WO1993020219A1

WO1993020219A1 - Control and/or prevention of binding of nf-kb/rel/dorsal (nrd) family proteins to dna

Info

Publication number: WO1993020219A1
Application number: PCT/US1993/003027
Authority: WO
Inventors: Warren J. Leonard; Michel B. Toledano
Original assignee: The Government Of The United States As Represented By The Secretary, Department Of Health And Human Services
Priority date: 1992-04-06
Filing date: 1993-04-01
Publication date: 1993-10-14
Also published as: AU4045193A

Abstract

A method for inhibiting, preventing or controlling binding of NRD family DNA-binding proteins to DNA. NRD family proteins which have an N-terminal region of homology and a DNA-binding region within the region of homology are modified by a method which comprises the mutation of the NRD protein in the DNA-binding region and includes the steps of replacing one or more amino acids in the DNA-binding region by an amino acid which is different from the normally-occuring replaced amino acid and not chemically analogous thereto. The improved NRD proteins so-obtained are such that gene activation can be controlled or inhibited.

Description

CONTROL AND/OR PREVENTION OF BINDING OF NF-XB/Rel/Dorsal (NRD) FAMILY PROTEINS TO DNA The present invention relates to a method of blocking proteins that influence gene expression from binding to DNA. Specifically, the present invention relates to the modification of NF-XB/Rel/Dorsal family proteins such as NF-KB p49 (p50B) (SEQ ID NO: 5) , p50 (SEQ ID NO: 4), p65 (SEQ ID NO: 1), c-Rel (SEQ ID NO: 6), v-Re Rel B (SEQ ID NO: 2) or Dorsal proteins (SEQ ID NO: 3) such that binding of the modified protein to DNA is controlled and/or prevented.

Background of the Invention NF-XB is an inducible transcription factor critical to the activation of many genes in different cel types (Lenardo and Baltimore, Cell 58, 227-229, 1989; Baeuerle, Bioch. Biophys. Acta, 1072, 63-80, 1991). In addition to its originally identified role in transcription of the immunoglobulin X light chain gene, NF-XB is required for expression of a number of virally encoded genes and cellular genes encoding cytokines, immunoregulatory cell surface proteins, and acute phase response proteins. NF-XB is formed by the non-covalent association of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) . Both p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: l)are homologous to the c-Rel (SEQ ID NO: 6) oncoprotein and the Drosophila dorsal gene product in a region spanning approximately 300 amino acids (Kieran et al.. Cell 62, 1007-1018, 1990; Ghosh et al.. Cell 62, 1019-1029, 1990; Nolan et al.. Cell 64, 961-969, 1991; Ruben et al.. Science 251, 1490-1493, 1991). Together, they form the Rel family of proteins (also called NRD proteins for the

? NF-XB/Rel/Dorsal family proteins) . More recently recognized members of this family include p49 (SEQ ID NO: 5) (p50B) (SEQ ID NO: 5) which is similar to p50 (SEQ ID NO: 4) in that both are derived from larger precursor proteins and can interact functionally with p65 (SEQ ID NO: 1) (Schmid et al. , Nature 352, 733-736, 1991) and Rel B (SEQ ID NO: 2) which has a region of high similarity to c-Rel (SEQ ID NO: 6) (Ryseck et al, Mol. and Cell. Biol, 12, 674-695, 1992.

In unstimulated cells, NF-XB is present in the cytoplasm in a latent form consisting of p50 (SEQ ID NO: 4) (or p49 (SEQ ID NO: 5)) and p65 (SEQ ID NO: 1), bound via p65 (SEQ ID NO: 1) to an inhibitor molecule, IXB (Baeuerle and Baltimore, Cell 53, 211-217, 1988; Science 242, 540-546, 1988, and Genes Dev. 3, 1689-1698, 1989). Cellular activation results in dissociation of NF-XB from IXB and its translocation to the nucleus as an active DNA binding complex. In vitro, p50 (SEQ ID NO: 4) , p65 (SEQ ID NO: 1) and c-Rel (SEQ ID NO: 6) can bind DNA as homodimers or as p50/p65 or p50/c-Rel heterodimers (Urban et al._r New Biol. 3, 279-288, 1991? Ballard et al., Cell 63, 803-814, 1990).

The amino acid sequences of NF-XB p49 (SEQ ID NO: 5) , p50 (SEQ ID NO: 4) , p65 (SEQ ID NO: 1) , Dorsal protein (SEQ ID NO: 3) and c-Rel (SEQ ID NO: 6) protein are known and are disclosed in the following citations respectively: Schmid et al. Nature 352, 733-736, 1991; Kieran et al, Cell 62, 1007-1018, 1990; Ruben et al, Science 251, 1490-1493, 1991; Steward, Science 238, 692- 694, 1987; and Brownell, Oncogne 4, 934-942, 1989. These references are incorporated in toto herein by reference.

It is an object of the present invention to characterize an N-terminal region of the Rel homology domain and show that this region is required for DNA binding and for the interaction of p65 (SEQ ID NO: 1) with IXB and to so provide a means to control and/or prevent the binding of these proteins to DNA.

Summary of the Invention NF-XB is a pleiotropic transcriptional activator. It has previously been reported that either oxidation or alkylation of NF-XB in vitro abrogates DNA binding (Toledano et al., Proc. Natl. Acad. Sci. USA 88, 4328-4332, 1991). Cys 62 of NF-XB p50 (SEQ ID NO: 4) mediates this effect and lies within a 40 amino acid region required for DNA binding but not for di erization Point mutations in this region confer a transdominant negative binding phenotype to p50 (SEQ ID NO: 4) . The region is highly conserved throughout the Rel family, an it has been determined that it is also critical for DNA binding of NF-XB p65 (SEQ ID NO: 1) . Replacement of the N-terminal region of p65 (SEQ ID NO: 1) with the corresponding region from p50 (SEQ ID NO: 4) changes the DNA binding specificity towards that of p50 (SEQ ID NO: 4) . Replacement of the N-terminal region of p65 (SEQ ID NO: 1) also decreases its sensitivity to the inhibitory molecule IXB.

These data indicate that the N-terminal region play important roles in determining the DNA binding specificity of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) It has been found that there is a sequence of amino acid located in the N-terminal region of NF-XB p50 (SEQ ID NO 4) which is highly conserved in all Rel family proteins. The control and/or prevention of binding of these protei to DNA can be obtained by substitution of at least one amino acid normally present in this "binding" region wit another amino acid.

Brief Description of the Figures

Fig. 1A shows the seven cysteine residues in p (SEQ ID NO: 4) .

Fig. IB shows EMSAs performed with wild type p (SEQ ID NO: 4) .

Fig. 1C shows an EMSA performed with a C(Cys)62S(Ser) translation product.

Fig. 2A shows the homology regions of NF-KB p5 (SEQ ID NO: 4), Dorsal (SEQ ID NO: 3), c-Rel (SEQ ID NO: 6) and NF-KB p65 (SEQ ID NO: 1).

Fig. 2B shows the binding activity of N-termi deletion constructs.

Fig. 2C shows the binding activities of mutant p50 (SEQ ID NO: 4) constructs.

Fig. 2D shows the binding activities of p65 (SEQ ID NO: 1) mutants.

Fig. 3A shows an EMSA performed with the Ig KB probe.

Fig. 3B shows EMSAs performed with various probes.

Fig. 4A shows coprecipitations of p50 (SEQ ID NO: 4) and c-Rel (SEQ ID NO: 6) constructs proving that p50 (SEQ ID NO: 4) mutants which cannot bind DNA are still capable of dimerization.

Figs. 4B and 4C are EMSAs relating to transdominant negative mutants of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) .

Fig. 5 shows EMSA relating to the sensitivity of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) constructs to dia ide and 1KB.

DETAILED DESCRIPTION OF THE FIGURES Fig. 1. (A) is a schematic depicting the seven cysteine residues in p50 (SEQ ID NO: 4) , three of which are conserved throughout the Rel family (shown in bold) .

In Fig. 1 (B) EMSAs performed with wild type p50 (SEQ ID NO: 4) (lanes 1-3), or p50 (SEQ ID NO: 4) mutants in which cysteines were individually replaced by serines: C(Cys)62S(Ser) (lanes 4-6), C(Cys)88S(Ser) (lanes 7-9), C(Cys)119S(Ser) (lanes 10-12), C(Cys)124S(Ser) (lanes 13- 15), C(Cys)171S(Ser) (lanes 16-18), C(Cys)262S(Ser) (lanes 19-21), C(Cys)273S(Ser) (lanes 22-24). DNA binding of samples not treated (lanes 1, 4, 7, 10, 13, 16, 19, 22), or treated with 25 M diamide (lanes 2, 5, 8, 11, 14, 17, 20, 23), or lO M NEM (N-ethylmaleimide) (lanes 3, 6, 9, 12, 15, 18, 21, 24). Because not all gels were run equivalent lengths, only the shifted bands are shown. All EMSAs were in keeping with those published in Toledano and Leonard (cited above) (1991) .

Fig. 1 (C) shows an EMSA performed with the C(Cys)62S(Ser) translation product and the Ig XB probe in the absence (lane 1) or presence (lane 2) of 25 mM diamide. Competition with excess unlabeled mutated (10, lane 3) or wild type (21, lane 4) Ig KB oligonucleotides which were added prior to the probe. Lane 5: binding wit the products from a wheat germ extract to which no mRNA was added, indicating that the wheat germ extracts contai low levels of endogneous XB binding activity.

Fig. 2. (A) depicts the homology between amino acids 43 to 88 of p50 (SEQ ID NO: 4) and corresponding parts of Dorsal (SEQ ID NO: 3), c-Rel (SEQ ID NO: 6) and p65 (SEQ ID NO: 1) . This region corresponds to the N- terminal part of the Rel homology region (3, 4, 6). Cys 62 is underlined; the amino acids in bold depict residues critical for DNA binding and those in italics are those whose substitution does not prevent binding (see panel C) .

Fig. 2. (B) shows the binding activities of N- terminal deletion constructs. Deletions were performed b oligonucleotide directed mutagenesis of p50 (SEQ ID NO: 4). Binding of control p50 (SEQ ID NO: 4) was "+". "-" refers to no detectable binding activity.

Fig. 2. (C) shows the binding activities of mutant p50 (SEQ ID NO: 4) constructs; and

Fig. 2. (D) shows the binding activities of p65 (SEQ ID NO: 1) mutants Y(Tyr)36A(Ala) and E(Ser)39I(Ile) .

Fig. 3. (A) is an EMSA performed with the Ig KB probe and reticulocyte lysate translation product of 1 μg of p50 (SEQ ID NO: 4) mRNA (lanes 1-11) or 1 μg of p65 (SEQ ID NO: 1) mRNA (lanes 12-22) or 1 μg of p50*p65 chimera mRNA (lanes 23-33) . Competition with increasing amount of AA (lanes 2-6, 13-17, 24-28) or BB (lanes 7-11, 18-22, 29-33) double stranded oligonucleotides. The fold molar excess of competitor over the labelled probe is indicated above the corresponding lanes.

Fig. 3 (B) is an EMSA performed with 20,000 (lanes 1, 5, 9) or 40,000 cpm (lanes 2, 6, 10) of labelled Ig XB probe or 20,000 (lanes 3, 7, 11) or 40,000 cpm (lanes 4, 8, 12) of IL-2Rα XB probe and reticulocyte lysate translation product of p50 (SEQ ID NO: 4) mRNA (lanes 1-4) , or p65 (SEQ ID NO: 1) mRNA (5-8) OR P50*P65 chimera (lanes 9-12) . Similar amounts of in vitro translated recombinant proteins were used in A and B, as determined by performing parallel translations labeled with [³⁵S]-methionine followed by analysis on SDS-PAGE (not shown) .

Fig. 4. (A) shows p50 (SEQ ID NO: 4) mutant which cannot bind DNA but which can dimerize with c-Rel (SEQ ID NO: 6) . Immunoprecipitations with anti-Rel (lanes 1-7) or 9E10 (lanes 8-10) were performed on translation products of lysates to which mRNA had been added corresponding to c-Rel (SEQ ID NO: 6) (lanes 1,9), pδONTmyc (lanes 2, 8), p50 (SEQ ID NO: 4) (lane 4), E631 (lane 6) , or combinations of c-Rel (SEQ ID NO: 6) + p50NTmyc (lanes 3 and 10), c-Rel (SEQ ID NO: 6) + p50(SEQ ID NO: 4) (lane 5) , c-Rel (SEQ ID NO: 6) + E631 (lane 7) . For co-translation reactions, we generally used 0.5 μg of the mRNA whose product was specific to the antibody used for the particular immunoprecipitation and 1 μg of the other mRNA species.

In Fig. 4. (B) is shown an EMSA performed with Ig XB probe and 3 μl of reticulocyte lysate translation product of 1 μg (lane 1), 2 μg (lane 2) or 2.5 μg (lane 3) of p50 (SEQ ID NO: 4) mRNA, or cotranslation product 1 μg of p50 (SEQ ID NO: 4) and 1 μg (lane 4) or 1.5 μg (lane 5) of E631 or 1 μg (lane 6) or 1.5 μg (lane 7) of Y(Tyr)60A(Ala) mRNA, or 1.5 μg of c-Jun mRNA (lane 8). Lane 9 represents the endogenous binding found when EMSAs were performed with 3 μl of reticulocyte lysate extract t which no mRNA was added (see arrow) .

In Fig. 4. (C) it is shown that the same region in p65 (SEQ ID NO: 1) is required for DNA binding but not for dimerization. An EMSA performed with the Ig KB probe and 3 μl of reticulocyte translation product of 1 μg of p50 (SEQ ID NO: 4) mRNA (lane 1), p65 (SEQ ID NO: 1) mRNA (lane 2), mutant p65 (SEQ ID NO: 1) E391 (lane 3), or cotranslation of 1 μg p50 (SEQ ID NO: 4) and 1 μg p65 (SE ID NO: 1) (lane 4) or 1 μg E391 (lane 5) . Specific and endogenous complexes are indicated.

Fig. 5. depicts the sensitivity of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) constructs to diamide and 1KB. EMSA performed with the Ig XB probe and reticulocyt lysate translation product of p65 (SEQ ID NO: 1) (lane 1- 3), p50*p65 chimera (lanes 4-6), C62S*p65 chimera (lanes 7-9), p65 (SEQ ID NO: 1) C38S mutant (lanes 10-12), p50 (SEQ ID NO: 4) (lanes 13, 14), and p65*p50 chimera (lanes 15, 16). Lysates were either untreated or treated with diamide 5 mM prior to the addition of probe, as indicated Note that the endogenous Ig KB binding activity present i the reticulocyte lysates (arrow) is also sensitive to the oxidation by diamide. The endogenous band is the lower band in lanes 1-12 and the upper band in lanes 13-16. Th effect of 1KB on binding was evaluated by adding 2 μl of reticulocyte lysate translation product of MAD-3 mRNA int binding reactions prior to the probe (lanes 2, 5, 8, 11, 14, 16). In those lanes, the Ig KB endogenous binding (arrow) generally decreases, rather than increasing proportionally to the amount of lysate. This is presumably a consequence of a specific inhibition by 1KB.

DETAILED DESCRIPTION OF THE INVENTION A limited region of the N-terminal part of the Rel protein homology domain of NF-KB p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: l) has been characterized as a DNA binding domain which determines the differential DNA binding specificities of these proteins. The corresponding homology regions of other Rel family proteins may also serve as the DNA binding domains of these proteins. Interestingly, the same region in p65 (SEQ ID NO: 1) appears to be involved in the interaction with 1KB suggesting that DNA binding and 1KB interaction are mutually exclusive functions of the same region of p65 (SEQ ID NO: 1) . This observation provides a new molecular mechanism for the control of transcription factor function through inhibition of DNA binding.

It has been previously demonstrated that NF-XB binding in vitro can be inactivated by alkylation and oxidation (Toledano and Leonard, PNAS, USA, 88, 4328-4333, 1991) and therefore, it has been hypothesized that a free sulfhydryl might be required for NF-KB binding and that modification of this residue by alkylation or oxidation abrogated binding. Such a sulfhydryl might be located in a domain important for dimerization and/or DNA binding. p50 (SEQ ID NO: 4) contains seven cysteines at amino acids 62, 88, 119, 124, 171, 262 and 273 (Fig. 1A) . The individual mutation of each cysteine to a serine was carried out. The substitution of cysteine 62 by serines denoted as C62S and similar nomenclature for other p50 mutants is used in this application for convenience. p50 (SEQ ID NO: 4) mutants were translated in vitro and assayed for DNA binding activity using electrophoretic mobility shift assays (EMSAs) . All mutants could bind the immunoglobulin (Ig) KB site (Fig. IB, lanes 1, 4, 7, 10, 13, 16, 19 and 22). However, one mutant, C62S, was at least partially resistant to treatment with diamide or Nrethylmaleimide (NEM) (Fig. IB, lanes 5 and 6; Fig. 1C, lane 2 vs. 1) , treatments which abrogated binding of wild type p50 (SEQ ID NO: 4) (Fig. IB, lanes 2, 3) and the other p50 mutants (lanes 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24).

The specificity of C(Cys)62S(Ser) binding was demonstrated by the ability of an unlabeled wild type (Fig. 1C, lane 4 vs. 1) but not a mutated KB (lane 3) oligonucleotide to inhibit binding.

Simultaneous substitution of Cys 62 plus any other cysteine in p50 (SEQ ID NO:- 4) with serines resulte in proteins with binding properties indistinguishable fro C62S (not shown) . Thus, Cys 62 as the only cysteine whic mediates p50 sensitivity to diamide and NEM. Further, replacement of Cys 62 by Ala (C62A) or Met (C62M) decreased binding activity (not shown) , suggesting that his residue lies within a DNA binding domain.

Cys 62 represents one of three cysteines conserved throughout the Rel family of proteins (shown in bold. Fig. 1A) , and is contained within an N-terminal region of extensive homology (Fig. 2A) . The high hydrophilicity of this region is consistent with the possibility that it is exposed on the protein surface and could interact with DNA. p50 mutants lacking the first 19, 32, 42, 47, o 54 amino acids (denoted Δ19NT, Δ32NT, etc. for N-Terminal deletions) were prepared and evaluated as to their bindin activities. Δ19NT, Δ32NT and Δ42NT had full binding activity, but Δ47NT and Δ54NT did not bind (Fig. 2B) . It is noteworthy that amino acid 43, which defines the N- terminus of the most truncated deletion mutant with binding activity, corresponds to the beginning of the Rel homology region.

Delineation of the putative DNA binding domain by C-terminal deletions was not possible due to the presence of a region, extending approximately from residu 200 to 399 which is required for dimerization and whose removal abrogates binding presumably as a result of the loss of ability to dimerize. The DNA binding domain was further analyzed by mutating conserved and/or charged residues in the vicinity of Cys 62. Mutants Y(Tyr)44A(Ala) , E(Glu)49A(Ala) , Q(Gln)50A(Ala) , R(Arg)54A(Ala) , S(Ser)66A(Ala) , G(Gly)72(Ala)A, Y(Tyr)82(Ala)A, and C(Cys)88(Ser)S retained full binding activity (Fig. 2C) . Mutants R(Arg)57(Ala)A, F(Phe)58(Alu) R(Arg)59(Ile)I, Y(Tyr)60(Ala)A, E(Glu)63(He)I, H(his)67(Ala)A, H(his)67(Cys)C, G(Gly)68(Ala)A, and L(leu)70(Ala)A did not bind DNA, even though similar amounts of wild type and mutant p50 proteins were synthesized in vitro as evaluated by analysis on SDS gels (not shown). G(Gly)64(Ala) had decreased but detectable binding activity. Binding was increased when Lys 52 was replaced by Ala (K(Lys)52(Ala)A or Val 61 was replaced by Glu (V(Val)61(Glu)E) , two residues present in the corresponding positions in Dorsal (SEQ ID NO: 3) . The fact that different mutations either decrease or increase binding is consistent with this region representing a DNA- binding domain. This putative DNA binding domain extends approximately from amino acids 54 to 72.

The role of the N-terminal region of p65 (SEQ ID NO: 1) in binding was also evaluated using mutants similar to those studied in p50. Analogous to the C(Cys)62(Ser)S retained full binding activity even following oxidation with diamide. Further Y(Tyr36(Ala)A and E(Glu)39(He)I (corresponding to Y(Tyr)60(Ala) and E(Glu)63(lie)I) had no detectable DNA binding activity (Fig. 2D and see below) . These results indicate that the N-terminal portion of the Rel homology region in p65 (SEQ ID NO: 1) is also essential for DNA binding.

Most NF-KB binding motifs consist of decameric sites of low symmetry. The canonical Ig KB site GGGACTTTCC (SEQ ID NO: 9) can be divided into two pentameric half sites denoted A (SEQ ID NO: 7) and B (SEQ ID NO: 8) half sites; Zabel et al.. J. Biol. Chem. 266, 252-260, 1991). Purified p50 (SEQ ID NO: 4) binds better to palindromes of the "A" half site (5'-GGGAC 01000-3') (SEQ ID NO: 7) whereas purified p65 (SEQ ID NO: 1) binds preferentially to palindromes of the "B" half site (5'- GGAAA TTTCC-3') (SEQ ID NO: 8) (Urban et al. Genes Dev. 4, 1975-1984, New Biol. 3,279-288, 1991 EMB0 J. 10, 1817-1825 1991) .

As a first step towards elucidating the contribution of the N-terminal regions to binding specificity, the AA and BB oligonucleotides were used to confirm binding differences between p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: l) translated in vitro. In these experiments, proteins were translated using rabbit reticulocyte lysates which contain much higher endogenous NF-KB binding activity than wheat germ extracts (Kieran e al.. Cell 62, 1007-1018 1990; see Figs. 1, 3, 4). The specific bands corresponding to the in vitro expressed recombinant proteins were identified by comparison with EMSAs performed with unprogrammed reticulocyte lysates. Whereas a 25 fold molar excess of the AA oligonucleotide competed p50 (SEQ ID NO: 4) binding to the Ig KB probe, the BB oligonucleotide was a much less effective competitor (Fig. 3A, lanes 2-6 vs. 7-11) . In contrast, competition of p65 (SEQ ID NO: 1) binding to the Ig KB probe with the AA and BB oligonucleotides yielded a pattern reciprocal to that seen with p50 (SEQ ID NO: 4) (lanes 13-17 vs. 18-22).

The binding specificities of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) were compared by using the Ig and IL-2Rα KB probe (lanes 3, 4, 7, 8). Thus, p50 (SEQ ID NO 4) and p65 (SEQ ID NO: 1) also differed in their binding specificities to these distinct sites.

The N-terminal regions in p50 and p65 were then investigated to determine if they were important for thei different DNA binding specificities. The N-terminal 58 amino acids of p65 were replaced with the N-terminal 82 amino acids of p50. This p50 boundary point was selected based on data indicating that the putative DNA binding domain did not extend beyond amino acid 82 (and probably not beyond amino acid 72) (Fig. 2) . p50 has a longer N- terminal extension p65 (42 versus 18 amino acids upstream of the Rel homology domain) ; hence the unequal replacemen of 58 amino acids with 82.

The resulting chimera (denoted p50*p65) had binding properties similar to that of p50 in that binding to the Ig KB probe could not be competed by the BB oligonucleotide (Fig. 3A, lanes 29-33) , but was at least partially inhibited by the AA oligonucleotide (lanes 24- 28) . Further, p50*p65 bound much better than p65 (SEQ I NO: 1) to the Il-2Rα KB probe (Fig. 3B, lanes 11 and 12 vs. 7 and 8) .

Conversely, when the N-terminal 82 amino acids of p50 were replaced by the N-terminal 58 amino acids of p65 (p65*p50 chimera) , the binding specificity changed towards that of p65 (SEQ ID NO: 1) (not shown) . These data established that the N-terminal regions characterized are required for DNA binding specificity to p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) .

Since dimerization is a prerequisite for NF-KB binding, investigation was made into whether the domain characterized was also essential for dimerization. The ability of p50 (SEQ ID NO: 4) constructs to form heterodimers with c-Rel (SEQ ID NO: 6) was evaluated. Wild type p50 (SEQ ID NO: 4) (see Fig. 1 legend) or E(Glu)63(He)I were translated with or without c-Rel (SEQ ID NO: 6) mRNA and the translation products were immunoprecipitated with a specific anti-Rel polyclonal rabbit antiserum, which does not recognize p50 (SEQ ID NO: 4) (Brownell et al. , Oncogene 4, 934-942, 1989). Precipitation of the c-Rel (SEQ ID NO: 6) primary translation product by anti-Rel is shown in Fig. 4A, lane 1. p50 (SEQ ID NO: 4) and E(Glu)63(He)I were precipitated by anti-Rel (see band c) if co-translated with c-Rel (SEQ ID NO: 6) but not in its absence (Fig. 4A, lanes 5 vs. 7 vs. 6) . Similar results were obtained with R(Arg)57(Ala)A, F(Phe)58(Ala)A, R(Arg)59(Ala)A, and Y(Tyr)60(Ala)A (data not shown).

To confirm the validity of the dimerization experiments, a modified version of p50 (SEQ ID NO: 4) , denoted pSONTmyc was used, which includes a 13 amino acid N-terminal extension derived from the human c-myc proto- oncogene product. This short peptide creates an epitope recognized by monoclonal antibody 9E10 (Pelham, EMBO J.7,913-918 1988). The resulting chimeric protein has a slightly slower migration on SDS gels. pSONTmyc was co- precipitated by anti-Rel when cotranslated with c-Rel (SEQ ID NO: 6) but not when translated alone (lane 8, band b) and c-Rel (SEQ ID NO: 6) was precipitated by 9E10 when cotranslated with pSONTmyc but not when translated alone (lane 10 vs. 9, band a).

It was next investigated if E(Glu)631(He) and Y(Tyr)60A(Ala) could behave as transdominant negative mutants (i.e. , whether these mutant constructs could inhibit wild type p50 (SEQ ID NO: 4) binding) . Co- translation of E(Glu)631(He) or Y(Tyr)60A(Ala) with p50 (SEQ ID NO: 4) mRNA resulted in dose-dependent inhibition of p50 (SEQ ID NO: 4) binding (Fig. 4B, in-dose dependent inhibition of p50 (SEQ ID NO: 4) binding (Fig. 4B, lanes 4-7 vs. lanes 2 and 3 in which additional wild type p50 (SEQ ID NO: 4) was co-translated instead of mutuant p50 (SEQ ID NO: 4)) .

As in the coprecipitation experiments, the transdominant negative effect was only observed when mutant and wild type mRNA were co-translated. Thus, there was no effect on the level of preformed binding activity endogenous to the reticulocyte lysate. Co-translation of p50 (SEQ ID NO: 4) with c-Jun mRNA did not decrease binding of p50 (SEQ ID NO:^4) to the Ig KB probe, indicating that the inhibition observed with E(Glu)631 (He) and Y(Tyr)60A(Ala) was specific. The ability of E(Glu)631 (He) and Y(Tyr)60A(Ala) to behave as transdominant negative mutants further confirm their ability to dimerize, demonstrating that the N-terminal region characterized was only a DNA binding domain. Analogous results were found with p65 mutant E(Glu)391 (He) (Fig. 4C) . This mutant cannot bind DNA (lane 3 vs. 2) and has a transdominant negative effect on p50 (SEQ ID NO: 4) binding (lane 5 vs. 4) .

In view of the importance of N-terminal region in DNA binding and because 1KB inhibits p65 (SEQ ID NO: 1) binding, it was examined whether the DNA binding domain o p65 (SEQ ID NO: 1) was involved in this inhibition. The MAD-3 cDNA was used which encodes a 40 kD protein with IKB-like activity (Haskill et al., Cell 65, 1281-1289 1991) . MAD-3 protein ability to inhibit the binding of various p50 and p65 constructs. As expected, MAD-3 completely inhibited p65 (SEQ ID NO: 1) binding (Fig. 5, lanes 2 vs. 1) .

Binding of the p50*p65 chimera was partially resistant to inhibition (lane 5 vs. 4) . Moreover, chi eric construct C62S*p65, identical to p50*p65 except that Cys 62 of p50 (SEQ ID NO: 4) was substituted to a Ser, was fully resistant to inhibition by MAD-3 (lane 8 vs. 7) . These data suggested the possible importance of the N-terminal part of p65 (SEQ ID NO: 1) for 1KB binding. They also suggested the possibility that a cysteine at position 38 of p65 (SEQ ID NO: 1) is required for full sensitivity to 1KB. The C(Cys)38S(Ser) mutant of p65 (SEQ ID NO: 1) was analyzed. Although a low level of resistance to 1KB was noted, potent inhibition was still observed (lane 11 vs. 10) , suggesting an important role for other residues in the interaction with 1KB. In contrast to the nearly complete resistance of p50 (SEQ ID NO: 4) to 1KB (lane 14 vs. 13) , the p65*p50 chimera was partially inhibited by 1KB (lane 16 vs. 15) . Thus, the N- terminal region of p65 is required for 1KB association, although it cannot be excluded that other region(s) of p65 also might contribute to this interaction.

An N-terminal portion of the Rel homology regio in p50 and p65 which is required for DNA binding but not for dimerization has not been identified. It is noteworthy that most of the residues which are important for p50 (SEQ ID NO: 4) binding (R(Arg)57, F(Phe)58, R(Arg)59, Y(Tyr)60, C(Cys)62, E(Glu)63, and G(Gly)68 are identical in p65 (SEQ ID NO: 1), c-Rel (SEQ ID NO: 6), an Dorsal (SEQ ID NO: 3) (Fig. 2A) , indicating that the same region may be involved in binding in all Rel family proteins. Since this region confers distinct specificities to p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) , it seems likely that amino acid differences in this domain contribute to the differential binding specificity of Rel family proteins.

DNA binding is influenced by the oxidation stat of Cys 62 in p50 (SEQ ID NO: 4) (or Cys 38 in p65 (SEQ ID NO: 1)), which is located within the DNA binding domain. * This situation is similar to that of c-Jun, in which a redox reactive cysteine was identified in the basic regio presumably used to contact DNA (Abate et al., Science 249 1157-1161 1990) . The data suggest that the naturally occurring cysteine at position 62 in p50 (SEQ ID NO: 4) (or position 38 in p65 (SEQ ID NO: 1)) must normally exis in a reduced form for binding. Either oxidation or alkylation of the free sulfhydryl may inhibit binding by steric hindrance and/or by the inability of the modified amino acid to make the proper contacts with the DNA. The nature of the oxidized form of Cys 62 is unknown but coul be a sulfonic acid as proposed for OxyR and c-Jun (Storz et al., Science 248, 189-194 1990; Abate et al, 1990). Cys 62 could be replaced by a serine, without loss of p50 (SEQ ID NO: 4) binding, but not by an alanine or methionine. This demonstrates that the serine hydroxyl, which is insensitive to oxidation, can replace the free sulfhydryl without apparent alteration of the DNA-protein interaction surface.

It has been demonstrated that the N-terminal region of p65 (SEQ ID NO: l) which is defined as a DNA binding domain also is required for interaction with 1KB interacts with p65 (SEQ ID NO: 1) . The data indicate tha IXB inhibits p65 (SEQ ID NO: 1) binding to DNA either by directly interacting with residues involved in DNA bindin or by sterically interfering with their access to DNA, a predict that DNA binding and IXB interaction are mutuall exclusive functions of this N-terminal region.

The data have established the biochemical basi for the differences in DNA binding specificities of p50 (SEQ ID NO: 4) and p65 (SEQ ID NO: 1) . The specificity p50 (SEQ ID NO: 4) for the more conserved ϊg XB "A" half site (SEQ ID NO: 7) and of p65 (SEQ ID NO: l) for the more degenerate "B" half site (SEQ ID NO: 8) (Zabel et al., J. Biol. Chem. 266, 252-260 1991; Urban et al.. Genes Dev. 4, 1975-1989 1990, New Biol. 3, 279-288 1991, EMBO J. 10, 1817-1825 1991; Fig. 3) suggests that different NF-XB sites can select among different heterodimers constituted by the association of p50 (SEQ ID NO: 4) with another member of the Rel family. Thus, the NF-XB system exemplifies the use of a combinatorial mechanism for the generation of increased diversity of DNA binding by the pair wise association of factors with distinct specificities. This may explain why different NF-XB sites are functionally distinct and why single sites can manifest different activities in distinct cell types. For example, the IL-2Rα expression in HTLV-1 transformed MT-2 T cells but not in Jurkat T cells, whereas the Ig XB site is active in both cell types. As shown in Fig. 3, the IL- 2Rα site can efficiently be bound by p50 (SEQ ID NO: 4) but not by p65 (SEQ ID NO: 1) . Assuming that the Ig XB site is transactivated by p50/p65 heterodimers in Jurkat cells, then p50/p65 heterodimers presumably cannot potently activate the IL-2Rα XB site. It is therefore interesting to postulate the existence of another partner of p50 (SEQ ID NO: 4) different than p65 (SEQ ID NO: 1) which is present in MT-2 cells but not jurkat cells. Such a protein could potentially explain the differential activity of the IL-2Rα KB site in these two different T cell lines.

Based upon the above findings, a method for the inhibition, prevention and/or control of the binding of the Rel family of DNA-binding proteins to DNA has been found. In this manner, activation of genes controlled by the Rel family proteins can be controlled. All Rel proteins have a significant area of homology in the N- terminal portion of these proteins. It is this homologous region in the N-terminal portion -of these proteins which provides DNA-binding sites. DNA-binding of the Rel family proteins can be controlled and/or prevented by mutation o one or more amino acids in the N-terminal homologous region of these proteins. Hence, for example, the p50 (SEQ ID NO: 4) N-terminal amino acid sequence in the area of amino acids 43 to 88 is homologous to the correspondin region of p65 (SEQ ID NO: 1) at amino acids 19 to 64. p5 mutant proteins in which up to the initial 42 N-terminal amino acids have been removed still bind to DNA and mutations in amino acid 72, 82 and 88 of p50 (SEQ ID NO: 4) do not affect binding. One can postulate from this information that mutations in the amino acid sequence in the N-terminal region of between amino acids 43 to 72 can prevent DNA binding. In fact, mutations in amino acids 54, 57, 58, 59, 60, 63, 64, 66, 67, 68 and 70 lead to los of binding of the protein to DNA. Mutations in the corresponding region of p65 (SEQ ID NO: 1) also leads to the loss of DNA-binding.

The present invention, hence, relates to a method of controlling or preventing the binding of a DNA- binding protein KB-DNA binding sites wherein said method comprises the mutation of a Rel family DNA-binding protei in the DNA-binding region by replacing one or more amino acids in the DNA-binding regions of the family of protein with an amino acid different from the normal protein amin acid. More specifically, the present invention relates t a method of controlling or preventing the binding of all Rel-family proteins, protein, to DNA said proteins having substantially homologous regions in the protein N-termina portions and a DNA-binding portion within the homologous region. This method is based on replacing one or more amino acids in the respective DNA-binding region of the protein with an amino acid different from the normally- occurring protein amino acid. Even more specifically, th method of the present invention relates to the prevention or controlling of DNA-binding of Rel family proteins including NF-KB p49 (SEQ ID NO: 5) (also called p50B) p50 (SEQ ID NO: 4) or p65 (SEQ ID NO: 1), Rel proteins or Dorsal protein (SEQ ID NO: 3) by replacing one or more of the R, F, Y, C, G, or E amino acid residues with another amino acid different from the replaced amino acid wherein said amino acids are located in the N-terminal, DNA- binding homologous regions of these proteins.

In the case of NF-KB p50 (SEQ ID NO: 4), the amino acid replacement within the N-terminal region between amino acids 43 and 72, preferably 50 to 71 more preferably 54 to 71. For NF-KB p65 (SEQ ID NO: 1), the amino acid replacement can be made in the N-terminal region between amino acids 19 and 48, more preferably between amino acids 26 to 47, even more preferably 30 to 47. As for Dorsal protein (SEQ ID NO: 3), replacement is in the N-terminal region between amino acids 48 and 77, more preferably between amino acids 55 and 76 even more preferably between 59 and 76. Replacement in c-Rel (SEQ ID NO: 6) can be made at amino acids 8 and 37. Experimental procedures Generation of mutant cDNAs p50 (SEQ ID NO: 4) constructs were generally derived from the 1680 bp Hind III to Xba I fragment (corresponding to amino acids 1 to 502) of the KBF1 cDNA (Kieran et al., Cell 62, 1017-1018 1990). However, in one case only, (Fig. 1, lane 1) , a p50 (SEQ ID NO: 4) construct corresponding to amino acids 1 to 399 (Hind III to Rsa I fragment) was used. The binding properties of p50₁.₃₉₉ and p50._|_₅₀₂ are indistinguishable (Kieran et al., 1990 and data not shown) . p65 (SEQ ID NO: 1) constructs were derived from a Bam HI fragment spanning the entire human cDNA coding sequence (Ruben et al., Science 251, 1490-1493, 1991). Single point mutation, N-terminal deletion, and the pSONTmyc constructs were prepared by site-directed mutagenesis as previously described in Toledano et al., PNAS USA 87, 1830-1834, 1990) using a Biorad kit. Following subcloning into pBluescript SK(-) (Stratagene) , mutant constructs were resequenced. Chimeras between p50 and p65 were prepared by a two step PCR method, as described by Higuchi et al., Nucl. Acids

Res. 16, 7351-7367, 1988.

In vitro transcription/translation

In vitro transcription was performed using constructs in which cDNAS were subcloned in pBluescript and the mRNA cap kit (Stratagene) . p50 (SEQ ID NO: 4) and chimeric constructs were linearized with Xba 1 and transcribed with T7 RNA polymerase. p65 (SEQ ID NO: 1) constructs were linearized with Hind III and transcribed with T3 RNA polymerase. The MAD-3 cDNA was transcribed from the pcDNAl plasmid (Haskill et al.. Cell 65, 1281- 1289, 1991). Translation reactions were performed in reticulocyte lysates except in Fig. 1, where wheat germ extracts were used. Reactions were performed according to the manufacturer's recommendations (Pro ega) . Although wheat germ extracts had lower endogenous NF-KB binding activity, the high variation in activity with different preparations resulted in our principally using reticulocyte lysates. DNA binding assays and EMSA

A 26 double stranded Ig KB or a 38 double stranded IL-2Rα-270/-237 oligonucleotides (Toledano et al. , 1990, cited above) were end-labeled with Klenow (BRL) using α-³²P-dCTP (3000 Ci/mmole, Amersham) . 20-40,000 cpm were used in binding reactions. The 26 bp long AA or BB oligonucleotides (Urban and Baeuerle, Genes Dev. 4, 1975- 1984, 1990) were used as unlabeled competitors where specified. Binding reactions were performed with 2 to 4 μl of translation product in 20 μl final volume of 10 mM tris-Cl pH 7.6, 100 mM NaCl, 1 mM EDTA, 5 mM DTT, 0.2% NP40, 10 μg/ml BSA and 1 μg poly(dl-dC) . Where indicated, diamide, NEM, or competitors were added 10 min prior to probe. In the binding inhibition assays, the MAD-3 primary translation product was added to the binding reaction prior to the sample containing the binding proteins. Following binding for 10 min at room temperature, samples were analyzed on native gels as previously described (Toledano et al., 1990, cited above) Immunoprecipitation

In vitro transcribed RNA were translated in reticulocyte lysates in the presence of Tran³⁵S-label (ICN) , and products were immunoprecipitated with anti-Rel antiserum (Brownell et al., Oncogene 4, 934-942, 1989) or anti-myc (9E10; Pelham EMBO/J.7, 913-918, 1988). Antibodies were bound to protein A-agarose beads in 50 mM Tris-Cl pH 7.4, 140 mM NaCl, 0.5% NP40, 1 mM PMSF, 10 μg/ml leupeptin prior to the addition of translation products. Beads were washed in the same buffer supplemented by 0.1% SDS and 0.4% sodium deoxycholate. Immunoprecipitates were analyzed by 8.75% SDS-PAGE. Sequence Analyses

Chou-Fasman analysis was performed using the GC Sequence Analysis Software Package.

All references which have been mentioned in thi application are specifically incorporated in toto by reference into this application.

SEQUENCE LIST

(1) GENERAL INFORMATION:

(i) APPLICANTS: LEONARD, WARREN; TOLEDANO, MICHEL

(ii) TITLE OF INVENTION: CONTROL AND/OR

PREVENTION OF BINDING OF NF-KB/REL/DORSAL (NRD) FAMILY PROTEINS TO DNA

(ϋi) NUMBER OF SEQUENCES: 9

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: MORGAN & FINNEGAN

(B) STREET: 345 PARK AVENUE

(C) CITY: NEW YORK

(D) STATE: NEW YORK

(E) COUNTRY: USA

(F) ZIP: 10154

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: FLOPPY DISK

(B) COMPUTER: IBM PC COMPATIBLE

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: WORDPERFECT 5.1

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 01-APR-1993

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/862,987

(B) FILING DATE: 06-APR-1992

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: DOROTHY R. AUTH

(B) REGISTRATION NUMBER: P-36,434

(C) REFERENCE/DOCKET NUMBER: 2026-4010 P

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212-758-4800

(B) TELEFAX: 212-751-6849

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 550

(B) TYPE: Amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No

(iv) ORIGINAL SOURCE: (A) ORGANISM: human

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: P65

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: non-DNA-binding subunit of NF-KB.

(X) PUBLICATION INFORMATION:

(A) AUTHORS: Ruben,S.M.; Dillon,P.J. ; Schreck,R. ; Henkel,T.; Chen,C.-H.; Maher,M. ; Baeuerle,P.A. ; Rosen,C.A.

(B) TITLE: A novel rel-related human cDNA that potentially encodes the 65 kDa subunit of NF-KB

(C) JOURNAL: Science

(D) VOLUME: 251

(E) ISSUE:

(F) PAGES: 1490-1493

(G) DATE: 1991

(H) DOCUMENT NUMBER:

(I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES:

( i) ^' SEQUENCE DESCRIPTION: SEQ ID NO. 1:

Met Asp Glu Leu Phe Pro Leu He Phe Pro Ala Glu Pro Ala

1 5 10

Gin Ala Ser Gly Pro Tyr Val Glu He He Glu Gin Pro Lys

15 20 25

Gin Arg Gly Met Arg Phe Arg Tyr Lys Cys Glu Gly Arg Ser

30 35 40

Ala Gly Ser He Pro Gly Glu Arg Ser Thr Asp Thr Thr Lys

45 50 55

Thr His Pro Thr He Lys He Asn Gly Tyr Thr Gly Pro Gly

60 65 70

Thr Val Arg He Ser Leu Val Thr Lys Asp Pro Pro His Arg

75 80

Pro His Pro His Glu Leu Val Gly Lys Asp Cys Arg Asp Gly 85 90 95 Phe Tyr Glu Ala Glu Leu Cys Pro Asp Arg Cys He His Ser

100 105 110

Phe Gin Asn Leu Gly He Gin Cys Val Lys Lys Arg Asp Leu

115 120 125

Glu Gin Ala He Ser Gin Arg He Gin Thr Asn Asn Asn Pro 130 135 140

Phe Gin Val Pro He Glu Glu Gin Arg Gly Asp Tyr Asp Leu

145 150

Asn Ala Val Arg Leu Cys Phe Gin Val Thr Val Arg Asp Pro 155 160 165

Ser Gly Arg Pro Leu Arg Leu Pro Pro Val Leu Pro His Pro

170 175 180

He Phe Asp Asn Arg Ala Pro Asn Thr Ala Glu Leu Lys He

185 190 195

Cys Arg Val Asn Arg Asn Ser Gly Ser Cys Leu Gly Gly Asp 200 205 210

Glu He Phe Leu Leu Cys Asp Lys Val Gin Lys Glu Asp He

215 220

Glu Val Tyr Phe Thr Gly Pro Gly Trp Glu Ala Arg Gly Ser 225 230 235

Phe Ser Gin Ala Asp Val His Arg Gin Val Ala He Val Phe

240 245 250

Arg Thr Pro Pro Tyr Ala Asp Pro Ser Leu Gin Ala Pro Val

255 260 265

Arg Val Ser Met Gin Leu Arg Arg Pro Ser Asp Arg Glu Leu 270 275 280

Ser Glu Pro Met Glu Phe Gin Tyr Leu Pro Asp Thr Asp Asp

285 290

Arg His Arg He Glu Glu Lys Arg Lys Arg Thr Tyr Glu Thr 295 300 305

Phe Lys Ser He Met Lys Lys Ser Pro Phe Ser Gly Pro Thr

310 315 320

Asp Pro Arg Pro Pro Pro Arg Arg He Ala Val Pro Ser Arg

325 330 335

Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gin Pro Tyr Pro 340 345 350

Phe Thr Ser Ser Leu Ser Thr He Asn Tyr Asp Glu Phe Pro

355 360

Thr Met Val Phe Pro Ser Gly-Arg Ser Ala Arg Pro Arg Leu 365 370 375

Gly Pro Ala Pro Pro Gin Val Leu Pro Gin Ala Pro Ala Pro

380 385 390

Ala Pro Ala Pro Ala Met Val Ser Ala Leu Ala Gin Ala Pro

395 400 405

Ala Pro Val Pro Val Leu Ala Pro Gly Pro Pro Gin Ala Val 410 415 420

Ala Pro Pro Ala Pro Lys Pro Thr Gin Ala Gly Glu Gly Thr

425 430

Leu Ser Glu Ala Leu Leu Gin Leu Gin Phe Asp Asp Glu Asp 435 440 445 Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala Val Phe

450 455 460

Thr Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gin Gin Leu

465 470 475

Leu Asn Gin Gly He Pro Val Ala Pro His Thr Thr Glu Pro 480 485 490

Met Leu Met Glu Tyr Pro Glu Ala He Thr Arg Leu Val Thr

495 500

Gly Ala Gin Arg Pro Pro Asp Pro Ala Pro Ala Pro Leu Gly 505 510 515

Ala Pro Gly Leu Pro Asn Gly Leu Leu Ser Gly Asp Glu Asp

520 525 530

Phe Ser Ser He Ala Asp Met Asp Phe Ser Ala Leu Leu Ser

535 540 545

Gin He Ser Ser 550

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 558

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

of h gh s milarity w th proteins of Rel family. (D) OTHER INFORMATION:

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Ryseck, R.-P.; Bull, P.;

Takamiya, M. ; Bours, V.; Siebenlist, U. ; Dobrzanski, P.; Bravo, R. (B) TITLE: RelB, a new Rel family transcription activator that can interact with p50-NF-K-B.

(C) JOURNAL: Mol. Cell. Biol.

(D) VOLUME: 12

(E) ISSUE:

(F) PAGES: 674-684

(G) DATE: FEB-1992 (H) DOCUMENT NUMBER: (I) FILING DATE:

(J) PUBLICATION DATE: (K) RELEVANT RESIDUES:

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

Met Pro Ser Arg Arg Ala Ala Arg Glu Ser Ala Pro Glu Leu

1 5 10

Gly Ala Leu Gly Ser Ser Asp Leu Ser Ser Leu Ser Leu Thr

15 20 25

Val Ser Arg Thr Thr Asp Glu Leu Glu He He Asp Glu Tyr

30 35 40

He Lys Glu Asn Gly Phe Gly Leu Val Gly Thr Gin Leu Ser

45 50 55

Glu Met Pro Arg Leu Val Pro Arg Gly Pro Ala Ser Leu Ser

60 65 ^" 70

Ser Val Thr Leu Gly Pro Ala Ala Pro Pro Pro Pro Ala Thr

75 80

Pro Ser Trp Ser Cys Thr Leu Gly Arg Leu Val Ser Pro Gly 85 90 95

Pro Cys Pro Arg Pro Tyr Leu Val He Thr Glu Gin Pro Lys

100 105 110

Gin Arg Gly Met Arg Phe Arg Tyr Glu Cys Glu Gly Arg Ser

115 120 125

Ala Gly Ser He Leu Gly Glu Ser Ser Thr Glu Ala Ser Lys 130 135 140

Thr Gin Pro Ala He Glu Leu Arg Asp Cys Gly Gly Leu Arg

145 150

Glu Val Glu Val Thr Ala Cys Leu Val Trp Lys Asp Trp Pro 155 160 165

His Arg Val His Pro His Ser Leu Val Gly Lys Asp Cys Thr

170 175 180

Asp Gly Val Cys Arg Val Arg Leu Arg Pro His Val Ser Pro

185 190 195

Arg His Ser Phe Asn Asn Leu Gly He Gin Cys Val Arg Lys 200 205 210

Lys Glu He Glu Ala Ala He Glu Arg Lys He Gin Leu Gly

215 220

He Asp Pro Tyr Asn Ala Gly Ser Leu Lys Asn His Gin Glu 225 230 235

Val Asp Met Asn Val Val Arg He Cys Phe Gin Ala Ser Tyr

240 245 250

Arg Asp Gin Gin Gly His Leu His Arg Met Asp Pro He Leu 255 260 265 Ser Glu Pro Val Tyr Asp Lys Lys Ser Thr Asn Thr Ser Glu

270 275 280

Leu Arg He Cys Arg He Asn Lys Glu Ser Gly Pro Cys Thr

285 290

Gly Gly Glu Glu Leu Tyr Leu Leu Cys Asp Lys Val Gin Lys 295 300 305

Glu Asp He Ser Val Val Phe Ser Thr Ala Ser Trp Glu Gly

310 315 320

Arg Ala Asp Phe Ser Gin Ala Asp Val His Arg Gin He Ala

325 330 335

He Val Phe Lys Thr Pro Pro Tyr Glu Asp Leu Glu He Ser 340 345 350

Glu Pro Val Thr Val Asn Val Phe Leu Gin Arg Leu Thr Asp

355 360

Gly Val Cys Ser Glu Pro Leu Pro Phe Thr Tyr Leu Pro Arg 365 370 375

Asp His Asp Ser Tyr Gly Val Asp Lys Lys Arg Lys Arg Gly

380 385 390

Leu Pro Asp Val Leu Gly Glu Leu Ser Ser Ser Asp Pro His

395 400 405

Gly He Glu Ser Lys Arg Arg Lys Lys Lys Pro Val Phe Leu 410 415 420

•Asp His Phe Leu Pro Gly His Ser Ser Gly Leu Phe Leu Pro

425 430

Pro Ser Ala Leu Gin Pro Ala Asp Ser Asp Phe Phe Pro Ala 435 440 445

Ser He Ser Leu Pro Gly Leu Glu Pro Pro Gly Gly Pro Asp

450 455 460

Leu Leu Asp Asp Gly Phe Ala Tyr Asp Pro Ser Ala Pro Thr

465 470 475

Leu Phe Thr Met Leu Asp Leu Leu Pro Pro Ala Pro Pro Leu 480 485 490

Ala Ser Ala Val Val Gly Ser Gly Gly Ala Gly Ala Thr Val

495 500

Val Glu Ser Ser Gly Pro Glu Pro Leu Ser Leu Asp Ser Phe 505 510 515

Ala Ala Pro Gly Pro Gly Asp Val Gly Thr Ala Ser Leu Val

520 525 530

Gly Ser Asn Met Phe Pro Asn Gin Tyr Arg Glu Ala Ala Phe

535 540 545

Gly Gly Gly Leu Leu Ser Pro Gly Pro Glu Ala Thr 550 555

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 678

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No

(iv) ORIGINAL SOURCE:

(A) ORGANISM: Drosophila melanogaster

(B) STRAIN: Oregon R

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE: embryo

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: Dorsal protein

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: D.melanogaster embryonic polarity (dorsal) protein containing region of high similarity with proteins of Rel family.

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Steward, R.

(B) TITLE: Dorsal, an embryonic poiarit gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel

(C) JOURNAL: Science

(D) VOLUME: 238

(E) ISSUE:

(F) PAGES: 692-694

(G) DATE: 1987

(H) DOCUMENT NUMBER:

(I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES:

( i) SEQUENCE DESCRIPTION: SEQ ID NO. 3:

Met Phe Pro Asn Gin Asn Asn Gly Ala Ala Pro Gly Gin Gly

1 5 10

Pro Ala Val Asp Gly Gin Gin Ser Leu Asn Tyr Asn Gly Leu

15 20 25

Pro Ala Gin Gin Gin Gin Gin Leu Ala Gin Ser Thr Lys Asn

30 35 40

Val Arg Lys Lys Pro Tyr Val Lys He Thr Glu Gin Pro Ala

45 50 55

Gly Lys Ala Leu Arg Phe Arg Tyr Glu Cys Glu Gly Arg Ser

60 65 70

Ala Gly Ser He Pro Gly Val Asn Ser Thr Pro Glu Asn Lys

75 80

Thr Tyr Pro Thr He Glu He Val Gly Tyr Lys Gly Arg Ala 85 90 95 Val Val Val Val Ser Cys Val Thr Lys Asp Thr Pro Tyr Arg

100 105 110

Pro His Pro His Asn Leu Val Gly Lys Glu Gly Cys Lys Lys

115 120 125

Gly Val Cys Thr Leu Glu He Asn Ser Glu Thr Met Arg Ala 130 135 140

Val Phe Ser Asn Leu Gly He Gin Cys Val Lys Lys Lys Asp

145 150

He Glu Ala Ala Leu Lys Ala Arg Glu Glu He Arg Val Asp 155 160 165

Pro Phe Lys Thr Gly Phe Ser His Arg Phe Gin Pro Ser Thr

170 175 180

He Asp Leu Asn Ser Val Arg Leu Cys Phe Gin Val Phe Met

185 190 195

Glu Ser Glu Gin Lys Gly Arg Phe Thr Ser Pro Leu Pro Pro 200 205 210

Val Val Ser Glu Pro He Phe Asp Lys Lys Ala Met Ser Asp

215 220

Leu Val He Cys Arg Leu Cys Ser Cys Ser Ala Thr Val Phe 225 230 235

Gly Asn Thr Gin He He Leu Leu Cys Glu Lys Val Ala Lys

240 245 250

Glu Asp He Ser Val Arg Phe Phe Glu Glu Lys Asn Gly Gin

255 260 265

Ser Val Trp Glu Ala Phe Gly Asp Phe Gin His Thr Asp Val 270 275 280

His Lys Gin Thr Ala He Thr Phe Lys Thr Pro Arg Tyr His

285 290

Thr Leu Asp He Thr Glu Pro Ala Lys Val Phe He Gin Leu 295 300 305

Arg Arg Pro Ser Asp Gly Val Thr Ser Glu Ala Leu Pro Phe

310 315 320

Glu Tyr Val Pro Met Asp Ser Asp Pro Ala His Leu Arg Arg

325 330 335

Lys Arg Gin Lys Thr Gly Gly Asp Pro Met His Leu Leu Leu 340 345 350

Gin Gin Gin Gin Lys Gin Gin Leu Gin Asn Asp His Gin Asp

355 360

Gly Arg Gin Thr Asn Met Asn Cys Trp Asn Thr Gin Asn He 365 370 375

Pro Pro He Lys Thr Glu Pro Arg Asp Thr Ser Pro Gin Pro

380 385 390

Phe Gly Leu Ser Tyr Arg Ala Pro Pro Glu Leu Thr Pro Ser

395 400 405

Pro Gin Pro Val Ser Pro Ser Ser Asn Tyr Asn His Asn Ser 410 415 420

Thr Pro Ser Pro Tyr Asn Met Ala Ser Ala Val Thr Pro Thr

425 430

Asn Gly Gin Gin Gin Leu Met Ser Pro Asn His Pro Gin Gin 435 440 445

Gin Gin Gin Gin Gin Gin Tyr Gly Ala Thr Asp Leu Gly Ser

450 455 460

Asn Tyr Asn Pro Phe Ala Gin Gin Val Leu Ala Gin Gin Gin 465 470 ^" 475 Gin His Gin Gin Gin Gin Gin Gin His Gin His Gin His Gin

480 485 490

Gin Gin His Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin

495 500

Glu Gin Gin Ser Leu Gin Phe His Ala Asn Pro Phe Gly Asn 505 510 515

Pro Gly Gly Asn Ser Trp Glu Ser Lys Phe Ser Ala Ala Ala

520 525 530

Val Ala Ala Ala Ala Pro Thr Ala Thr Gly Ala Ala Pro Ala

535 540 545

Asn Gly Asn Ser Asn Asn Leu Ser Asn Leu Asn Asn Pro Phe 550 555 560

Thr Met His Asn Leu Leu Thr Ser Gly Gly Gly Pro Gly Asn

565 570

Ala Asn Asn Leu Gin Trp Asn Leu Thr Thr Asn His Leu His 575 580 585

Asn Gin His Thr Leu His Gin Gin Gin Gin Leu Gin Gin Gin

590 595 600

Gin Gin Gin Gin Tyr Asp Asn Thr Ala Pro Thr Asn Asn Asn

605 610 615

Ala Asn Leu Asn Asn Asn Asn Asn Asn Asn Asn Thr Ala Gly 620 625 630

Asn Gin Ala Asp Asn Asn Gly Pro Thr Leu Ser Asn Leu Leu

635 640

Ser Phe Asp Ser Gly Gin Leu Val His He Asn Ser Glu Asp 645 650 655

Gin Gin He Leu Arg Leu Asn Ser Glu Asp Leu Gin He Ser

660 665 670

Asn Leu Ser He Ser Thr 675

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 969

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(A) NAME/KEY: P50 or KBF1

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: DNA-binding subunit of NF-K-B

(X) PUBLICATION INFORMATION:

(A) AUTHORS: Kieran, M. ; Blank, v.; Logeat, F. ; Bandekerckhove, J. ; Lottspeich, F. ; Le Bail, 0.; Urban, M.B.; Kourilsky, P.; Baeuerle, P.A. ,* Israel, A.

(B) TITLE: The DNA binding subunit of NF- KB is identical to factor KBF1 and homologous to the rel oncogene product.

(C) JOURNAL: Cell

(D) VOLUME: 62

(E) ISSUE:

(F) PAGES: 1007-1018

(G) DATE: 07-SEP-1990 (H) DOCUMENT NUMBER: (I) FILING DATE: . (J) PUBLICATION DATE: (K) RELEVANT RESIDUES:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 4:

Met Ala Glu Asp Asp Pro Tyr Leu Gly Arg Pro Glu Gin Met

1 5 10

Phe His Leu Asp Pro Ser Leu Thr His Thr He Phe Asn Pro

15 20 25

Glu Val Phe Gin Pro Gin Met Ala Leu Pro Thr Ala Asp Gly

30 35 40

Pro Tyr Leu Gin He Leu Glu Gin Pro Lys Gin Arg Gly Phe

45 50 55

Arg Phe Arg Tyr Val Cys Glu Gly Pro Ser His Gly Gly Leu

60 65 70

Pro Gly Ala Ser Ser Glu Lys Asn Lys Lys Ser Tyr Pro Gin

75 80

Val Lys He- Cys Asn Tyr Val Gly Pro Ala Lys Val He Val 85 90 95

Gin Leu Val Thr Asn Gly Lys Asn He His Leu His Ala His

100 105 110

Ser Leu Val Gly Lys His Cys Glu Asp Gly He Cys Thr Val

115 120 125

Thr Ala Gly Pro Lys Asp Met Val Val Gly Phe Ala Asn Leu 130 135 140

Gly He Leu His Val Thr Lys Lys Lys Val Phe Glu Thr Leu

145 150

Glu Ala Arg Met Thr Glu Ala Cys He Arg Gly Tyr Asn Pro 155 160 165

Gly Leu Leu Val His Pro Asp Leu Ala Tyr Leu Gin Ala Glu 170 175 180 Gly Gly Gly Asp Arg Gin Leu Gly Asp Arg Glu Lys Glu Leu

185 190 195

He Arg Gin Ala Ala Leu Gin Gin Thr Lys Glu Met Asp Leu 200 205 210

Ser Val Val Arg Leu Met Phe Thr Ala Phe Leu Pro Asp Ser

215 _ 220

Thr Gly Ser Phe Thr Arg Arg Leu Glu Pro Val Val Ser Asp 225 230 235

Ala He Tyr Asp Ser Lys Ala Pro Asn Ala Ser Asn Leu Lys

240 245 250

He Val Arg Met Asp Arg Thr Ala Gly Cys Val Thr Gly Gly

255 260 265

Glu Glu He Tyr Leu Leu Cys Asp Lys Val Gin Lys Asp Asp 270 275 280

He Gin He Arg Phe Tyr Glu Glu Glu Glu Asn Gly Gly Val

285 290

Trp Glu Gly Phe Gly Asp Phe Ser Pro Thr Asp Val His Arg 295 300 305

Gin Phe Ala He Val Phe Lys Thr Pro Lys Tyr Lys Asp He

310 315 320

Asn He Thr Lys Pro Ala Ser Val Phe Val Gin Leu Arg Arg

325 330 335

Lys Ser Asp Leu Glu Thr Ser Glu Pro Lys Pro Phe Leu Tyr 340 345 350

Tyr Pro Glu He Lys Asp Lys Glu Glu Val Gin Arg Lys Arg

355 360

Gin Asn Leu Met Pro Asn Phe Ser Asp Ser Phe Gly Gly Gly 365 370 375

Ser Gly Ala Gly Ala Gly Gly Gly Gly Met Phe Gly Ser Gly

380 385 390

Gly Gly Gly Gly Gly Thr Gly Ser Thr Gly Pro Gly Tyr Ser

395 400 405

Phe Pro His Tyr Gly Phe Pro Thr Tyr Gly Gly He Thr Phe 410 415 420

His Pro Gly Thr Thr Lys Ser Asn Ala Gly Met Lys His Gly

425 430

Thr Met Asp Thr Glu Ser Lys Lys Asp Pro Glu Gly Cys Asp 435 440 445

Lys Ser Asp Asp Lys Asn Thr Val Asn Leu Phe Gly Lys Val

450 455 460

He Glu Thr Thr Glu Gin Asp Gin Glu Pro Ser Glu Ala Thr

465 ,470 475

Val Gly Asn Gly Glu Val Thr Leu Thr Tyr Ala Thr Gly Thr 480 485 490

Lys Glu Glu Ser Ala Gly Val Gin Asp Asn Leu Phe Leu Glu

495 500

Lys Ala Met Gin Leu Ala Lys Arg His Ala Asn Ala Leu Phe 505 510 515

Asp Tyr Ala Val Thr Gly Asp Val Lys Met Leu Leu Ala Val

520 525 530

Gin Arg His Leu Thr Ala Val Gin Asp Glu Asn Gly Asp Ser

535 540 545

Val Leu His Leu Ala He He His Leu His Ser Gin Leu Val 550 555 560 Arg Asp Leu Leu Glu Val Thr Ser Gly Leu He Ser Asp Asp

565 570

He He Asn Met Arg Asn Asp Leu Tyr Gin Thr Pro Leu His

575 580 585

Leu Ala Val He Thr Lys Gin Glu Asp Val Val Glu Asp Leu

590 595 600

Leu Arg Ala Gly Ala Asp Leu Ser Leu Leu Asp Arg Leu Gly

605 610 615

Asn Ser Val Leu His Leu Ala Ala Lys Glu Gly His Asp Lys 620 625 630

Val Leu Ser He Leu Leu Lys His Lys Lys Ala Ala Leu Leu

635 640

Leu Asp His Pro Asn Gly Asp Gly Leu Asn Ala He His Leu 645 650 655

Ala Met Met Ser Asn Ser Leu Pro Cys Leu Leu Leu Leu Val

660 665 670

Ala Ala Gly Ala Asp Val Asn Ala Gin Glu Gin Lys Ser Gly

675 680 685

Arg Thr Ala Leu His Leu Ala Val Glu His Asp Asn He Ser 690 695 700

Leu Ala Gly Cys Leu Leu Leu Glu Gly Asp Ala His Val Asp

705 710

Ser Thr Thr Tyr Asp Gly Thr Thr Pro Leu His He Ala Ala 715 720 725

Gly Arg Gly Ser Thr Arg Leu Ala Ala Leu Leu Lys Ala Ala

730 735 740

Gly Ala Asp Pro Leu Val Glu Asn Phe Glu Pro Leu Tyr Asp

745 750 755

Leu Asp Asp Ser Trp Glu Asn Ala Gly Glu Asp Glu Gly Val 760 765 770

Val Pro Gly Thr Thr Pro Leu Asp Met Ala Thr Ser Trp Gin

775 780

Val Phe Asp He Leu Asn Gly Lys Pro Tyr Glu Pro Glu Phe 785 790 795

Thr Ser Asp Asp Leu Leu Ala Gin Gly Asp Met Lys Gin Leu

800 805 810

Ala Glu Asp Val Lys Leu Gin Leu Tyr Lys Leu Leu Glu He

815 820 825

Pro Asp Pro Asp Lys Asn Trp Ala Thr Leu Ala Gin Lys Leu 830 835 840

Gly Leu Gly He Leu Asn Asn Ala Phe Arg Leu Ser Pro Ala

845 850

Pro Ser Lys Thr Leu Met Asp Asn Tyr Glu Val Ser Gly Gly 855 860 865

Thr Val Arg Glu Leu Val Glu Ala Leu Arg Gin Met Gly Tyr

870 875 880

Thr Glu Ala He Glu Val He Gin Ala Ala Ser Ser Pro Val

885 890 895

Lys Thr Thr Ser Gin Ala His Ser Leu Pro Leu Ser Pro Ala 900 905 910

Ser Thr Arg Gin Gin He Asp Glu Leu Arg Asp Ser Asp Ser

915 920

Val Cys Asp Thr Gly Val Glu Thr Ser Phe Arg Lys Leu Ser 925 930 935 Phe Thr Glu Ser Leu Thr Ser Gly Ala Ser Leu Leu Thr Leu

940 945 950

Asn Lys Met Pro His Asp Tyr Gly Gin Glu Gly Pro Leu Glu

955 960 965

Gly Lys He

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 447

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No

(iv) ORIGINAL SOURCE:

(A) ORGANISM: bovine

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: (H) CELL LINE:

(I) ORGANELLE: spleen

(ix) FEATURE:

(A) NAME/KEY: p49

(B) LOCATION:

(C) IDENTIFICATION METHOD: by homology with p50 and K B binding proteins

(D) OTHER INFORMATION:

(X) PUBLICATION INFORMATION:

(A) AUTHORS: Schmid, R.M. ; Perkins, M.D.; Duckett, C.S.; Andrews, P.C.; Nabel, G.J.

(B) TITLE: Cloning of an NF-KB subunit which stimulates HIV transcription in synergy with p65.

(C) JOURNAL: Nature

(D) VOLUME: 352

(E) ISSUE:

(F) PAGES:733-736

(G) DATE: 22-AUG-1991 (H) DOCUMENT NUMBER: (I) FILING DATE:

(J) PUBLICATION DATE: (K) RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO . 5 :

Met Glu Ser Cys Tyr Asn Pro Gly Leu Asp Gly He He Glu

1 5 10

Tyr Asp Asp Phe Lys Leu Asn Ser Ser He Val Glu Pro Lys

15 20 25

Glu Pro Ala Pro Glu Thr Ala Asp Gly Pro Tyr Leu Val He

30 35 40

Val Glu Gin Pro Lys Gin Arg Gly Phe Arg Phe Arg Tyr Gly

45 50 55

Cys Glu Gly Pro Ser His Gly Gly Leu Pro Gly Ala Ser Ser

60 65 70

Glu Lys Gly Arg Lys Thr Tyr Pro Thr Val Lys He Cys Asn

75 80

Tyr Glu Gly Pro Ala Lys He Glu Val Asp Leu Val Thr His 85 90 95

Ser Asp Pro Pro Arg Ala His Ala His Ser Leu Val Gly Lys

100 105 110

Gin Cys Ser Glu Leu Gly He Cys Ala Val Ser Val Gly Pro

115 120 125

Lys Asp Met Thr Ala Gin Phe Asn Asn Leu Gly Val Leu His 130 135 140

Val Thr Lys Lys Asn Met Met Gly Thr Met He Gin Lys Leu

145 150

Gin Arg Gin Arg Leu Arg Ser Arg Pro Gin Gly Leu Thr Glu 155 160 165

Ala Glu Gin Arg Glu Leu Glu Gin Glu Ala Lys Glu Leu Lys

170 175 180

Lys Val Met Asp Leu Ser He Val Arg Leu Arg Phe Ser Ala

185 190 195

Phe Leu Arg Ala Ser Asp Gly Ser Phe Ser Leu Pro Leu Lys 200 205 210

Pro Val Thr Ser Gin Pro He His Asp Ser Lys Ser Pro Gly

215 220

Ala Ser Asn Leu Lys He Ser Arg Met Asp Lys Thr Ala Gly 225 230 235

Ser Val Arg Gly Gly Asp Glu Val Tyr Leu Leu Cys Asp Lys

240 245- 250

Val Gin Lys Asp Asp He Glu Val Arg Phe Tyr Glu Asp Asp

255 260 265

Glu Asn Gly Trp Gin Ala Phe Gly Asp Phe Ser Pro Thr Asp 270 275 280

Val His Lys Gin Tyr Ala He Val Phe Arg Thr Pro Pro Tyr

285 290

His Lys Met Lys He Glu Arg Pro Val Thr Val Phe Leu Gin 295 300 305

Leu Lys Arg Lys Arg Gly Gly Asp Val Ser Asp Ser Lys Gin

310 315 320

Phe Thr Tyr Tyr Pro Leu Val Glu Asp Lys Glu Glu Val Gin

325 330 335

Arg Lys Arg Arg Lys Ala Leu Pro Thr Phe Ser Gin Pro Phe 340 345 350

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 619

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: No

(iv) ORIGINAL SOURCE:

(A) ORGANISM: human

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE: lymphoma

(H) CELL LINE: Daudi Burkett (I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: c-rel or hc-rel

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION: homology to rel protein family

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Brownell, E. ; Mettereder, N Rice, N.R.

(B) TITLE: A human rel proto-oncogene cD containing an Alu fragment as a potential coding exon.

(C) JOURNAL: Oncogene

(D) VOLUME: 4 (E) ISSUE:

(F) PAGES: 934-941

(G) DATE: 1989

(H) DOCUMENT NUMBER:

(I) FILING DATE:

(J) PUBLICATION DATE:

(K) RELEVANT RESIDUES:

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6:

Met Ala Ser Gly Ala Tyr Asn Pro Tyr He Glu He He Glu

1 5 10

Gin Pro Arg Gin Arg Gly Met Arg Phe Arg Tyr Lys Cys Glu

15 20 25

Gly Arg Ser Ala Gly Ser He Pro Gly Glu His Ser Thr Asp

30 35 40

Asn Asn Arg Thr Tyr Pro Ser He Gin He Met Asn Tyr Tyr

45 50 55

Gly Lys Gly Lys Val Arg He Thr Leu Val Thr Lys Asn Asp

60 65 70

Pro Tyr Lys Pro His Pro His Asp Leu Val Gly Lys Asp Cys

75 80

Arg Asp Gly Tyr Tyr Glu Ala Glu Phe Gly Gin Glu Arg Arg 85 90 95

Pro Leu Phe Phe Gin Asn Leu Gly He Arg Cys Val Lys Lys

100 105 110

Lys Glu Val Lys Glu Ala He He Thr Arg He Lys Ala Gly

115 120 125

He Asn Pro Phe Asn Val Pro Glu Lys Gin Leu Asn Asp He 130 135 140

Glu Asp Cys Asp Leu Asn Val Val Arg Leu Cys Phe Gin Val

145 150

Phe Leu Pro Asp Glu His Gly Asn Leu Thr Thr Ala Leu Pro 155 160 165

Pro Val Val Ser Asn Pro He Tyr Asp Asn Arg Ala Pro Asn

170 175 180

Thr Ala Glu Leu Arg He Cys Arg Val Asn Lys Asn Cys Gly

185 190 195

Ser Val Arg Gly Gly Asp Glu—Ile Phe Leu Leu Cys Asp Lys 200 205 210

Val Gin Lys Asp Asp He Glu Val Arg Phe Val Leu Asn Asp

215 220

Trp Glu Ala Lys Gly He Phe Ser Gin Ala Asp Val His Arg 225 230 235

Gin Val Ala He Val Phe Lys Thr Pro Pro Tyr Cys Lys Ala

240 245 250

He Thr Glu Pro Val Thr Val Lys Met Gin Leu Arg Arg Pro

255 260 265

Ser Asp Gin Glu Val Ser Glu Ser Met Asp Phe Arg Tyr Leu 270 275 280

Pro Asp Glu Lys Asp Thr Tyr Gly Asn Lys Ala Lys Lys Gin

285 290

Lys Thr Thr Leu Leu Phe Gin Lys Leu Cys Gin Asp His Val 295 300 305 Glu Thr Gly Phe Arg His Val Asp Gin Asp Gly Leu Glu Leu

310 315 320

Leu Thr Ser Gly Asp Pro Pro Thr Leu Ala Ser Gin Ser Ala

325 330 335

Gly He Thr Val Asn Phe Pro Glu Arg Pro Arg Pro Gly Leu 340 345 350

Leu Gly Ser He Gly Glu Gly Arg Tyr Phe Lys Lys Glu Pro

355 360

Asn Leu Phe Ser His Asp Ala Val Val Arg Glu Met Pro Thr 365 370 375

Gly Val Ser Ser Gin Ala Glu Ser Tyr Tyr Pro Ser Pro Gly

380 385 390

Pro He Ser Ser Gly Leu Ser His His Ala Ser Met Ala Pro

395 400 405

Leu Pro Ser Ser Ser Trp Ser Ser Val Ala His Pro Thr Pro 410 415 420

Arg Ser Gly Asn Thr Asn Pro Leu Ser Ser Phe Ser Thr Arg

425 430

Thr Leu Pro Ser Asn Ser Gin Gly He Pro Pro Phe Leu Arg 435 440 445

He Pro Val Gly Asn Asp Leu Asn Ala Ser Asn Ala Cys He

450 455 460

Tyr Asn Asn Ala Asp Asp He Val Gly Met Glu Ala Ser Ser

465 470 475

Met Pro Ser Ala Asp Leu Tyr Gly He Ser Asp Pro Asn Met 480 485 490

Leu Ser Asn Cys Ser Val Asn Met Met Thr Thr Ser Ser Asp

495 500

Ser Met Gly Glu Thr Asp Asn Pro Arg Leu Leu Ser Met Asn 505 510 515

Leu Glu Asn Pro Ser Cys Asn Ser Val Leu Asp Pro Arg Asp

520 525 530

Leu Arg Gin Leu His Gin Met Ser Ser Ser Ser Met Ser Ala

535 540 545

Gly Ala Asn Ser Asn Thr Thr Val Phe Val Ser Gin Ser Asp 550 555 560

Ala Phe Glu Gly Ser Asp Phe Ser Cys Ala Asp Asn Ser Met

565 570

He Asn Glu Ser Gly Pro Ser Asn Ser Thr Asn Pro Asn Ser 575 580 585

His Gly Phe Val Gin Asp Ser Gin Tyr Ser Gly He Gly Ser

590 595 600

Met Gin Asn Glu Gin Leu Ser Asp Ser Phe Pro Tyr Glu Phe

605 610 615

Phe Gin Val

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL: No

(ix) FEATURE:

(A) NAME/KEY: "A" half of Ig-KB binding site

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 7: GGGAC

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL: No

(ix) FEATURE:

(A) NAME/KEY: "B" half of Ig-KB binding site

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 8: GGAAA

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: Unknown

(ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL: No (ix) FEATURE: (A) NAME/KEY: canonical Ig-KB binding site

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9: GGGACTTTCC 1

Claims

We claim:

1. A method for inhibiting, preventing or controlling of binding of NRD family proteins to DNA, said proteins having an N-terminal region of homology and a DNA-binding region within the region of homology, the method comprising the mutation of the DNA-binding region of the proteins and includes the steps of replacing one or more amino acids in the DNA-binding region by an amino acid which is different from the normally-occurring replaced amino acid and not chemically analogous thereto.

2. A method according the claim 1, wherein the protein is NF-KB p49 (p50B) (SEQ ID NO: 5) , NF-KB p50 (SEQ ID NO: 4) , NF-KB p65 (SEQ ID NO: 1) , c-Rel protein (SEQ ID NO: 6) , v-Rel protein, Dorsal protein (SEQ ID NO: 3) or Rel-B protein (SEQ ID NO: 2) .

^'3. A method according to claim 1, comprising replacing one or more of the arginine, phenylalanine", tyrosine, cysteine, or glycine residues occurring in the N-terminal DNA-binding homologous region of the NRD protein.

4. A method according to claim 2, wherein the NRD protein is NFKB p50 (SEQ ID NO: 4) and an amino acid in the N-terminal region between amino acids 43 and 72 is replaced.

5. A method according to claim 4, wherein an amino acid in the N-terminal region between amino acids 50 to 71 is replaced.

6. A method according to claim 4, wherein an amino acid in the N-terminal region between amino acids 54 to 71 is replaced.

7. A method according to claim 2, wherein the NRD protein is Dorsal protein (SEQ ID NO: 3) and an amino acid in the N-terminal region between amino acids 48 and 77 is replaced.

8. A method according to claim 7, wherein an amino acid in the N-terminal region between amino acids 55 to 76 is replaced.

9. A method according to claim 7, wherein an amin acid in the N-terminal region between amino acids 59 to is replaced.

10. A method according to claim 2, wherein the NRD protein is NF-KB p65 (SEQ ID NO: 1) and an amino acid i the N-terminal region between amino acids 19 to 48 is replaced.

11. A method according to claim 10, wherein an ami acid in the N-terminal region between amino acids 26 to is replaced.

12. A method according to claim 10, wherein an ami acid in the N-terminal region between amino acids 30 to is replaced.

13. In an NRD family protein having an N-terminal DNA binding region, an improved NRD protein wherein DNA binding and gene activation is controlled or inhibited said improved protein being a mutated NRD protein wherei one or more amino acids in the N-terminal DNA-binding region is replaced with a different amino acid.

14. An improved NRD protein according to claim 13, wherein the protein is NF-KB p49 (SEQ ID NO: 5) , NF-KB p (SEQ ID NO: 4), NF-KB p65 (SEQ ID NO: 1), c-Rel (SEQ ID NO: 6) protein, v-Rel protein. Dorsal protein (SEQ ID NO 3) or Rel-B protein (SEQ ID NO: 2) .

15. An improved NRD protein according to claim 13, wherein one or more of the arginine, phenylalanine, tyrosine, cysteine, or glycine residues occurring in the N-terminal DNA-binding homologous region of the protein replaced.

16. An improved protein according to claim 14, wherein the protein is NF-KB p50 (SEQ ID NO: 4) and an amino acid in the N-terminal region between amino acids and 72 is replaced.

17. An improved protein according to claim 16, wherein an amino acid in the N-terminal region between amino acids 50 to 71 is replaced-.

18. An improved protein according to claim 16, wherein an amino acid in the N-terminal region between amino acids 54 to 71 is replaced.

19. An improved protein according to claim 14, wherein the protein is Dorsal protein (SEQ ID NO: 3) and an amino acid in the N-terminal region between amino acids 48 to 77 is replaced.

20. An improved protein according to claim 19, wherein an amino acid in the N-terminal region between amino acids 55 to 76 is replaced.

21. An improved protein according to claim 19, wherein an amino acid in the N-terminal region between amino acids 59 to 76 is replaced.

22. An improved protein according to claim 14, wherein the protein is NF-KB p65 (SEQ ID NO: 1) and an amino acid in the N-terminal region between amino acids 19 to 48 is replaced.

23. An improved protein according to claim 22, wherein an amino acid in the N-terminal region between amino acids 26 to 47 is replaced.

24. An improved protein according to claim 22, wherein an amino acid in the N-terminal region between amino acids 30 to 47 is replaced.