard Vv, et al., occlusion in rats, Br. J. Pharmacol pigs or rabbits 1994, 113, 869-876.

Thiemermann C, et al., Proc Natl Acad

Sci U S A. 1997, 94(2):679-83.

Zingarelli B, et al., Cardiovasc Res. 1997, 36(2):205-15.

Langendorff heart See below for model in rats or description 40 rabbits

Septic shock Endotoxin shock in Szabo C, et al., J rats Clin Invest, 1997, 100(3):723-35. 45

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EERE carrageenan-induced |Exp Med. 1997, multiple organ 186(7):1041-9. failure in rats or Cuzzocrea S, et al. mice Eur J Pharmacol. 1998, 342(1):67-76.

CTT ER Ee collagen-induced Proc Natl Acad Sci arthritis in rats or |U S A. 1998, mice 95(7):3867-72.

Ba = alloxan-induced or Diabetes 1983, 32: obesity-associated 316-318.Masiello P et al., Diabetologia 1985, 28: 683-686 .Shimabukuro

M et al., J Clin

Invest 1997, 100: 290-295, see below 1999, 75(1), 91-100. a) NMDA excitotoxicity model

Glutamate is the most important excitory neurotransmitter in the brain. Under normal conditions, glutamate is secreted into the synaptic cleft and stimulates the post-synaptic glutamate receptors, specifically the glutamate receptors of the “NMDA” and “AMPA” types. This stimulation plays a significant part in numerous functions of the brain, including learning, memory and motor control.

Under the conditions of acute and chronic neurodegeneration (e.g. stroke), however, there is a great increase in the presynaptic glutamate secretion, resulting in excessive stimulation of the receptors. This leads to death of the cells stimulated in this way. These increased glutamate activities occur in a number of neurological disorders or psychological disturbances and lead to states of overexcitation or toxic effects in the central nervous system (CNS) but also in the peripheral nervous system. Thus, glutamate is involved in a large number of neurodegenerative 40 disorders, in particular neurotoxic disturbances following hypoxia, anoxia, ischemia and after lesions like those occurring after stroke and trauma, and stroke, Alzheimer’s disease,

Huntington'’s disease, amyotrophic lateral sclerosis (ALS; “Lou

Gehring’s disease”), cranial trauma, spinal cord trauma, 45 peripheral neuropathies, AIDS dementia and Parkinson’s disease.

Another disease in which glutamate receptors are important is

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- 0050/49790 epilepsy (cf. Brain Res Bull 1998; 46(4):281-309, Eur

Neuropsychopharmacol 1998, 8(2):141-52.).

Glutamate effects are mediated through various receptors. One of these receptors is called the NMDA (N-methyl-D-aspartate) receptor after a specific agonist (Arzneim.Forschung 1990, 40, 511-514; TIPS, 1990, 11, 334-338; Drugs of the Future 1989, 14, 1059-1071). N-Methyl-D-aspartate is a strong agonist of a particular class of glutamate receptors (”NMDA” type). stimulation of the NMDA receptor leads to influx of calcium into the cell and the generation of free radicals. The free radicals lead to DNA damage and activation of PARP. PARP in turn causes cell death through depletion of high-energy phosphates (NAD and

ATP) in the cell. This explains the toxicity of NMDA. Treatment of animals with NMDA can therefore be regarded as a model of the abovementioned disorders in which excitotoxicity is involved.

Because of the importance of glutamate receptors in neurodegeneration, many pharmacological approaches to date have been directed at specific blocking of precisely these receptors.

However, because of their importance in normal stimulus conduction, these approaches have proved to be problematic (side effects). In addition, stimulation of the receptors is an event which takes place very rapidly so that administration of the receptors often comes too late (“time window” problem). Thus there is a great need for novel principles of action and inhibitors of NMDA-related neurotoxicity.

Protection against cerebral overexcitation by excitatory amino acids (NMDA antagonism in mice) can be regarded as adequate proof of the activity of a pharmacological effector of PARP in disorders based on excitotoxicity. Intracerebral administration of excitatory amino acids (EAA) induces such massive overexcitation that it leads within a short time to convulsions and death of the animals (mice).

In the present case there was unilateral intracerebroventricular administration of 10 ul of a 0.035% strength aqueous NMDA solution 120 minutes after intraperitoneal (i.p.) administration of the 40 test substance. These symptoms can be inhibited by systemic, e.g. intraperitoneal, administration of centrally acting drugs. Since excessive activation of EAA receptors in the central nervous system plays an important part in the pathogenesis of various neurological disorders, information can be gained from the 45 detected EAA antagonism in vivo about possible therapeutic utilizability of the substances for such CNS disorders. An ED50 at which 50% of the animals are, due to preceding i.p.

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Pt 44

Ts administration of the measured substance, free of symptoms with a fixed dose of NMDA was determined as a measure of the activity of the substances. 9 5 b) Langendorf heart model (model for myocardial arrest)

Male Sprague-Dawley rats (bodyweight 300-400 g; origin Janvier,

Le Genest-St-Isle, France) were used for the test. The rats were treated orally by gavage with the active substance or placebo (volume: 5 ml/kg). 50 minutes later, heparin is administered intraperitoneally (Liquemin N Roche, 125 IU/animal in 0.5 ml). .The animals are anesthesized with Inactin® T133 (thiobetabarbital sodium 10%), fixed on the operating table, tracheotomized and ventilated with a “Harvard ventilatory pump” (40 beats/min, 4.5 ml/beat). Thoracotomy was followed by immediate catheterization of the aorta, removal of the heart and immediate retrograde perfusion. The hearts were perfused with a constant pressure of 75 mmHg, which is achieved using a “Gilson Miniplus 2 perfusion pump”. Composition of the perfusate (mmol/l): NaCl 118,

KCl 4.7, CaCl x 2 H,0 2.52, MgSOgq x 7 H;0 1.64, NaHCO; 24.88,

KHPO4 1.18, glucose 11. The temperature is kept at 37°C throughout the experiment. Functional parameters were continuously recorded using a “Gould 4-channel recorder”.

Measurements were made of the left-ventricular pressure (LVP; ’ mmHg), LVEDP (mmHg), enzyme release (creatine kinase, mU/ml/qg), coronary flow rate (ml/min), HR (pulse rate, min-l). The left-ventricular pressure was measured using a liquid-filled latex balloon and a Statham23 Db pressure transducer. The volume of the balloon was initially adjusted to reach an LVEDP (left-ventricular end-diastolic pressure) of about 12 mmHg. The

DP [sic]/dtpax (maximum pumping force) is derived from the pressure signal using a differentiator module. The heart rate was calculated from the pressure signal. The flow rate was determined using a drop counter (BMT Messtechnik GmbH Berlin). After an equilibration time of 20 minutes, the hearts were subjected to a 30-minute global ischemia by stopping the perfusate supply while keeping the temperature at 37°C. During the following 60-minute reperfusion period, samples of the perfusate were taken after 3, 5, 10, 15, 30, 45 and 60 min for analysis of creatine kinase (CK) 40 activity. Means and standard deviations for the measured parameters were analyzed statistically (Dunnett test). The significance limit was p=0.05.

The experiment on rabbit hearts was carried out similarly. Male 45 white New Zealand rabbits (obtained from: Interfauna) were used.

The hearts were prepared as described above for the rat model.

The perfusion pressure was set at a maximum of 60 mmHg and the

M/40043 : ’

- 0050/49790 ) flow rate at about 25ml/min. The equilibration time was about 30 min. The substance was administered by infusion directly upstream of the heart. 15 min after starting the infusion, a 30-minute global ischemia was caused by stopping the flow while maintaining the temperature of the heart. A 30-minute reperfusion followed. Perfusate was taken for investigation of CK activity before administration of the substance, after 15 min and at various times (5, 10, 15, 20, 30 min) during the reperfusion. The following parameters were measured: LVP (mmHg), LVEDP, LVdP/dt,

PP (mmHg), HR (pulse rate; beats/min), CK activity (U/min/g heart weight). : c) Animal model for acute kidney failure

The protective effect of intravenous administration of PARP inhibitors (4 days) on the kidney function of rats with postischemic acute kidney failure was investigated.

Male Sprague-Dawley rats (about 330 g at the start of the experiments; breeder: Charles River) were used. 10-15 animals were employed per experimental group. Administration of active substance/placebo took place continuously with an osmotic micropump into the femoral vein. Orbital blood was taken (1.5 ml of whole blood) under inhalation anesthesia with enflurane (Ethrane Abbot, Wiesbaden).

After the initial measurements (blood sample) and determination of the amount of urine excreted in 24h, the rats were anesthetized (“Nembutal”, pentobarbital sodium, Sanofi CEVA; 50mg/kg i.p., volume injected 1.0 ml/kg) and fastened on a heatable operating table (37°C). 125 IU/kg heparin (Liguemin N,

Roche) were administered i.v. into the caudal vein. The abdominal cavity was opened and the right kidney was exposed. The branching-off renal artery was exposed and clamped off superiorly using bulldog clamps (Diefenbach 38mm). The left renal artery was likewise exposed and clamped off (superiorly, about half way to the kidney). During the operation, an osmotic micropump was implanted into the femoral vein. The intestine was reinserted and the fluid loss was compensated with luke-warm 0.9% NaCl. The 40 animals were covered with a moist cloth and kept warm under red light. After 40 min, the appearance of the kidneys was recorded, and the clamps were removed, first the right then the left. The intestine was put back and 2 drops of antibiotic (Tardomyocel,

Bayer) were added. The abdominal wall was closed with sterile cat 45 gut (Ethicon No.4) and treated once more with 1 drop of antibiotic. The epidermis was sutured with sterile Ethibond Exel (Ethicon) No.3/0, and the suture was sprayed with Nebacetin N

M/40043

- 0050/49790 ) (Yamanouchi) wound spray. A tenth of a daily dose of drug/placebo is given as i.v. bolus.

Samples and blood were taken for investigating biochemical parameters in the serum and urine: Na, K, creatinine, protein (only in urine), on days 1, 2 and 4 of the experiment. In addition, the feed and water consumption, bodyweight and urine volume were recorded. After 14 days, the animals were sacrificed and the kidneys were assessed.

The assessment excluded all animals which died of an infarct during the experiment or showed an infarct at necropsy on day 14.

The creatinine clearance and the fractional sodium excretion were calculated as kidney function parameters, comparing treated animals with control and sham. d) In vitro model for radiosensitization (tumor therapy)

MCF-7-cells (human breast carcinoma) were cultivated in

Dulbecco’s modified Eagle’s medium with 10% heat-inactivated FCS and 2 mM L-glutamine. Cells were seeded out overnight in cell densities of 100, 1000 or 10,000 cells per well in a 6-well plate and then exposed to ionizing radiation with a dose in the range from 0 to 10 Gy (137Cs, Shepard Mark, model I-68A, dose rate 3.28 Gy/min). 10 days after the irradiation, the experiment was assessed, counting colonies with fifty cells as positive. e) Model of stroke (focal cerebral ischemia; MCA (middle cerebral artery) occlusion in the rat

Focal ischemia was achieved by cauterization of the right distal

MCA in Sprague-Dawley or Long-Evans rats. The rats can be treated with modulators of the proteins according to the invention before or after the start of the MCA occlusion. As a rule, doses of 1-10 mg/kg are chosen (bolus administration), where appropriate followed by a continuous infusion of 0.5-5 mg/kg/h. The rats are anesthetized with halothane in a mixture of 70% nitrogen and 30% oxygen (4% for induction and 0.8-1.2% during the operation). The body temperature was measured rectally throughout and was kept 40 constant at 37.5°C + 0.5°C by means of a controllable heating blanket. Also measured where appropriate via a tail vein catheter were the arterial pressure, arterial pH, PaO, and PaCO,. The focal ischemia was then carried out by the method of Chen et al. (Stroke 17: 738-743; 1986) or Liu et al. (Am. J. Physiol. 256: 45 H589-593; 1989) by means of permanent cauterization of the distal part of the right MCA. After completion of the operation, the animals were kept in a warm environment for a further 24 hours.

M/40043 :

: AMENDED SHEET y | They are then sacrificed using CO, and decapitated. The brains are hal removed without delay, shock-frozen (dry ice or liquid nitrogen) and stored at -80°C. The brains are cut into slices 0.02 mm thick, each twentieth section being used for the subsequent analysis. ® 5 The corresponding sections are stained with cresyl violet (Nissl staining). An alternative possibility is to stain with TTC (2,3,5-triphenyltetraztolium chloride). The infarct volume can then be assessed under the microscope. A computer-assisted image analysis software can be used for accurate quantification (J.

Cereb. Blood Flow Metabol. 10: 290-293; 1990). f) Septic shock

Groups each of 10 male C57/BL mice (body weight 18-20 g) are treated with LPS (lipopolysaccharide from E. coli, LDjgq = mg/animal i.v.) plus galactosamine (20 mg/animal i.v.). The substance to be tested is administered i.p. or i.v. for three consecutive days (for example 1-10 mg/kg), the first dose being given 30 minutes after the LPS administration. The mortality rate 20 is determined every 12 h. An alternative possibility is also to administer the substance in several doses distributed over the days. g) Determination of altered gene expression in aging cells

Aging of cells is stimulated by changing cell culture media from complete medium to medium with a reduced serum concentration and is then analyzed by quantitative PCR or Northern blot (Linskens et al., Nucleic Acids Res. 1995; 23(16): 3244-51). Collagen or elastin can, for example, act as typical markers for aging of the skin. Use is made of human fibroblasts or fibroblast cell lines able to reflect aging of the skin. Modulators of the proteins according to the invention are added to the medium and their effect on the alteration in gene expression is observed. An increased elastin production can be observed in cells with an aging process reduced by the modulators. "Comprises/comprising" when used in this specification is 40 taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps or components or groups thereof. 45

M/40043

LR 0050/49790 : <Q 48

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BASF Aktiengesellschaft (B) STREET: - (C) CITY: Ludwigshafen (E) COUNTRY: Germany (F) POSTAL CODE: 67065 (ii) TITLE OF INVENTION: Novel Poly ADP Ribose Polymerase Genes (iii) NUMBER OF SEQUENCES: 28 (iv) COMPUTER-READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1843 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: (F) TISSUE TYPE: brain (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..1715 (D) OTHER INFORMATION: /product= "Poly ADP Ribose

Polymerase”

oo 0050/49790 ) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CC ATG GCG GCG CGG CGG CGA CGG AGC ACC GGC GGC GGC AGG GCG AGA 47

Met Ala Ala Arg Arg Arg Arg Ser Thr Gly Gly Gly Arg Ala Arg 1 5 10 15

GCA TTA AAT GAA AGC AAA AGA GTT AAT AAT GGC AAC ACG GCT CCA GAA 95

Ala Leu Asn Glu Ser Lys Arg Val Asn Asn Gly Asn Thr Ala Pro Glu

GAC TCT TCC CCT GCC AAG AAA ACT CGT AGA TGC CAG AGA CAG GAG TCG 143

Asp Ser Ser Pro Ala Lys Lys Thr Arg Arg Cys Gln Arg Gln Glu Ser 40 45

AAA AAG ATG CCT GTG GCT GGA GGA AAA GCT AAT AAG GAC AGG ACA GAA 191

Lys Lys Met Pro Val Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu 50 55 60

GAC AAG CAA GAT GAA TCT GTG AAG GCC TTG CTG TTA AAG GGC AAA GCT 239

Asp Lys Gln Asp Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala 65 70 75

CCT GTG GAC CCA GAG TGT ACA GCC AAG GTG GGG AAG GCT CAT GTG TAT 287

Pro Val Asp Pro Glu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr 80 85 90 95

TGT GAA GGA AAT GAT GTC TAT GAT GTC ATG CTA AAT CAG ACC AAT CTC 335

Cys Glu Gly Asn Asp Val Tyr Asp Val Met Leu Asn Gln Thr Asn Leu 100 105 110

CAG TTC AAC AAC AAC AAG TAC TAT CTG ATT CAG CTA TTA GAA GAT GAT 383

Gln Phe Asn Asn Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu Asp Asp 115 120 125

GCC CAG AGG AAC TTC AGT GTT TGG ATG AGA TGG GGC CGA GTT GGG AAA 431

Ala Gln Arg Asn Phe Ser Val Trp Met Arg Trp Gly Arg Val Gly Lys 130 135 140

ATG GGA CAG CAC AGC CTG GTG GCT TGT TCA GGC AAT CTC AAC AAG GCC 479

Met Gly Gln His Ser Leu Val Ala Cys Ser Gly Asn Leu Asn Lys Ala 145 150 155

AAG GAA ATC TTT CAG AAG AAA TTC CTT GAC AAA ACG AAA AAC AAT TGG 527

Lys Glu Ile Phe Gln Lys Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp 160 165 170 175

GAA GAT CGA GAA AAG TTT GAG AAG GTG CCT GGA AAA TAT GAT ATG CTA 575

-o 0050/49790 ) Glu Asp Arg Glu Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu 180 185 190

CAG ATG GAC TAT GCC ACC AAT ACT CAG GAT GAA GAG GAA ACA AAG AAA 623

Gln Met Asp Tyr Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys Lys 195 200 205

GAG GAA TCT CTT AAA TCT CCC TTG AAG CCA GAG TCA CAG CTA GAT CTT 671

Glu Glu Ser Leu Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu Asp Leu 210 215 220

CGG GTA CAG GAG TTA ATA AAG TTG ATC TGT AAT GTT CAG GCC ATG GAA 719

Arg Val Gln Glu Leu Ile Lys Leu Ile Cys Asn Val Gln Ala Met Glu 225 230 235

GAA ATG ATG ATG GAA ATG AAG TAT AAT ACC AAG AAA GCC CCA CTT GGG 767

Glu Met Met Met Glu Met Lys Tyr Asn Thr Lys Lys Ala Pro Leu Gly 240 245 250 255

AAG CTG ACA GTG GCA CAA ATC AAG GCA GGT TAC CAG TCT CTT AAG AAG 815

Lys Leu Thr Val Ala Gln Ile Lys Ala Gly Tyr Gln Ser Leu Lys Lys 260 265 270

ATT GAG GAT TGT ATT CGG GCT GGC CAG CAT GGA CGA GCT CTC ATG GAA 863

Ile Glu Asp Cys Ile Arg Ala Gly Gln His Gly Arg Ala Leu Met Glu 275 280 285

GCA TGC AAT GAA TTC TAC ACC AGG ATT CCG CAT GAC TTT GGA CTC CGT 911

Ala Cys Asn Glu Phe Tyr Thr Arg Ile Pro His Asp Phe Gly Leu Arg 290 295 300

ACT CCT CCA CTA ATC CGG ACA CAG AAG GAA CTG TCA GAA AAA ATA CAA 959

Thr Pro Pro Leu Ile Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln 305 310 315

TTA CTA GAG GCT TTG GGA GAC ATT GAA ATT GCT ATT AAG CTG GTG AAA 1007

Leu Leu Glu Ala Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys 320 325 330 335

ACA GAG CTA CAA AGC CCA GAA CAC CCA TTG GAC CAA CAC TAT AGA AAC 1055

Thr Glu Leu Gln Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg Asn 340 345 350

CTA CAT TGT GCC TTG CGC CCC CTT GAC CAT GAA AGT TAC GAG TTC AAA 1103

Leu His Cys Ala Leu Arg Pro Leu Asp His Glu Ser Tyr Glu Phe Lys 355 360 365

- 0050/49790

GTG ATT TCC CAG TAC CTA CAA TCT ACC CAT GCT CCC ACA CAC AGC GAC 1151

Val Ile Ser Gln Tyr Leu Gln Ser Thr His Ala Pro Thr His Ser Asp 370 375 380

TAT ACC ATG ACC TTG CTG GAT TTG TTT GAA GTG GAG AAG GAT GGT GAG 1199

Tyr Thr Met Thr Leu Leu Asp Leu Phe Glu Val Glu Lys Asp Gly Glu 385 390 395

AAA GAA GCC TTC AGA GAG GAC CTT CAT AAC AGG ATG CTT CTA TGG CAT 1247

Lys Glu Ala Phe Arg Glu Asp Leu His Asn Arg Met Leu Leu Trp His 400 405 410 415

GGT TCC AGG ATG AGT AAC TGG GTG GGA ATC TTG AGC CAT GGG CTT CGA 1295

Gly Ser Arg Met Ser Asn Trp Val Gly Ile Leu Ser His Gly Leu Arg 420 425 430

ATT GCC CCA CCT GAA GCT CCC ATC ACA GGT TAC ATG TTT GGG AAA GGA 1343

Ile Ala Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met Phe Gly Lys Gly 435 440 445

ATC TAC TTT GCT GAC ATG TCT TCC AAG AGT GCC AAT TAC TGC TTT GCC 1391

Ile Tyr Phe Ala Asp Met Ser Ser Lys Ser Ala Asn Tyr Cys Phe Ala 450 455 460

TCT CGC CTA AAG AAT ACA GGA CTG CTG CTC TTA TCA GAG GTA GCT CTA 1439

Ser Arg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser Glu Val Ala Leu 465 470 475

GGT CAG TGT AAT GAA CTA CTA GAG GCC AAT CCT AAG GCC GAA GGA TTG 1487

Gly Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro Lys Ala Glu Gly Leu 480 485 490 495

CTT CAA GGT AAA CAT AGC ACC AAG GGG CTG GGC AAG ATG GCT CCC AGT 1535

Leu Gln Gly Lys His Ser Thr Lys Gly Leu Gly Lys Met Ala Pro Ser 500 505 510

TCT GCC CAC TTC GTC ACC CTG AAT GGG AGT ACA GTG CCA TTA GGA CCA 1583

Ser Ala His Phe Val Thr Leu Asn Gly Ser Thr Val Pro Leu Gly Pro 515 520 525

GCA AGT GAC ACA GGA ATT CTG AAT CCA GAT GGT TAT ACC CTC AAC TAC 1631

Ala Ser Asp Thr Gly Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr 530 535 540

AAT GAA TAT ATT GTA TAT AAC CCC AAC CAG GTC CGT ATG CGG TAC CTT 1679

Asn Glu Tyr Ile Val Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu cs 0050/49790 545 550 555

TTA AAG GTT CAG TTT AAT TTC CTT CAG CTG TGG TGA ATGTTGATAT 1725

Leu Lys Val Gln Phe Asn Phe Leu Gln Leu Trp * 560 565 570

TAAATAAACC AGAGATCTGA TCTTCAAGCA AGAAAATAAG CAGTGTTGTA CTTGTGAATT 1785

TTGTGATATT TTATGTAATA AAAACTGTAC AGGTCTAAAA AAAAAAAAAA AAAAAAAA 1843 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 571 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Ala Arg Arg Arg Arg Ser Thr Gly Gly Gly Arg Ala Arg Ala 1 5 10 15

Leu Asn Glu Ser Lys Arg Val Asn Asn Gly Asn Thr Ala Pro Glu Asp

Ser Ser Pro Ala Lys Lys Thr Arg Arg Cys Gln Arg Gln Glu Ser Lys 40 45

Lys Met Pro Val Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu Asp 50 55 60

Lys Gln Asp Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala Pro 65 70 75 80

Val Asp Pro Glu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr Cys 85 90 95 . Glu Gly Asn Asp Val Tyr Asp Val Met Leu Asn Gln Thr Asn Leu Gln 100 105 110

Phe Asn Asn Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu Asp Asp Ala 115 120 125

Gln Arg Asn Phe Ser Val Trp Met Arg Trp Gly Arg Val Gly Lys Met 130 135 140 os 0050/49790

X ; 53 ) Gly Gln His Ser Leu Val Ala Cys Ser Gly Asn Leu Asn Lys Ala Lys 145 150 155 160

Glu Ile Phe Gln Lys Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp Glu 165 170 175

Asp Arg Glu Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu Gln 180 185 190

Met Asp Tyr Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys Lys Glu 195 200 205

Glu Ser Leu Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu Asp Leu Arg 210 215 220

Val Gln Glu Leu Ile Lys Leu Ile Cys Asn Val Gln Ala Met Glu Glu 225 230 235 240

Met Met Met Glu Met Lys Tyr Asn Thr Lys Lys Ala Pro Leu Gly Lys 245 250 255

Leu Thr val Ala Gln Ile Lys Ala Gly Tyr Gln Ser Leu Lys Lys Ile 260 265 270

Glu Asp Cys Ile Arg Ala Gly Gln His Gly Arg Ala Leu Met Glu Ala 275 280 285

Cys Asn Glu Phe Tyr Thr Arg Ile Pro His Asp Phe Gly Leu Arg Thr 290 295 300

Pro Pro Leu Ile Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln Leu 305 310 315 320

Leu Glu Ala Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys Thr 325 330 335

Glu Leu Gln Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg Asn Leu 340 345 350

His Cys Ala Leu Arg Pro Leu Asp His Glu Ser Tyr Glu Phe Lys Val 355 360 365

Ile Ser Gln Tyr Leu Gln Ser Thr His Ala Pro Thr His Ser Asp Tyr 370 375 380

Thr Met Thr Leu Leu Asp Leu Phe Glu Val Glu Lys Asp Gly Glu Lys

El 0050/49790 : ® 54 385 390 395 400

Glu Ala Phe Arg Glu Asp Leu His Asn Arg Met Leu Leu Trp His Gly 405 410 415

Ser Arg Met Ser Asn Trp Val Gly Ile Leu Ser His Gly Leu Arg Ile 420 425 430

Ala Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met Phe Gly Lys Gly Ile 435 440 445

Tyr Phe Ala Asp Met Ser Ser Lys Ser Ala Asn Tyr Cys Phe Ala Ser 450 455 460

Arg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser Glu Val Ala Leu Gly 465 470 475 480

Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro Lys Ala Glu Gly Leu Leu 485 490 495

Gln Gly Lys His Ser Thr Lys Gly Leu Gly Lys Met Ala Pro Ser Ser 500 505 510

Ala His Phe Val Thr Leu Asn Gly Ser Thr val Pro Leu Gly Pro Ala 515 520 525

Ser Asp Thr Gly Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr Asn 530 535 540

Glu Tyr Ile Val Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu Leu 545 550 555 560

Lys Val Gln Phe Asn Phe Leu Gln Leu Trp * 565 570 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2265 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

Ea 0050/49790 . @ ss ) (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: (F) TISSUE TYPE: uterus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 242..1843 (D) OTHER INFORMATION:/product= "Poly ADP Ribose

Polymerase” (x1) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TGGGACTGGT CGCCTGACTC GGCCTGCCCC AGCCTCTGCT TCACCCCACT GGTGGCCAAA 60

TAGCCGATGT CTAATCCCCC ACACAAGCTC ATCCCCGGCC TCTGGGATTG TTGGGAATTC 120

TCTCCCTAAT TCACGCCTGA GGCTCATGGA GAGTTGCTAG ACCTGGGACT GCCCTGGGAG 180

GCGCACACAA CCAGGCCGGG TGGCAGCCAG GACCTCTCCC ATGTCCCTGC TTTTCTTGGC 240

C ATG GCT CCA AAG CCG AAG CCC TGG GTA CAG ACT GAG GGC CCT GAG 286

Met Ala Pro Lys Pro Lys Pro Trp Val Gln Thr Glu Gly Pro Glu 575 580 585

AAG AAG AAG GGC CGG CAG GCA GGA AGG GAG GAG GAC CCC TTC CGC TCC 334

Lys Lys Lys Gly Arg Gln Ala Gly Arg Glu Glu Asp Pro Phe Arg Ser 590 595 600

ACC GCT GAG GCC CTC AAG GCC ATA CCC GCA GAG AAG CGC ATA ATC CGC 382

Thr Ala Glu Ala Leu Lys Ala Ile Pro Ala Glu Lys Arg Ile Ile Arg 605 610 615

GTG GAT CCA ACA TGT CCA CTC AGC AGC AAC CCC GGG ACC CAG GTG TAT 430

Val Asp Pro Thr Cys Pro Leu Ser Ser Asn Pro Gly Thr Gln Val Tyr 620 625 630

GAG GAC TAC AAC TGC ACC CTG AAC CAG ACC AAC ATC GAG AAC AAC AAC 478

Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr Asn Ile Glu Asn Asn Asn 635 640 645 650

AAC AAG TTC TAC ATC ATC CAG CTG CTC CAA GAC AGC AAC CGC TTC TTC 526

Asn Lys Phe Tyr Ile Ile Gln Leu Leu Gln Asp Ser Asn Arg Phe Phe 655 660 665

ACC TGC TGG AAC CGC TGG GGC CGT GTG GGA GAG GTC GGC CAG TCA AAG 574

Thr Cys Trp Asn Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys

Ey 0050/49790 .® 56 670 675 680

ATC AAC CAC TTC ACA AGG CTA GAA GAT GCA AAG AAG GAC TTT GAG AAG 622

Ile Asn His Phe Thr Arg Leu Glu Asp Ala Lys Lys Asp Phe Glu Lys 685 690 695

AAA TTT CGG GAA AAG ACC AAG AAC AAC TGG GCA GAG CGG GAC CAC TTT 670

Lys Phe Arg Glu Lys Thr Lys Asn Asn Trp Ala Glu Arg Asp His Phe 700 705 710

GTG TCT CAC CCG GGC AAG TAC ACA CTT ATC GAA GTA CAG GCA GAG GAT 718

Val Ser His Pro Gly Lys Tyr Thr Leu Ile Glu Val Gln Ala Glu Asp 715 720 725 730

GAG GCC CAG GAA GCT GTG GTG AAG GTG GAC AGA GGC CCA GTG AGG ACT 766

Glu Ala Gln Glu Ala Val Val Lys Val Asp Arg Gly Pro Val Arg Thr 735 740 745

GTG ACT AAG CGG GTG CAG CCC TGC TCC CTG GAC CCA GCC ACG CAG AAG 814

Val Thr Lys Arg Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln Lys 750 755 760

CTC ATC ACT AAC ATC TTC AGC AAG GAG ATG TTC AAG AAC ACC ATG GCC 862

Leu Ile Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn Thr Met Ala 765 770 775

CTC ATG GAC CTG GAT GTG AAG AAG ATG CCC CTG GGA AAG CTG AGC AAG 910

Leu Met Asp Leu Asp Val Lys Lys Met Pro Leu Gly Lys Leu Ser Lys 780 785 790

CAA CAG ATT GCA CGG GGT TTC GAG GCC TTG GAG GCG CTG GAG GAG GCC 958

Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu Ala 795 800 805 810

CTG AAA GGC CCC ACG GAT GGT GGC CAA AGC CTG GAG GAG CTG TCC TCA 1006

Leu Lys Gly Pro Thr Asp Gly Gly Gln Ser Leu Glu Glu Leu Ser Ser 815 820 825

CAC TTT TAC ACC GTC ATC CCG CAC AAC TTC GGC CAC AGC CAG CCC CCG 1054

His Phe Tyr Thr val Ile Pro His Asn Phe Gly His Ser Gln Pro Pro 830 835 840

CCC ATC AAT TCC CCT GAG CTT CTG CAG GCC AAG AAG GAC ATG CTG CTG 1102

Pro Ile Asn Ser Pro Glu Leu Leu Gln Ala Lys Lys Asp Met Leu Leu 845 850 855

GTG CTG GCG GAC ATC GAG CTG GCC CAG GCC CTG CAG GCA GTC TCT GAG 1150

Ea 0050/49790 - 9 57

Val Leu Ala Asp Ile Glu Leu Ala Gln Ala Leu Gln Ala Val Ser Glu 860 865 870

CAG GAG AAG ACG GTG GAG GAG GTG CCA CAC CCC CTG GAC CGA GAC TAC 1198

Gln Glu Lys Thr Val Glu Glu Val Pro His Pro Leu Asp Arg Asp Tyr 875 880 885 890

CAG CTT CTC AAG TGC CAG CTG CAG CTG CTA GAC TCT GGA GCA CCT GAG 1246

Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser Gly Ala Pro Glu 895 900 905

TAC AAG GTG ATA CAG ACC TAC TTA GAA CAG ACT GGC AGC AAC CAC AGG 1294

Tyr Lys Val Ile Gln Thr Tyr Leu Glu Gln Thr Gly Ser Asn His Arg 910 915 920

TGC CCT ACA CTT CAA CAC ATC TGG AAA GTA AAC CAA GAA GGG GAG GAA 1342

Cys Pro Thr Leu Gln His Ile Trp Lys Val Asn Gln Glu Gly Glu Glu 925 930 935

GAC AGA TTC CAG GCC CAC TCC AAA CTG GGT AAT CGG AAG CTG CTG TGG 1390

Asp Arg Phe Gln Ala His Ser Lys Leu Gly Asn Arg Lys Leu Leu Trp 940 945 950

CAT GGC ACC AAC ATG GCC GTG GTG GCC GCC ATC CTC ACT AGT GGG CTC 1438

His Gly Thr Asn Met Ala Val Val Ala Ala Ile Leu Thr Ser Gly Leu 955 960 965 970

CGC ATC ATG CCA CAT TCT GGT GGG CGT GTT GGC AAG GGC ATC TAC TTT 1486

Arg Ile Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe 975 980 985

GCC TCA GAG AAC AGC AAG TCA GCT GGA TAT GTT ATT GGC ATG AAG TGT 1534

Ala Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Ile Gly Met Lys Cys 990 995 1000

GGG GCC CAC CAT GTC GGC TAC ATG TTC CTG GGT GAG GTG GCC CTG GGC 1582

Gly Ala His His val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu Gly 1005 1010 1015

AGA GAG CAC CAT ATC AAC ACG GAC AAC CCC AGC TTG AAG AGC CCA CCT 1630

Arg Glu His His Ile Asn Thr Asp Asn Pro Ser Leu Lys Ser Pro Pro 1020 1025 1030

CCT GGC TTC GAC AGT GTC ATT GCC CGA GGC CAC ACC GAG CCT GAT CCG 1678

Pro Gly Phe Asp Ser Val Ile Ala Arg Gly His Thr Glu Pro Asp Pro 1035 1040 1045 1050

ER 0050/49790 - LJ 58 ) ACC CAG GAC ACT GAG TTG GAG CTG GAT GGC CAG CAA GTG GTG GTG CCC 1726

Thr Gln Asp Thr Glu Leu Glu Leu Asp Gly Gln Gln Val val Val Pro 1055 1060 1065

CAG GGC CAG CCT GTG CCC TGC CCA GAG TTC AGC AGC TCC ACA TTC TCC 1774

Gln Gly Gln Pro Val Pro Cys Pro Glu Phe Ser Ser Ser Thr Phe Ser 1070 1075 1080

CAG AGC GAG TAC CTC ATC TAC CAG GAG AGC CAG TGT CGC CTG CGC TAC 1822

Gln Ser Glu Tyr Leu Ile Tyr Gln Glu Ser Gln Cys Arg Leu Arg Tyr 1085 1090 1095

CTG CTG GAG GTC CAC CTC TGA GTGCCCGCCC TGTCCCCCGG GGTCCTGCAA 1873

Leu Leu Glu Val His Leu * 1100 1105

GGCTGGACTG TGATCTTCAA TCATCCTGCC CATCTCTGGT ACCCCTATAT CACTCCTTTT 1933

TTTCAAGAAT ACAATACGTT GTTGTTAACT ATAGTCACCA TGCTGTACAA GATCCCTGAA 1993

CTTATGCCTC CTAACTGAAA TTTTGTATTC TTTGACACAT CTGCCCAGTC CCTCTCCTCC 2053

CAGCCCATGG TAACCAGCAT TTGACTCTTT ACTTGTATAA GGGCAGCTTT TATAGGTTCC 2113

ACATGTAAGT GAGATCATGC AGTGTTTGTC TTTCTGTGCC TGGCTTATTT CACTCAGCAT 2173

AATGTGCACC GGGTTCACCC ATGTTTTCAT AAATGACAAG ATTTCCTCCT TTAAAAAAAA 2233

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 2265 (2) INFORMATION FOR SEQ ID NO: 4: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 534 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala Pro Lys Pro Lys Pro Trp Val Gln Thr Glu Gly Pro Glu Lys 1 5 10 15

Lys Lys Gly Arg Gln Ala Gly Arg Glu Glu Asp Pro Phe Arg Ser Thr

Ala Glu Ala Leu Lys Ala Ile Pro Ala Glu Lys Arg Ile Ile Arg Val

ER 0050/49790

X | 59 40 45

Asp Pro Thr Cys Pro Leu Ser Ser Asn Pro Gly Thr Gln Val Tyr Glu 50 55 60

Asp Tyr Asn Cys Thr Leu Asn Gln Thr Asn Ile Glu Asn Asn Asn Asn 65 70 75 80

Lys Phe Tyr Ile Ile Gln Leu Leu Gln Asp Ser Asn Arg Phe Phe Thr 85 90 95

Cys Trp Asn Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys Ile 100 105 110

Asn His Phe Thr Arg Leu Glu Asp Ala Lys Lys Asp Phe Glu Lys Lys 115 120 125

Phe Arg Glu Lys Thr Lys Asn Asn Trp Ala Glu Arg Asp His Phe Val 130 135 140

Ser His Pro Gly Lys Tyr Thr Leu Ile Glu Val Gln Ala Glu Asp Glu 145 150 155 160

Ala Gln Glu Ala val val Lys Val Asp Arg Gly Pro Val Arg Thr val 165 170 175

Thr Lys Arg Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln Lys Leu 180 185 190

Ile Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn Thr Met Ala Leu 195 200 205

Met Asp Leu Asp Val Lys Lys Met Pro Leu Gly Lys Leu Ser Lys Gln 210 215 220

Gln Ile Ala Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu Ala Leu 225 230 235 240

Lys Gly Pro Thr Asp Gly Gly Gln Ser Leu Glu Glu Leu Ser Ser His 245 250 255

Phe Tyr Thr Val Ile Pro His Asn Phe Gly His Ser Gln Pro Pro Pro 260 265 270

Ile Asn Ser Pro Glu Leu Leu Gln Ala Lys Lys Asp Met Leu Leu Val 275 280 285 oo 0050/49790 ) Leu Ala Asp Ile Glu Leu Ala Gln Ala Leu Gln Ala Val Ser Glu Gln 290 295 300

Glu Lys Thr val Glu Glu Val Pro His Pro Leu Asp Arg Asp Tyr Gln 305 310 315 320

Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser Gly Ala Pro Glu Tyr 325 330 335

Lys Val Ile Gln Thr Tyr Leu Glu Gln Thr Gly Ser Asn His Arg Cys 340 345 350

Pro Thr Leu Gln His Ile Trp Lys Val Asn Gln Glu Gly Glu Glu Asp 355 360 365

Arg Phe Gln Ala His Ser Lys Leu Gly Asn Arg Lys Leu Leu Trp His 370 375 380

Gly Thr Asn Met Ala Val val Ala Ala Ile Leu Thr Ser Gly Leu Arg 385 390 395 400

Ile Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala 405 410 415

Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Ile Gly Met Lys Cys Gly 420 425 : 430

Ala His His Val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu Gly Arg 435 440 445

Glu His His Ile Asn Thr Asp Asn Pro Ser Leu Lys Ser Pro Pro Pro 450 455 460

Gly Phe Asp Ser Val Ile Ala Arg Gly His Thr Glu Pro Asp Pro Thr 465 470 475 480

Gln Asp Thr Glu Leu Glu Leu Asp Gly Gln Gln Val Val Val Pro Gln 485 490 495

Gly Gln Pro Val Pro Cys Pro Glu Phe Ser Ser Ser Thr Phe Ser Gln 500 505 510

Ser Glu Tyr Leu Ile Tyr Gln Glu Ser Gln Cys Arg Leu Arg Tyr Leu 515 520 525

Leu Glu val His Leu *

ER 0050/49790 ) (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2265 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: (F) TISSUE TYPE: uterus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 221..1843 (D) OTHER INFORMATION: /product= "Poly ADP Ribose

Polymerase” (x1) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TGGGACTGGT CGCCTGACTC GGCCTGCCCC AGCCTCTGCT TCACCCCACT GGTGGCCAAA 60

TAGCCGATGT CTAATCCCCC ACACAAGCTC ATCCCCGGCC TCTGGGATTG TTGGGAATTC 120

TCTCCCTAAT TCACGCCTGA GGCTCATGGA GAGTTGCTAG ACCTGGGACT GCCCTGGGAG 180

GCGCACACAA CCAGGCCGGG TGGCAGCCAG GACCTCTCCC ATG TCC CTG CTT TTC 235

Met Ser Leu Leu Phe 535

TTG GCC ATG GCT CCA AAG CCG AAG CCC TGG GTA CAG ACT GAG GGC CCT 283

Leu Ala Met Ala Pro Lys Pro Lys Pro Trp Val Gln Thr Glu Gly Pro 540 545 550 555

GAG AAG AAG AAG GGC CGG CAG GCA GGA AGG GAG GAG GAC CCC TTC CGC 331

Glu Lys Lys Lys Gly Arg Gln Ala Gly Arg Glu Glu Asp Pro Phe Arg 560 565 570

TCC ACC GCT GAG GCC CTC AAG GCC ATA CCC GCA GAG AAG CGC ATA ATC 379

Ser Thr Ala Glu Ala Leu Lys Ala Ile Pro Ala Glu Lys Arg Ile Ile 575 580 585

BE 0050/49790

L 62 ) CGC GTG GAT CCA ACA TGT CCA CTC AGC AGC AAC CCC GGG ACC CAG GTG 427

Arg Val Asp Pro Thr Cys Pro Leu Ser Ser Asn Pro Gly Thr Gln Val 590 595 600

TAT GAG GAC TAC AAC TGC ACC CTG AAC CAG ACC AAC ATC GAG AAC AAC 475

Tyr Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr Asn Ile Glu Asn Asn 605 610 615

AAC AAC AAG TTC TAC ATC ATC CAG CTG CTC CAA GAC AGC AAC CGC TTC 523

Asn Asn Lys Phe Tyr Ile Ile Gln Leu Leu Gln Asp Ser Asn Arg Phe 620 625 630 635

TTC ACC TGC TGG AAC CGC TGG GGC CGT GTG GGA GAG GTC GGC CAG TCA 571

Phe Thr Cys Trp Asn Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser 640 645 650

AAG ATC AAC CAC TTC ACA AGG CTA GAA GAT GCA AAG AAG GAC TTT GAG 619

Lys Ile Asn His Phe Thr Arg Leu Glu Asp Ala Lys Lys Asp Phe Glu 655 660 665

AAG AAA TTT CGG GAA AAG ACC AAG AAC AAC TGG GCA GAG CGG GAC CAC 667

Lys Lys Phe Arg Glu Lys Thr Lys Asn Asn Trp Ala Glu Arg Asp His 670 675 680

TTT GTG TCT CAC CCG GGC AAG TAC ACA CTT ATC GAA GTA CAG GCA GAG 715

Phe Val Ser His Pro Gly Lys Tyr Thr Leu Ile Glu Val Gln Ala Glu 685 690 695

GAT GAG GCC CAG GAA GCT GTG GTG AAG GTG GAC AGA GGC CCA GTG AGG 763

Asp Glu Ala Gln Glu Ala Val Val Lys Val Asp Arg Gly Pro Val Arg 700 705 710 715

ACT GTG ACT AAG CGG GTG CAG CCC TGC TCC CTG GAC CCA GCC ACG CAG 811

Thr Val Thr Lys Arg Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln 720 725 730

AAG CTC ATC ACT AAC ATC TTC AGC AAG GAG ATG TTC AAG AAC ACC ATG 859

Lys Leu Ile Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn Thr Met 735 740 745

GCC CTC ATG GAC CTG GAT GTG AAG AAG ATG CCC CTG GGA AAG CTG AGC 907

Ala Leu Met Asp Leu Asp Val Lys Lys Met Pro Leu Gly Lys Leu Ser 750 755 760

AAG CAA CAG ATT GCA CGG GGT TTC GAG GCC TTG GAG GCG CTG GAG GAG 955

Lys Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu

ER 0050/49790 - LC 63 765 770 775

GCC CTG AAA GGC CCC ACG GAT GGT GGC CAA AGC CTG GAG GAG CTG TCC 1003

Ala Leu Lys Gly Pro Thr Asp Gly Gly Gln Ser Leu Glu Glu Leu Ser 780 785 790 795

TCA CAC TTT TAC ACC GTC ATC CCG CAC AAC TTC GGC CAC AGC CAG CCC 1051

Ser His Phe Tyr Thr Val Ile Pro His Asn Phe Gly His Ser Gln Pro 800 805 810

CCG CCC ATC AAT TCC CCT GAG CTT CTG CAG GCC AAG AAG GAC ATG CTG 1099

Pro Pro Ile Asn Ser Pro Glu Leu Leu Gln Ala Lys Lys Asp Met Leu 815 820 825

CTG GTG CTG GCG GAC ATC GAG CTG GCC CAG GCC CTG CAG GCA GTC TCT 1147

Leu Val Leu Ala Asp Ile Glu Leu Ala Gln Ala Leu Gln Ala Val Ser 830 835 840

GAG CAG GAG AAG ACG GTG GAG GAG GTG CCA CAC CCC CTG GAC CGA GAC 1195

Glu Gln Glu Lys Thr Val Glu Glu Val Pro His Pro Leu Asp Arg Asp 845 850 855

TAC CAG CTT CTC AAG TGC CAG CTG CAG CTG CTA GAC TCT GGA GCA CCT 1243

Tyr Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser Gly Ala Pro 860 865 870 875

GAG TAC AAG GTG ATA CAG ACC TAC TTA GAA CAG ACT GGC AGC AAC CAC 1291

Glu Tyr Lys Val Ile Gln Thr Tyr Leu Glu Gln Thr Gly Ser Asn His 880 885 890

AGG TGC CCT ACA CTT CAA CAC ATC TGG AAA GTA AAC CAA GAA GGG GAG 1339

Arg Cys Pro Thr Leu Gln His Ile Trp Lys Val Asn Gln Glu Gly Glu 895 900 905

GAA GAC AGA TTC CAG GCC CAC TCC AAA CTG GGT AAT CGG AAG CTG CTG 1387

Glu Asp Arg Phe Gln Ala His Ser Lys Leu Gly Asn Arg Lys Leu Leu 910 915 920

TGG CAT GGC ACC AAC ATG GCC GTG GTG GCC GCC ATC CTC ACT AGT GGG 1435

Trp His Gly Thr Asn Met Ala Val Val Ala Ala Ile Leu Thr Ser Gly 925 930 935

CTC CGC ATC ATG CCA CAT TCT GGT GGG CGT GTT GGC AAG GGC ATC TAC 1483

Leu Arg Ile Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr 940 945 950 955

TTT GCC TCA GAG AAC AGC AAG TCA GCT GGA TAT GTT ATT GGC ATG AAG 1531

,. 0050/49790

Phe Ala Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Ile Gly Met Lys 960 965 970

TGT GGG GCC CAC CAT GTC GGC TAC ATG TTC CTG GGT GAG GTG GCC CTG 1579

Cys Gly Ala His His Val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu 975 980 985

GGC AGA GAG CAC CAT ATC AAC ACG GAC AAC CCC AGC TTG AAG AGC CCA 1627

Gly Arg Glu His His Ile Asn Thr Asp Asn Pro Ser Leu Lys Ser Pro 990 995 1000

CCT CCT GGC TTC GAC AGT GTC ATT GCC CGA GGC CAC ACC GAG CCT GAT 1675

Pro Pro Gly Phe Asp Ser Val Ile Ala Arg Gly His Thr Glu Pro Asp 1005 1010 1015

CCG ACC CAG GAC ACT GAG TTG GAG CTG GAT GGC CAG CAA GTG GTG GTG 1723

Pro Thr Gln Asp Thr Glu Leu Glu Leu Asp Gly Gln Gln Val Val Val 1020 1025 1030 1035

CCC CAG GGC CAG CCT GTG CCC TGC CCA GAG TTC AGC AGC TCC ACA TTC 1771

Pro Gln Gly Gln Pro Val Pro Cys Pro Glu Phe Ser Ser Ser Thr Phe 1040 1045 1050

TCC CAG AGC GAG TAC CTC ATC TAC CAG GAG AGC CAG TGT CGC CTG CGC 1819

Ser Gln Ser Glu Tyr Leu Ile Tyr Gln Glu Ser Gln Cys Arg Leu Arg 1055 1060 1065

TAC CTG CTG GAG GTC CAC CTC TGA GTGCCCGCCC TGTCCCCCGG GGTCCTGCAA 1873

Tyr Leu Leu Glu Val His Leu * 1070 1075

GGCTGGACTG TGATCTTCAA TCATCCTGCC CATCTCTGGT ACCCCTATAT CACTCCTTTT 1933

TTTCAAGAAT ACAATACGTT GTTGTTAACT ATAGTCACCA TGCTGTACAA GATCCCTGAA 1993

CTTATGCCTC CTAACTGAAA TTTTGTATTC TTTGACACAT CTGCCCAGTC CCTCTCCTCC 2053

CAGCCCATGG TAACCAGCAT TTGACTCTTT ACTTGTATAA GGGCAGCTTT TATAGGTTCC 2113

ACATGTAAGT GAGATCATGC AGTGTTTGTC TTTCTGTGCC TGGCTTATTT CACTCAGCAT 2173

AATGTGCACC GGGTTCACCC ATGTTTTCAT AAATGACAAG ATTTCCTCCT TTAAAAAAAA 2233

AAAAAAAAAA AAAAAAAAAA AAAAADAAAA AA 2265 (2) INFORMATION FOR SEQ ID NO: 6:

oo 0050/49790

KX 65 ) (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 541 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Ser Leu Leu Phe Leu Ala Met Ala Pro Lys Pro Lys Pro Trp Val 1 5 10 15

Gln Thr Glu Gly Pro Glu Lys Lys Lys Gly Arg Gln Ala Gly Arg Glu

Glu Asp Pro Phe Arg Ser Thr Ala Glu Ala Leu Lys Ala Ile Pro Ala 40 45

Glu Lys Arg Ile Ile Arg Val Asp Pro Thr Cys Pro Leu Ser Ser Asn 50 55 60

Pro Gly Thr Gln Val Tyr Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr 65 70 75 80

Asn Ile Glu Asn Asn Asn Asn Lys Phe Tyr Ile Ile Gln Leu Leu Gln 85 90 95

Asp Ser Asn Arg Phe Phe Thr Cys Trp Asn Arg Trp Gly Arg Val Gly 100 105 110

Glu Val Gly Gln Ser Lys Ile Asn His Phe Thr Arg Leu Glu Asp Ala 115 120 125

Lys Lys Asp Phe Glu Lys Lys Phe Arg Glu Lys Thr Lys Asn Asn Trp 130 135 140

Ala Glu Arg Asp His Phe Val Ser His Pro Gly Lys Tyr Thr Leu Ile 145 150 155 160

Glu Val Gln Ala Glu Asp Glu Ala Gln Glu Ala Val Val Lys Val Asp 165 170 175

Arg Gly Pro Val Arg Thr val Thr Lys Arg Val Gln Pro Cys Ser Leu 180 185 190

Asp Pro Ala Thr Gln Lys Leu Ile Thr Asn Ile Phe Ser Lys Glu Met 195 200 205

- 0050/49790

KX | 66

Phe Lys Asn Thr Met Ala Leu Met Asp Leu Asp Val Lys Lys Met Pro 210 215 220

Leu Gly Lys Leu Ser Lys Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu 225 230 235 240

Glu Ala Leu Glu Glu Ala Leu Lys Gly Pro Thr Asp Gly Gly Gln Ser 245 250 255

Leu Glu Glu Leu Ser Ser His Phe Tyr Thr Val Ile Pro His Asn Phe 260 265 270

Gly His Ser Gln Pro Pro Pro Ile Asn Ser Pro Glu Leu Leu Gln Ala 275 280 285

Lys Lys Asp Met Leu Leu Val Leu Ala Asp Ile Glu Leu Ala Gln Ala 290 295 300

Leu Gln Ala Val Ser Glu Gln Glu Lys Thr Val Glu Glu Val Pro His 305 310 315 320

Pro Leu Asp Arg Asp Tyr Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu 325 330 335

Asp Ser Gly Ala Pro Glu Tyr Lys Val Ile Gln Thr Tyr Leu Glu Gln 340 345 350

Thr Gly Ser Asn His Arg Cys Pro Thr Leu Gln His Ile Trp Lys Val 355 360 365

Asn Gln Glu Gly Glu Glu Asp Arg Phe Gln Ala His Ser Lys Leu Gly 370 375 380

Asn Arg Lys Leu Leu Trp His Gly Thr Asn Met Ala Val Val Ala Ala 385 390 395 400

Ile Leu Thr Ser Gly Leu Arg Ile Met Pro His Ser Gly Gly Arg Val 405 410 415

Gly Lys Gly Ile Tyr Phe Ala Ser Glu Asn Ser Lys Ser Ala Gly Tyr 420 425 430

Val Ile Gly Met Lys Cys Gly Ala His His Val Gly Tyr Met Phe Leu 435 440 445

Gly Glu val Ala Leu Gly Arg Glu His His Ile Asn Thr Asp Asn Pro

Ea 0050/49790 450 455 460

Ser Leu Lys Ser Pro Pro Pro Gly Phe Asp Ser Val Ile Ala Arg Gly 465 470 475 480

His Thr Glu Pro Asp Pro Thr Gln Asp Thr Glu Leu Glu Leu Asp Gly 485 490 495

Gln Gln Val Val Val Pro Gln Gly Gln Pro Val Pro Cys Pro Glu Phe 500 505 510

Ser Ser Ser Thr Phe Ser Gln Ser Glu Tyr Leu Ile Tyr Gln Glu Ser 515 520 525

Gln Cys Arg Leu Arg Tyr Leu Leu Glu Val His Leu * 530 535 540 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1740 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA ’ (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:112..1710 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CCCGGCTTTC ACTTTTTCTG CTGCCTCGGG GAACACCTCG AGCCAACTGC TTCCTAACTC 60

AGGGTGGGCE GAACTGACGG GATCTAAGCT TCTGCATCTC TGAGGAGAAC C ATG GCT 117

Met Ala

CCA AAA CGA AAG GCC TCT GTG CAG ACT GAG GGC TCC AAG RAG CAG CGa 165

Pro Lys Arg Lys Rle Ser Vel Gln Thr Glu Gly Ser Lys Lys Gln Arg 545 550 535

Ea 0050/49790

KX | 68 i CAR GGG ACA GAG GAG GAG GAC AGC TTC CGG TCC ACT GCC GAG GCT CTC 213

Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu Ala Leu 560 565 570 575

AGA GCA GCA CCT GCT GAT AAT CGG GTC ATC CGT GTC GAC CCC TCA TGT 261

Arg Ala Ala Pro Rla Asp Asn Arg Vel Ile Arg Val Asp Pro Ser Cys 580 585 590

CCA TTC AGC CGG AAC CCC GGG ATA CAG GTC CAC GAG GAC TAT GAC TGT 309

Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His Glu Asp Tyr Asp Cys 595 600 605

ACC CTG RAC CAG ACC AAC ATC GGC AAC AAC AAC AAC AAG TTC TAT ATT 357

Thr Leu 2sn Gln Thr Asn Ile Gly Asn Asn Asn Asn Lys Phe Tyr Ile 610 615 620

LTC CAA CTG CTG GAG GAG GGT AGT CGC TTC TTC TGC TGG AAT CGC TGG 405

Ile Gln Leu Leu Glu Glu Gly Ser Arg Phe Phe Cys Trp Asn Arg Top 625 # 630 635

GGC CGC GTG GGA GAG GTG GGC CAG AGC AAG ATG AAC CAC TTC ACC TGC 453

Gly arg Val Gly Glu Val Gly Gln Ser Lys Met Asn His Phe Thr Cys 640 645 650 655

CTG GAA GAT GCA AAG AAG GAC TTT AAG AAG AAA TTT TGG GAG AAG ACT 501

Leu Glu Asp kla Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu Lys Thr 660 665 670

AAL RAC AAR TGG GAG GAG CGG GAC CGT TTT GTG GCC CAG CCC AAC AAG 549

Lys Asn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln Pro Asn Llys 675 680 685

TACT ACA CTT ATA GAA GTC CAG GGA GAA GCA GAG AGC CAA GAG GCT GTA 587

Tyr Thr Leu Ile Glu Vel Gln Gly Glu Ala Glu Ser Gln Glu Ala Val €S0 695 700

GTG AAG GCC TTR TCT CCC CAG GTG GAC AGC GGC CCT GTG AGG ACC GTG 645

Val Lys klza Leu Ser Pro Gln Val Asp Ser Gly Pro Val Arg Thr Val 705 710 715

GTC AAG CCC TGC TCC CTA GAC CCT GCC ACC CAG AAC CTT ATC ACC AAC 693

Val Lys Pro Cys Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile Thr Asn 720 725 730 735

ATC TTC AGC ARA GAG ATG TTC AAG AAC GCA ATG ACC CTC ATG AAC CTG 741

Ile Phe Ser Lys Glu Met Phe Lys Asn Ala Met Thr Leu Met Asn Leu 740 745 750

GAT GTG AAG AAG ATG CCC TTG GGA AAG CTG ALC AAG CAG CAG ATT GCC 789 sp Val Lys Lys Met Pro Leu Gly Lys Leu Thr Lys Gln Gln Ile Ala 755 760 765

CGT GGC TTC GAG GCC TTG GAZ GCT CTA GAG GAG GCC ATG AAA AAC CCC B37

Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu Ala Me<+ Lys Asn Pro 770 775 780

ACR GGG GAT GGC CAG AGC CTG GAA GAG CTC TCC TCC TGC TTC TAC ACT 885

Thr Gly Asp Gly Gln Ser Leu Glu Glu Leu Ser Ser Cys Phe Tyr Thr 785 790 795

GTC ATC CCA CAC AAC TTC GGC CGC AGC CGa ccC CCG CCC ATC AAC TCC 933

Val Ile Pro His Asn Phe Gly Arc Ser Arg Pro Pro Pro Ile Asn Ser oo 0050/49790

XK | 69 . 800 1-1) B10 815

CCT GAT GTG CTT CAG GCC AAG AAG GAC ATG CTG CTG GTC CTA GCG GAC S81

Pro Asp Val Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu Ala Asp 820 B25 830

ATC GAG TTG GCG CAG ACC TTG CAG GCA GCC CCT GGG GAG GAG GAG GAG 1029

Ile Glu Leu Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu Glu Glu Glu

B35 840 845

AAA GTG GAA GAG GTG CCA CAC CCA CTG GAT CGA GAC TAC CAG CTC CTC 1077

Lys Val Glu Glu Val Pro His Pro Leu Asp Arg Asp Tyr Gln Leu Leu 850 855 860

AGG TGC CAG CTT CAR CTG CTG GAC TCC GGG GAG TCC GAG TAC AAG GCA 1125

Arg Cys Gln Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu Tyr Lys Ala 865 870 875

ATA CAG ACC TAC CTG AAA CAG ACT GGC AAC AGC TAC AGG TGC CCA AAC 1173

Ile Gln Thr Tyr Leu Llys Gln Thr Gly Asn Ser Tyr Arg Cys Pro Asn 880 88S _. BBO 895

CTG CGG CAT GTT TGG AAA GTG AAC CGA GAA GGG GAG GGA GAC AGG TTC 1221

Leu Arg His Val Trp Lys Val Asn Arg Glu Gly Glu Gly Asp Arg Phe 900 905 S10

CAG GCC CAC TCC AAA CTG GGC AAT CGG AGG CTG CTG TGG CAC GGC ACC 1269

Gln Ala His Ser Lys Leu Gly Asn Arg Arg Leu Leu Trp His Gly Thr 915 920 925

AAT GTG GCC GTG GTG GCT GCC ATC CTC ACC AGT GGG CTC CGA ATC ATG 1317 asn Val Ala Val Val 2la ala Ile Leu Thr Ser Glv Leu Arg Ile Met 830 935 840

CCA CAC TCG GGT GGT CGT GTT GGT AAG GGT ATT TAT TTT GCC TCT GAG 1365

Pro Eis Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe ala Ser Glu 945 950 955

AAC AGC AAG TCA GCT GGC TAT GTT ACC ACC ATG CAC TCT GGG GGC CAC 1413

Asn Ser Lys Ser Ala Gly Tyr Val Thr Thr Met His Cys Gly Gly Eis 960 965 S870 975

CAG GTG GGC TAC ATG TTC CTG GGC GAG GTG GCC CTC GGC AAA GAG CAC 1461

Gln Vel Gly Tyr Met Phe Leu Gly Glu Val Ala Leu Gly Lvs Glu His 980 985 990

CAC ATC ACC ATC GAT GAC CCC AGC TTG AAG AGT CCA CCC CCT GGC TTT 1509

Eis Ile Thr Ile Asp Asp Pro Ser Leu Lys Ser Pro Pro Pro Gly Phe 995 1000 1005

GAC AGC GTC ATC GCC CGA GGT CAR ACC GAG CCG GAT CCC GCC CAG GAC 1557

Asp Ser Val Ile Ala Arg Gly Gin Thr Glu Pro Asp Pro Ala Gln Asp 1010 1015 1020

ATT GAR CTT GAA CTG GAT GGG CAG CZZ GTC GTC CGTG CCC CAn GGC CCG 1605

Ile Glu Leu Glu Leu Asp Gly Gln Pro Val Val Val Pro Gln Gly Pro 1025 1030 1035

CCT GTG CAG TGC CCG TCA TTC AAA AGC TCC AGC TTC AGC CAG AGT GAx 1653

Pro Val Gln Cys Pro Ser Phe Lys Ser Ser Ser Phe Ser Gln Ser Glu 1040 1045 1050 1055

TAC CTC ATA TAC AAG GAG AGC CAG TGT CGC CTG CGC TAC CTG CTG GAG 1701

Tyr Leu Ile Tyr Lys Glu Ser Gln Cys Arg Leu Arg Tyr leu Leu Glu

-o 0050/49790 @® 70

To. 1060

ATT CAC CTC TAAGCTGCTT Gceereeer,

A

Ile His Leu GGTCCAAGCC 1740 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 533 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met rla Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lvs Lys 1 5 10 15

Gln Arg Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu

Ala Leu Arg Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val Asp Pro 40 45

Ser Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His Glu Asp Tyr 50 55 60 4sp Cys Thx Leu Asn Gln Thr Asn Ile Gly Asn Asn Asn Asn Lys Phe £5 70 75 80

Tyr Ile Ile Gln Leu Leu Glu Glu Gly Ser Arg Phe Phe Cys Trp Asn 85 90 95

Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys Met Asn His Phe 100 105 110

Thr Cys Leu Glu Asp Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu 115 120 125

Lys Thr Lys Rsn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln Pro 130 135 140

Zsn Lys Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu Ser Gln Glu 143 150 155 160

Alia Val Vel Lys Ala Leu Ser Pro Gln Val Asp Ser Gly Pro Val Arg 165 170 175

Thr Val Vel Lys Pro Cvs Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile 180 185 190 . Thr 2sn Ile Phe Ser Lys Glu Met Phe Lys Asn Ala Met Thr Leu Met: 185 200 205

Asn Leu Asp Val Lys lys Met Pro Leu Gly Lys Leu Thr Lys Gln Gln 210 215 220

Ile Ala Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu Ala Met Lys 223 230 235% 240

Asn Pro Thr Gly Asp Gly Gln Ser Leu Glu Glu Leu Ser Ser Cvs Phe 245 250 255

Tyr Thr Val Ile Pro His Asn Phe Gly Arg Ser Arg Pro Pro Pro Ile 260 265 270 oo 0050/49790 9 n

Co Asn Ser Pro Asp Val Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu 215; 280 285 2la Asp Ile Glu Leu Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu Glu 290 295 300

Glu Glu lys val Glu Glu Val Pro His Pro Leu Asp Arg Asp Tyr Gln 305 310 313 320

Leu Leu Arg Cys Gln Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu Tyr 325 330 335

Lys Ala Ile Gln Thr Tyr Leu Lys Gln Thr Gly Asn Ser Tyr Arg Cys 340 345 350

Pro Asn Leu Arg Ris Val Trp lys Val Asn Arg Glu Gly Glu Gly Asp 355 360 365

A-g Phe Gln Ala His Ser Lys Leu Gly Asn Arg Acg Leu Leu Trp His 370 375 380

Gly Thr Asn Val Ala Val Val Ala Ala Ile Leu Thr Ser Gly leu Arg 385 380 398 400

Ile Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala 405 410 415

Ser Glu 2sn Ser Lys Ser Ala Gly Tyr Val Thr Thr Met His Cys Gly 420 425 430

Gly His Gln Val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu Gly Lys 435 440 445

Glu His His Ile Thr Ile Asp Asp Pro Ser Leu Lys Ser Pro Pro Pro 450 485 460

Gly Phe Asp Ser Val Ile Ala Arg Gly Gln Thr Glu Pro Asp Pro Ala 465 470 475 480

Gin Asp Ile Glu Leu Glu Leu Asp Gly Gln Pro Val Val val Pro Gln 485 490 495

Gly Pro Pro Val Gln Cvs Pro Ser Phe lvs Ser Ser Ser Phe Ser Gln 500 505 510

Ser Glu Tyr Leu Ile Tyr Lys Glu Ser Gln Cys Arg Leu Arg Tyr Leu 515 520 525

Leu Glu Ile Eis Leu 530 (2) INFORMATION FOR SEQ ID NO: 9: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1587 base pairs (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

Ea 0050/49790 . ¢ 72 ) (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..1584 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

ATG GCT CCR AAA CGR AAG GCC TCT GTG CAG ACT GAG GGC TCC AAG AAG 48

Met kle Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lys Lys 535 540 545

CAG CGA CAR GGG ACA GAG GAG GAG GAC AGC TTC CGG TCC ACT GCC GAG 96

Gin Arg Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu 550 555 560 565

GCT CTC AGA GCA GCA CCT GCT GAT AAT CGG GTC ATC CGT GTG GAC CCC 144

Ala Leu Arg Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val Asp Pro 570 575 580

TCA TGT CCA TTC AGC CGG AAC CCC GGG ATA CAG GTC CAC GAG GAC TAT 192

Ser Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His Glu Asp Tyr 585 590 595

GAC TGT ACC CTG AAC CAG ACC AAC ATC GGC AAC AAC AAC AAC AAC TTC 240 hsp Cys Thr Leu Asn Gln Thr Asn Ile Gly Asn Asn Asn Asn Lys Phe 600 605 610

TAT ATT ATC CAA CTG CTG GAG GAG GGT AGT CGC TTC TTC TGC TGS AAT 288

Tyr Ile Ile Gln Leu Leu Glu Glu Gly Ser Arg Phe Phe Cys Trp Asn 615 620 625

CGC TGG GGC CGC GTG GGA GAG GTG GGC CAG AGC AAG ATG AAC CAC TTC 336

Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys Met Asn His Phe €30 635 640 645

ACC TGC CTG GAA GAT GCA AAG ARG GAC TTT AAG AAG AAA TTT TGG GAG 384

Thr Cys Leu Glu Asp Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu €50 655 660

AAG ACT AAA AAC RAR TGG GAG GAG CGG GAC CCT TTT GTG GCC CAG CCC 432

Lys Thr Lys 2sn Lys Trp Glu Glu Arg Asp Arg Phe Val ala Gln Pro 665 670 675

AAC RAG TAC ACA CTT ATA GAA GTC CAG GGA GAA GCA GAG AGC CAA GAG 480

Asn Lys Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu Ser Gln Glu 680 685 630

GCT GTA GTG AAG GTG GAC AGC GGC CCT GTG AGG ACC GTG GTC AAG CCC 528

Ala Val Val Lys Vel Asp Ser Gly Pro Val Arg Thr Val val Lys Pro 695 700 705

TGC TCC CTA GAC CCT GCC ACC CAG AAC CTT ATC ACC AAC ATC TTC AGC 576

Cys Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile Thr Asn Ile Phe Ser 710 ‘ 715 720 725

ARR GAG ATG TTC AAG AAC GCA ATG ACC CTC 2TG RAC CTC GAT GTG AAG 624

Lys Glu Met Phe Lys Asn Ala Met Thr Leu Met Asn Leu Asp Val Lys 730 735 740

AAG ATG CCC TTG GGA AAG CTG ACC AAG CAG CAG ATT GCC CGT GGC TTC 672

Lys.Met Pro Leu Gly Lys Leu Thr Lys Gln Gln Ile ala Arg Gly Phe 745 750 755

Ea 0050/49790 ® 73 i - GAG GCC TTG GAA GCT CTA GAG GAG GCC ATG AAA AAC CCC ACA GGG GAT 720

Glu Ale Leu Glu Ala Leu Glu Glu Ala Met Lys Asn Pro Thr Gly asp 760 765 770

GGC CAG AGC CTG GAA GAG CTC TCC TCC TGC TTC TAC ACT GTC ATC CCa 768

Gly Gln Ser Leu Glu Glu Leu Ser Ser Cys Phe Tyr Thr val Ile Pro 775 780 785

CAC AAC TTC GGC CGC AGC CGA CCC CCG CCC ATC AAC TCC CCT GAT GTG B16

His Asn Phe Gly Arg Ser Arg Pro Pro Pro Ile Asn Ser Pro Asp Val 790 795 800 BOS

CTT CAG GCC AAG AAG GAC ATG CTG CTG GTG CTA GCG GAC ATC GAG TTG 864

Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu Ala Asp Ile Glu Leu 810 815 820

GCG CAG ACC TTG CAG GCA GCC CCT GGG GAG GAG GAG GAG AAA GTG GAA S12

Ala Gln Thr Leu Gln Ala 2la Pro Gly Glu Glu Glu Glu Lys Val Glu 825 830 835

GAG GTG CCA CAC CCA CTG GAT CGA GAC TAC CAG CTC CTC AGG TGC CAG 960

Glu Val Pro His Pro Leu Asp Arg Asp Tyr Gln Leu Leu Arg Cys Gin 840 845 850

CTT CAA CTG CTG GAC TCC GGG GAG TCC GAG Tal AAG GCR ATA CAG ACC 1008

Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu Tv: Lys Ala Ile Gln Thr 855 860 B65

TAC CTG AAR CAG ACT GGC AAC AGC TAC AGS TGC CCA ARC CTG CGG CaT 1056

Tyr Leu Lys Gln Thr Gly Asn Ser Tyr Arg Cys Pro Asn Leu Arg His

B70 875 B80 BBS

GTT TGG AAA GTG AAC CGA GAA GGG GAG GGA GAC AGG TTC CAG GCC Cac 1104

Val Trp Lys Val Asn Arg Glu Gly Glu Gly 2sp Ac-g Phe Gln Ala His 890 BSS 900

TCC AAR CTG GGC ART CGG AGG CTG CTC TGC CAC GGC ACC AAT GTG GCC 1152

Ser Lys Leu Gly Asn Arg Arg Leu Leu Trp His Gly Thr Asn Val ala 805 910 9.5

STG GTG GCT GCC ATC CTC ACC AGT GGG CTC CGA ATC ATG CCA CAC TCG 1200

Val Vel Ala Ala Ile Leu Thr Ser Gly Leu Arg Ile Met Pro His Ser 920 825 930

GGT GGT CGT GTT GGC AAG GGT ATT TAT TTT GCC TCT GAG AAC AGC AAG 1248

Gly Gly Arg val Gly Lys Gly Ile Tyr Phe Ala Ser Glu Asn Ser Lys 835 8940 945

TCA GCT GGC TAT ,GTT ACC ACC ATG CAC TGT GGG GGC CAC CaG GTG GGC 1296

Sex la Gly Tyr ‘Val Thr Thr Met Eis Cys Gly Gly His Gln val Gly 250 935 960 965

TAC ATG TTC CTG GGC GAG GTG GCC CTC GGC AAA GAG CAC CAC ATC ACC 1344

Tyz Met Phe Leu Gly Glu Val ala Leu Gly Lys Glu Bis His Ile Thr 870 875 S80

ATC GAT GAC CCC AGC TTG AAG AGT CCA CCC CCT GGC TTT GAC AGC GTC 1392

Ile Asp Asp Pro Ser Leu lys Ser Pro Pro Pro Gly Phe asp Ser Val

Ses 990 995

ATC GCC CGX GGC CAA ACC GAG CCG GAT CCC GCC CAG GAC ATT Gaa CTT 1440

I-e Ala Arg Gly Gln Thr Glu Pro Asp Pro Ala Gln Asp Ile Glu Leu 1000 1005 1010 N no 0050/49790 ® 74 a GAA CTG GAT GGG CAG CCG GIG GTG GTG CCC CAA GGC CCG CCT GTG CAG 1488 i Glu Leu Asp Gly Gln Pro Val Val Val Pro Gln Gly Pro Pro Val Gln 1015 1020 1025

TGC CCG TCA TTC ARA AGC TCC AGC TTC AGC CAG AGT GAA TAC CTC ATA 1536

Cys Pro Ser Phe Lys Ser Ser Ser Phe Ser Gln Ser Glu Tyr Leu Ile 1030 1035 1040 1045

TAC AAG GAG AGC CAG TGT CGC CTG CGC TAC CTG CTG GAG ATT CAC CTC 1584

Tyr Lys Glu Ser Gln Cys Arg Leu Arg Tyr Leu Leu Glu Ile His Leu 1050 1055 1060

TAA 1587 (2) INFORMATION FOR SEQ ID NO: 10: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 528 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Ala Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lys Lys 1 5 10 15

Gln Arg Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu

Ala Leu Arg Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val Asp Pro 40 45

Ser Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His Glu Asp Tyr 50 55 60

Asp Cys Thr Leu Asn Gln Thr Asn Ile Gly Asn Asn Asn Asn Lys Phe €5 70 75 80

Tyr Ile Ile Gln Leu Leu Glu Glu Gly Ser Arg Phe Phe Cys Trp Asn 85 90 85

Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys Met Asn Eis Phe 100 105 110

Thr Cys Leu Glu Asp Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu 115 120 125

Lys Thr Lys Asn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln Pro 130 135 140

Asn Lys Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu Ser Gln Glu 145 150 155 160

Ale Val Val Lys Val Asp Ser Gly Pro Val Arg Thr Val val Lys Pro 165 170 175

Cys Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile Thr Asn Ile Phe Ser 180 185 190

Lys Glu Met Phe Lys Asn Xla Met Thr Leu Met Asn Leu Asp Val Lys 185 200 205

Lys Met Pro Leu Gly Lys Leu Thr Lvs Gln Gln Ile 2la Arg Gly Phe 210 215 - 220

-o. 0050/49790 @® 5

Glu Ala Leu Glu Ala Leu Glu Glu Ala Met Lys Asn Pro Thr Gly Asp 225 230 235% 240

Gly Gln Ser Leu Glu Glu Leu Ser Ser Cys Phe Tyr Thr Val lle Pro 245 250 255

Bis Asn Phe Gly Arg Ser Arg Pro Pro Proc Ile Asn Ser Pro Asp Val 260 265 270

Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu Ala Asp Ile Glu Leu 275 280 285

Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu Glu Glu Glu Lys Val Glu 290 295 300

Glu Val Pro Eis Pro Leu Asp Arg Asp Tyr Gln Leu Leu Arg Cys Gln 305 310 315 320

Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu Tyr Lys Ala Ile Gln Thr 325 330 335

Tyr Leu Lys Gln Thr Gly Asn Ser Tyr Arg Cys Pro Asn Leu Arg Eis 340 345 350 val Trp Lys Val Asn Arg Glu Gly Glu Gly Asp Arg Phe Gln Ala His 355 360 365

Ser Lys Leu Gly Asn Arg Arg Leu Leu Trp His Gly Thr Asn Val Ala 370 375 380 val val Ala 2la Ile Leu Thr Ser Gly Leu Arg Ile Met Pro His Ser 385 390 395 400

Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala Ser Glu Asn Ser Lys 405 410 415

Ser Ala Gly Tyr Val Thr Thr Met His Cys Gly Gly Eis Gln val Gly 420 425 430

Ty: Met Phe Leu Gly Glu Val Ala Leu Gly Lys Glu His His Ile Thr 435 440 445

Ile Asp Asp Pro Ser Leu lys Ser Pro Pro Pro Gly Phe Asp Ser Val 450 455 460

Ile Rla Arg Gly Gln Thr Glu Pro Asp Pro Zla Gln Asp Ile Glu Leu 4€5 470 475 480

Glu Leu Asp Gly Gln Pro Val Val Val Pro Gln Gly Pro Pro Val Gln 485 490 495

Cvs Pro Ser Phe lvs Ser Ser Ser Phe Ser Gln Ser Glu Tyr Leu lle 500 505 510

Tyr Lys Glu Ser Gln Cys Arg Len Arg Tyr Leu Leu Glu Ile Eis Leu 515 520 525

Claims (1)

1. , 0080/49100/49790 . [A AMENDED SHEET HK ( 76 : ® 1. A poly(ADP-ribose) polymerase (PARP) homolog from human or non-human mammals, which has an amino acid sequence which has a) a functional NAD* binding domain and b) no zinc finger sequence motif of the general formula © CXaCXnBX,C oC in which m is an integral value of 28 or 30, and the X radicals are, independently of one another, any amino acid.

   2. A PARP homolog as claimed in claim 1, wherein the functional NAaD* binding domain comprises one of the following general sequence motifs: PXn(S/T)GX3GKGIYFA, (S/T)XGLR(I/V)XPXn(S/T)GX3GKGIYFA or LLWHG(S/T)X7IL(5/T)XGLR(I/V)XPXy(S/T)GX3GKGIYFAX3SKSAXY in which : n is an integral value from 1 to 5, and the X radicals are, independently of one another, any amino acid.

   3. A PARP homolog as claimed in either of the preceding claims, comprising at least another one of the following part-sequence motifs: . LXgNX2YX2QLLX (D/E)X10/11WGRVG, ~ AX3FXKX4KTXNXWXsFX3PXK, : QXL(1/L)X,IXgMX;oPLGKLX30IXcL, FYTXIPHXFGX3 PP; and .. .KX3LX2LXDIEXAX,L, : in which the-X radicals are, independently of one another, any amino acid.

   40 .

   4. A PARP homolog as claimed in any of the preceding claims, selected from human PARP homologs, which has the amino acid sequence shown in SEQ ID NO: 2 (human PARP2) or SEQ ID NO: 4 or 6 (human PARP3 type 1 or 2); or murine PARP homologs which 45 have the amino acid sequence shown in SEQ ID NO:8 (mouse PARP long form) or SEQ ID No:10 (mouse PARP short form). ]

   AMENDED SHEET / 0050/49100/49790 : “ - | 77

   5. a binding partner with Specificity for paRrp homologs ag claimed in any of the Preceding claims, selected from C 2) antibodies and fragments thereof, b) Protein-ljke compounds which interact with a bPart-sequence of the Protein, ang Cc) low molecular weight effectors which modulate the catalytic parp activity or another biological function of a PARP molecule.

   6. A nucleic acid comprising 2) a nucleotide sequence coding for at least one pagrp homolog ag claimed in any of claims 1 to 4, or the complementary nucleotide sequence thereof; b) a nucleotide Sequence which hybridizes with a Sequence ag Specified in a) under stringent conditions; or C) nucleotide Sequences which are derived fron the nucleotide Sequences defined in a) and b) through the degeneracy of the genetic code,

   7. A nucleic acid as claimed jn claim 6, comprising a) nucleotides +3 to +1715 shown in SEQ ID NO:1; b) nucleotides +242 to +1843 shown in SEQ ID NO:3; C) nucleotides +221 to +1843 shown in SEQ ID NO:5; d) nucleotides +112 to +1710 shown in SEQ 1p NO:7; or €) nucleotides +1 to +1584 shown in seq 1p NO:9.

   8. An expression cassette comprising, under the genetic control of ‘at least ope regulatory nucleotide Sequence, at least one nucleotide Sequence as claimeq in either of claims 6 and 7.

   9. a recombinant vector comprising at least one expression cassette as claimed in claim 8.

   10. a recombinant microorganism comprising at least one recombinant vector as claimed ip claim 9,

   11. a transgenic mammal comprising a vector as claimed ip claim 9. 40

   12. a PARP-deficient mammal or PARP-deficient eukaryotic cell, in which functional expression of at least one gene which codes for a parp homolog ag claimed in any of claims 1 to 4 jg inhibited,

   ; 0080/491200/49790 AMENDED SHEET

   13. An in vitro detection method for PARP inhibitors, which © Vvomprises @ a) incubating an unsupported or supported polyADP-ribosylatable target with a reaction mixture comprising al) a PARP homolog as claimed in any of claims 1 to 4, a2) a PARP activator; and a3) a PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected; . b) carrying out the polyADP ribosylation reaction; and c¢) determining the polyADP ribosylation of the target qualitatively or quantitatively.

   14. A method as claimed in claim 13, wherein the PARP homolog is preincubated with the PARP activator and the PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected, before the polyADP ribosylation reaction is carried out.

   15. A method as claimed in either of claims 13 and 14, wherein the polyADP-ribosylatable target is a histone protein.

   16. A method as claimed in any of claims 13 to 15, wherein the PARP activator is activated DNA.

   17. A method as claimed in any of claims 13 to 16, wherein the polyADP ribosylation reaction is started by adding NAD*.

   18. A method as claimed in any of claims 13 to 17, wherein the polyADP ribosylation of the supported target is determined using anti-poly (ADP-ribose) antibodies.

   19. A method as claimed in any of claims 13 to 17, wherein the unsupported target is labeled with an acceptor fluorophore.

   20. A method as claimed in claim 19, wherein the polyADP ribosylation of the unsupported target is determined using anti-poly (ADP-ribose) antibody which is labeled with a donor 40 fluorophore which is able to transfer energy to the acceptor fluorophore.

   21. A method as claimed in either of claims 19 and 20, wherein the target is biotinylated histone, and the acceptor 45 fluorophore is coupled thereto via avidin or streptavidin.

   ) 0050/491200/49790 AMENDED SHEET

   22.% A method ag claimed in either of claims 20 ang 21, wherein the anti-poly (ADP-ribose) antibody carries a europium Cryptate as donor fluorophore. ® 5 23. an in vitro Screening method for binding partners for a paRrp molecule, which comprises al) immobilizing at least one parp homolog as claimed in any of claims 1 to 4 on a support; bl) contacting the immobilized parp homolog with an analyte in which at least one binding partner is Suspected; and cl) determining, where appropriate after an incubation period, analyte constituents bound to the immobilized PARP homolog;

   24. An in vitro screening method for binding partners for a PARP molecule, which comprises az) immobilizing on a support an analyte which comprises at least one possible binding partner for a pParp molecule; b2) contacting the immobilized analyte with at least one PARP homolog as claimed in any of claims 1 to 4 for which a binding partner is sought; and c2) examining the immobilized analyte, where appropriate ‘ after an incubation period, for binding of the PARP homolog.

   25. A method for the qualitative or quantitative determination of nucleic acids encoding a PARP homolog as claimed in any of claims 1 to 4, which comprises a) incubating a biological sample with a defined amount of an exogenous nucleic acid as claimed in either of claims 6 and 7, hybridizing under stringent conditions, determining the hybridizing nucleic acids and, where appropriate, comparing with a standard; or b) incubating a biological sample with a pair of oligonucleotide Primers with specificity for a pagrp homolog-encoding nucleic acid, amplifying the nucleic acid, determining the amplification product and, where appropriate, comparing with a standard. 40

   26. A method for the qualitative or quantitative determination of a PARP homolog as claimed in any of claims 1 to 4, which comprises a) incubating a biological sample with a binding partner 45 specific for a paRrp homolog, b) detecting the binding partner/PARP complex and, where appropriate, J

   0050/4910 / / 0/49750 AMENDED SHEET oo 80 Cc) comparing the result with a standard. PY 27. A method as claimed in claim 26, wherein the binding partner is an antibody or a binding fragment thereof, which carries a detectable label where appropriate.

   28. A method as claimed in any of claims 25 to 27 for diagnosing energy deficit-mediated illnesses.

   29. A method for determining the efficacy of PARP effectors, which comprises a) incubating a PARP homolog as claimed in any of claims 1 to 4 with an analyte which comprises an effector of a physiological or pathological PARP activity; removing the effector again where appropriate; and b) determining the activity of the PARP homolog, where appropriate after adding substrates or cosubstrates.

   30. A gene therapy composition, which comprises in a vehicle acceptable for gene therapy a nucleic acid construct which a) comprises an antisense nucleic acid against a coding nucleic acid as claimed in either of claims 6 and 7; or b) a ribozyme against a nucleic acid as claimed in either of claims 6 and 7; or c) codes for a specific PARP inhibitor.

   31. A pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one PARP protein as claimed in any of claims 1 to 4, at least one PARP binding partner as claimed in claim 5 or at least one coding nucleotide sequence as claimed in claim 6 or 7.

   32. The use of low molecular weight PARP binding partners as claimed in claim 5 for producing a pharmaceutical composition for the diagnosis or therapy of pathological states in the development. and/or progress of which at least one PARP protein, or a polypeptide derived therefrom, is involved.

   33. The use of low molecular weight PARP binding partners as 40 claimed in claim 5 for producing a pharmaceutical composition for the diagnosis or therapy of pathological states mediated by an energy deficit. 45 Tn

   / AMENDED SHEET

   34. A PARP homolog as claimed in any one of claims 1 to 4, substantially as herein before described or exemplified.

   35. A PARP homolog including any new inventive integer or combination of integers, substantially as herein described.

   36. A binding partner as claimed in claim 5, substantially as herein before described or exemplified.

   37. A binding partner including any new inventive integer or combination of integers, substantially as herein described.

   38. A nucleic acid as claimed in any one of claims 6 and 7, substantially as herein before described or exemplified.

   39. A nucleic acid including any new inventive integer or combination of integers, substantially as herein described.

   40. An expression cassette as claimed in claim 8, substantially as herein before described or exemplified.

   41. An expression cassette including any new inventive integer or combination of integers, substantially as herein described.

   42. A recombinant vector as claimed in claim 9, substantially as herein before described or exemplified. ]

   / AMENDED SHEET

   43. A recombinant vector including any new inventive integer or combination of integers, substantially as herein described.

   44. A recombinant microorganism as claimed in claim 10, substantially as herein before described or exemplified.

   45. A recombinant microorganism including any new inventive integer or combination of integers, substantially as herein described.

   46. A method according to the invention for in vitro detecting PARP inhibitors, substantially as herein before described or exemplified.

   47. A method for in vitro detecting PARP inhibitors including any new inventive integer or combination of integers, substantially as herein described.

   48. A screening method according to the invention for binding partners for a PARP molecule, substantially as herein before described or exemplified. 49, A screening method for binding partners for a PARP molecule including any new inventive integer or combination of integers, substantially as herein described. ]

   4 AMENDED SHEET

   50. A method according to the invention for qualitative or quantitative determination of nucleic acids encoding a PARP homolog, substantially as herein before described or exemplified.

   51. A method for qualitative or quantitative determination of nucleic acids encoding a PARP homolog including any new inventive integer or combination of integers, substantially as herein described.

   52. A method according to the invention for determining the efficacy of PARP effectors, substantially as herein before described or exemplified.

   53. A method for determining the efficacy of PARP effectors including any new inventive integer or combination of integers, substantially as herein described.

   54. A composition as claimed in any one of claims 30 to 31, substantially as herein before described or exemplified.

   55. A composition according to the invention including any new inventive integer or combination of integers, substantially as herein described.

   56. Use of low molecular weight PARP binding partners as claimed in any one of claims 32 and 33, substantially as herein before described or exemplified. ‘

   4 AMENDED SHEET , ® }

   57. Use of low molecular weight PARP binding partners including any new inventive integer or combination of integers, substantially as herein described.

   58. A pharmaceutical composition as claimed in claim 31 whenever supplied with instructions for the use thereof in the treatment of pathological states relating to PARP protein or polypeptide derived therefrom or pathological states mediated by an energy defict.

   59. A composition as claimed in claim 58 when the instructions are in printed or written form.

   60. A composition as claimed in claim 59 supplied in a package or container having the said instructions provided therein or thereon.