CELL ABLATION USING ΓΛANS-SPLICING RIBOZYMES

Field of the Invention

The present invention is directed to novel trans- splicing ribozymes and methods of cell ablation using these ribozymes.

BRIEF DESCRIPTION OF THE BACKGROUND ART

I. Group I Introns

RNA molecules with catalytic activity are called ribozymes or RNA enzymes (Cech, T.R. , Ann . Rev .

Biochem . 59:543-568 (1990). The Tetrahymena thermophila precursor rRNA contains an intron (a ribozyme) capable of catalyzing its own excision. This ribozyme is one of a class of structurally related Group I introns. The splicing activity of the modified T . thermophila intron requires the presence of a guanosine cofactor and a divalent cation, either Mg ⁺⁺ or Mn ⁺⁺ , and occurs via two sequential transesterification reactions (Figure 1). First, a free guanosine is bound to the ribozyme and its 3' hydroxyl group is positioned to attack the phosphorus atom at the 5' splice site. The guanosine is covalently attached to the intron sequence and the 5' exon is released. Second, the phosphodiester bond located at the 3' splice site undergoes attack from the newly freed 3' hydroxyl group of the 5' exon, resulting in production

of the ligated exon sequences. The excised intron subsequently undergoes a series of transesterification reactions, involving its 3 ¹ hydroxyl group and internal sequences, resulting in the formation of shortened circular forms.

These successive reactions are chemically similar and appear to occur at a single active site. The reactions of self-splicing are characterized by the formation of alternative RNA structures as differing RNA chains are each brought to form similar conformations around the highly conserved intron. Splicing requires the alignment of the intron-exon junctions across a complementary sequence termed the "internal guide sequence" or IGS. The first cleavage at the 5' splice site requires the formation of a base-paired helix (PI) between the IGS and sequences adjacent the splice site. The presence of a U:G "wobble" base-pair within this helix defines the phosphodiester bond that will be broken in the catalytic reaction of the ribozyme. After cleavage of this bond, a portion the PI helix is displaced and a new helix, PIO, is formed due to complementarity between the IGS and sequences adjacent the 3' splice site. An invariant guanosine residue precedes the phosphodiester at the 3' splice site, similar to the portion of the PI sequence that it is displacing. Thus, ligation of the exons occurs in a reverse of the first cleavage reaction but where new exon sequences have been substituted for those of the intron. It may be noted that intron circularization reactions subsequent to exon ligation also involve fcase-pairing of 5' sequences across the IGS, and attack mediated by the 3' hydroxyl group of the intron's terminal guanine residue (Been, M.D. et al. , "Selection Of Circularizaton Sites In A Group I IVS RNA Requires

Multiple Alignments Of An Internal Template-Like Sequence," Ceil 50:951 (1987)).

II. Catalytic Activities In order to better define the structural and catalytic properties of the Group I introns, exon sequences have been stripped from the "core" of the T. thermophila intron. Cech, T.R. et al . , WO 88/04300, describes at least three catalytic activities possessed by the Tetrahymena intron ribozyme: (1) a dephosphorylating activity, capable of removing the 3' terminal phosphate of RNA in a sequence-specific manner, (2) an RNA polymerase activity (nucleotidyl transferase) , capable of catalyzing the conversion of oligoribonucleotides to polyribonucleotides, and (3) a sequence-specific endoribonuclease activity.

Isolated ribozyme activities can interact with substrate RNAs in tranε , and these interactions characterized. For example, when truncated forms of the intron are incubated with sequences corresponding to the 5' splice junction, the site undergoes guanoεien- dependent cleavage in mimicry of the first step in splicing. The substrate and endoribonucleolytic intron RNAs base-pair to form helix PI, and cleavage occurs after a U:G base-pair at the 4th-6th position.

Phylogenetic comparisons and mutational analyses- indicate that the nature of the sequences immediately adjacent the conserved uracil residue at the 5 ¹ splice site are unimportant for catalysis, provided the base- pairing of helix PI is maintained (Doudna, J.A. et al . ,

Proc . Natl . Acad . Sci . USA 86 : 7402-7406 (1989)).

The sequence requirements for 3' splice-site selection appear to lie mainly within the structure of the intron itself, including helix P9.0 and the following guanoεine residue which delineates the 3' intron boundary. However, flanking sequences within the

3' exon are required for the formation of helix PIO and efficient splicing, as shown by mutational analysis

(Suh, E.R. et al . , Mol . Cell . Biol . 10:2960-2965

(1990)). In addition, oligonucleotides have been ligated in trans, using a truncated form of the intron, and "external" guide sequence and oligonucleotides which had been extended by a 5' guanosine residue. The substrate oligonucleotides corresponding to 3' exon sequences were aligned solely by the formation of P10- like helices on an external template, prior to ligation

(Doudna, J.A. et al . , Nature 339:519-522 (1989)).

The cleavage activity of ribozymes has been targeted to specific RNAε by engineering a discrete

"hybridization" region into the ribozyme, such hybridization region being capable of specifically hybridizing with the desired RNA. For example,

Gerlach, W.L. et al. , EP 321,201, constructed a ribozyme containing a sequence complementary to a target RNA. Increasing the length of this complementary sequence increased the affinity of this sequence for the target. However, the hybridizing and cleavage regions of this ribozyme were integral parts of each other. Upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme cleaved the target. It was suggested that the ribozyme would be useful for the inactivation or cleavage of target RNA in vivo , such as for the treatment of human diseases characterized by the production of a foreign host's RNA. However, ribozyme- directed trans-splicing, (as oppoεed to trans-cleivage) waε not described or suggested.

The endoribonuclease activities (the cleavage activities) of variouε- naturally-occurring ribozymes have been extenεively εtudied. Analysis of the structure and sequence of these ribozymes haε indicated that certain nucleotideε around the cleavage site are

highly conserved but flanking sequences are not so conserved. This information haε lead to the design of ' novel endoribonuclease activities not found in nature.

For example, Cech and others have constructed novel * 5 ribozymes with altered substrate sequence specificity (Cech, T.R. et al . , WO 88/04300; Koizumi, M. et al . , FEBS Lett . 228 : 228-230 (1988); Koizumi, M. et al . , FEBS Lett . 239:285-288 (1988); Haseloff, J. et al . , Nature 334:585-591 (1987); and Heus, H.A. et al . , Nucl . Acidε

10 Res . 18:1103-1108 (1990)). From early studies of the self-cleaving plant viroidε and satellite RNAε (Buzayan, J.M. et al . , Proc . Natl . Acad . Sci . USA 83:8859-8862 (1986), guidelineε for the deεign of ribozy eε that are capable of cleaving other RNA

15 moleculeε in trans in a highly sequence specific have been developed (Haseloff, J. et al . , Nature 334:585-591

(1988)). However, these constructs were unable to catalyze efficient, targeted trans-splicing reactions.

The joining of exons contained on separate RNAs,

20 that is, trans-splicing, occurs in nature for both snRNP-mediated and self-catalyzed group I and group II introns. In trypanoso e and Caenorhabditiε eleqanε mRNAs, common 5' leader sequenceε are transcribed from separate genes and spliced to the 3' portions of the

25 mRNAs (Agabian, N., Cell 61:1157-1160 (1990); Hirsh, D. et al . , Mol . Biol . Rep . 14:115 (1990). These small "spliced leader" RNAs (slRNAε) consist of the 5' exon fused to sequences that can functionally substitute for Ul snRNA in mammalian εnRNP-splicing extracts.

30 Also, both the group I and group II self-splicing introns are capable of exon ligation in trans in artificial systems (Been, M.D. et al . , Cell 47:207-216 (1986); Galloway-Salvo, J.L. et al . , J . Mol . Biol . 211:537-549 (1990); Jacquier, A. et al . , Science

35 234:1099-1194 (1986); and Jarrell, K.A. et al . , Mol .

Cell Biol . 8:2361-2366 (1988)). Trans-splicing occurs

in vivo for group II introns in split genes of chloroplasts (Kohchi, T. et al . , Nucl . Acids Res . 16:10025-10036 (1988)), and has been shown for a group I intron in an artificially split gene in Escherichia coli (Galloway-Salvo, J.L. et al . , J. Mol . Biol .

211:537-549 (1990)). In the latter case, a bacteriophage T4 thymidylate synthase gene (td) containing a group I intron was divided at the loop connecting the intron helix P6a. Transcripts of the td gene segments were shown to undergo trans-splicing in vitro, and to rescue dysfunctional E. coli host cells. Known base-pairings (P3, P6 and P6a) and possible tertiary interactions between the intron segments, allowed correct assembly and processing of the gene halves.

In vitro, the Tetrahymena ribozyme is capable of catalyzing the trans-splicing of single-stranded model oligoribonucleotide substrateε. Four components were necessary: ribozyme, 3' single-stranded RNA, 5' exon and GTP. A shortened form of the Tetrahymena ribozyme

(L-21 Seal IVS RNA) , starting at the internal guide sequence and terminating at U ₄₀₉ has been used in such a reaction (Flanegan, J.B. et al . , J. Cell . Biochem . (Supp. ) 12 part D: 28 (1988)). Attack by GTP at the 5' splice site released the 5' exon which was then ligated by the ribozyme to the 3' exon in a transesterification reaction at the 3 ¹ splice site.

The in vivo use of ribozymes as an alternative to the use of antisense RNA for the targeting and destruction of specific RNAs haε been propoεed

(Gerlach, W.L. et al., EP321,201; Cotten, M. , Trends

Biotechnol . 8:174-178 (1990); Cotten, M. et al . , EMBO

J. 8:3861-3866 (1989); Sarver, N. et al . , Science

247:1222-1225 (1990)). For example, expreεεion of a ribozyme with catalytic endonucleolytic activity towards an RNA expressed during HIV-l infection haε

been suggested as a potential therapy against human immunodeficiency virus type 1 (HIV-l) infection (Sarver, N. et al .. Science 247:1222-1225 (1990); Cooper, M., CDC AIDS Weekly, April 3, 1989, page 2; Rossi, J.J., Abstract of Grant No. 1R01AI29329 in

Dialog's Federal Research in Progress File 265). However, such attempts have not yet been successful.

In a study designed to investigate the potential use of ribozymes as therapeutic agents in the treatment of human immunodeficiency virus type 1 (HIV-l) infection, ribozymes of the hammerhead motif (Hutchins, C.J. et al . , Nucl . Acids Res . 14:3627 (1986); Keese, P. et al . , in Viroidε and Viroid-Like Pathogenε, J.S. Se ancik, ed. , CRC Press, Boca Raton, FL, 1987, pp. 1- 47) were targeted to the HIV-l gag transcripts.

Expression of the gag-targeted ribozyme in human cell cultures resulted in a decrease (but not a complete disappearance of) the level of HIV-l gag RNA and in antigen p24 levels (Sarver, N. et al . , Science 247:1222-1225 (1990)). Thus, the medical effectiveness of Sarver's ribozyme waε limited by itε low efficiency since any of the pathogen's RNA that escapes remains a problem for the host.

Another problem with in vivo ribozyme applications is that a high ribozyme to substrate ratio is required for ribozyme inhibitory function in nuclear extracts and it haε been difficult to achieve such ratios. Cotton et ai. achieved a high ribozyme to substrate ration by microinjection of an expresεion caεεette containing a ribozyme-producing gene operably linked to a strong tRNA promoter (a polymerase III promoter) in frog oocytes, together with εubεtrate RNA that containε the cleavage sequence for the ribozyme (Cotton, M. et al . , EMBO J. 8:3861-3866 (1989).- However, microinjection iε not an appropriate method of delivery in multicellular organiεmε.

The in vivo activity of ribozymes designed against mRNA coding for Eεcherichia coli /S-galactosidase haε been reported (Chuat, J.-C. et al . , Biochem . Biophyε . Res . Commun . 162 :1025-1029 (1989)). However, this activity was only observed when the ribozyme and target were transfected into bacterial cells on the same molecule. Ribozyme activity was inefficient when targeted against an mRNA transcribed from a bacterial F episome that possessed the target part of the β- galactosidase gene.

Thus, current technological applicationε of ribozyme activitieε are limited to thoεe which propose to utilize a ribozyme'ε cleavage activity to destroy the activity of a target RNA. Unfortunately, such applications often require complete destruction of all target RNA molecules, and/or relatively high ribozyme:subεtrate ratioε to enεure effectiveneεε and thiε has been difficult to achieve. Most importantly, the modified ribozymes of the art are not capable of efficient, directed trans-splicing.

Accordingly, a need exists for the development of highly efficient ribozymes and ribozyme expreεεion syεte ε. Eεpecially, the art doeε not deεcribe an effective meanε in which to destroy anexisting RNA sequence or to alter the coding sequence of an exiεting

RNA by the trans-εplicing of a new RNA εequence into a hoεt'ε RNA.

SUMMARY OF THE INVENTION

Recognizing the potential for the design of novel ribozymes, and cognizant of the need for highly efficient methods to alter the genetic characteriεtics of higher eukaryoteε in vivo , the inventorε have investigated the use of ribozymeε to alter the genetic information of native RNA'ε in vivo. These efforts

have culminated in the development of highly effective trans-splicing ribozymes, and guidelines for the engineering thereof.

According to the invention, there is first provided an RNA or DNA molecule, such molecule encoding a trans-splicing ribozyme, such ribozyme being capable of efficiently splicing a new 3' exon sequence into any chosen target RNA sequence in a highly precise manner, in vitro or in vivo, and such molecule being novel in the ability to accomodate, any chosen target RNA or 3' exon sequences, and in the addition of a complementary sequence which enhances the specificity of such ribozyme.

According to the invention, there is also provided an RNA or DNA molecule, such molecule encoding a ribozyme, the sequence for such ribozyme being a fusion RNA, εuch fuεion RNA providing a firεt RNA sequence that is sufficient for targeting such ribozyme to hybridize to a target RNA, and further a second RNA sequence, such second RNA sequence capable of being transposed into the target RNA, and εuch second RNA sequence encoding an RNA sequence foreign to the targeted RNA sequence.

According to the invention, there is further provided an RNA or DNA molecule, εuch molecule encoding a ribozyme, the sequence for such ribozyme being a fusion RNA as described above, the first RNA sequence provided by the fusion RNA being a sequence for targeting such RNA molecule to hybridize to GAL4 RNA, and the second RNA εequence of the fuεion RNA providing the coding sequence of the A chain of diphtheria toxin (DTA) .

According to the invention, there is also provided an RNA or DNA molecule, εuch molecule encoding a confor ationally diεrupted ribozyme of the invention, a pro-ribozyme, εuch pro-ribozyme being substrate-

activated, that is, such pro-ribozyme possessing neglible or no self-cleavage or trans-splicing activity, until being reactived by specific interaction with target RNA. According to the invention, there is further provided an RNA or DNA molecule containing a ribozyme or pro-ribozyme expression cassette, such cassette being capable of being stably maintained in a host, or inserted into the genome of a host, and such cassette providing the sequence of a promoter capable of func¬ tioning in such host, operably linked to the sequence of a ribozyme or pro-ribozyme of the invention.

According to the invention, there is further provided an RNA or DNA molecule containing a ribozyme or pro-ribozyme expression cassette, such cassette being capable of being stably inserted into the genome of a host, such ribozyme expression cassette providing the sequence of a GA 4-responsive promoter operably linked to the sequence of a ribozyme or pro-ribozyme of the invention.

According to the invention, there is further provided a method for in-vitro trans-splicing, such method comprising the steps of (1) providing a ribozyme or pro-ribozyme of the invention and an appropriate substrate for such ribozyme in vitro , (2) further providing in vitro reaction conditions that promote the desired catalytic activity of such ribozyme or pro- ribozyme; and (3) allowing such ribozyme or pro- ribozyme to react with such substrate under such conditions.

According to the invention, there is further provided a method for in vivo trans-splicing, such method comprising the steps of (1) providing an RNA or DNA molecule of the invention to a host cell, (2) expressing the ribozyme or pro-ribozyme encoded by such molecule in εuch host cell, (3) expreεεing a εubεtrate

of such ribozyme or pro-ribozyme in such host cell, and (4) allowing such ribozyme or pro-ribozyme to react with such substrate in such host cell.

According to the invention, there is further provided a method for inactivating the activity of a target RNA, εuch method comprising (1) providing a ribozyme or pro-ribozyme of the invention, such ribozyme or pro-ribozyme being catalytically active against such target RNA, (2) providing such target RNA, and (3) providing conditions that allow such ribozyme or pro-ribozyme to expresε itε catalytic activity towardε such target RNA.

According to the invention, there is further provided a method for providing a desired genetic sequence to a host cell in vivo, such method comprising

(1) providing a ribozyme or pro-ribozyme of the invention to a desired host cell, such ribozyme or pro- ribozyme being catalytically active against a target RNA in such host cell, (2) providing such ribozyme or pro-ribozyme encoding εuch deεired genetic sequence, and (3) providing conditionε that allow εuch ribozyme or pro-ribozyme to trans-εplice such desired genetic sequence into the sequence of the target RNA.

According to the invention, there is further provided a method for cell ablation in multicellular plantε and animalε, εuch method comprising providing a ribozyme or pro-ribozyme of the invention to a any host cell, and especially into a fertilized embryonic hoεt cell, such ribozyme or pro-ribozyme encoding the sequence of a gene toxic to such host cell and such ribozyme or pro-ribozyme being capable of trans- splicing with a desired target in such hoεt cell.

According to the invention, there is further provided a method for engineering male or female sterility in agronomically important plant species, εuch method comprising the ablation of any cell

necessary for fertility using a ribozyme or pro- ribozyme of the invention.

According to the invention, there is further provided a method of immunizing plants against plant pathogens, such method comprising the construction of transgenic plants capable of expressing a plant pathogen-specific fusion ribozyme or pro-ribozyme of the invention, and εuch ribozyme or pro-ribozyme being capable of ablating any host cell infected with such pathogen.

According to the invention, there is further provided a transformed, pathogen-resistant microorganism, such microorganism being resistant to a desired pathogen, such microorganism being transformed with a ribozyme or pro-ribozyme of the invention and such ribozyme or pro-ribozyme providing a catalytic activity that targets a nucleic acid molecule expresεed by such pathogen.

According to the invention, there is further provided a viral pathogen capable of delivering a desired ribozyme or pro-ribozyme activity to a desired host, such ribozyme or pro-ribozyme activity being delivered by a ribozyme or pro-ribozyme of the invention.

DESCRIPTION OF THE FIGURES

Figure 1 is a diagram of the mechanism of ribozyme splicing of the group I intron. Figure 2 is a diagram of structure of the (A)

Tetrahymena thermophila rRNA intron; (B) Target mRNA and trans-splicing ribozyme or pro-ribozyme of the invention.

Figure 3(A) is a diagram of the design of a CAT- LacZ α-peptide trans-splicing ribozyme; (B) is the complete DNA coding sequence of the CAT-LacZ ribozyme.

Figure 4 presentε the εequenceε of cucumber mosaic virus (CMV) RNA 4 trans-splicing ribozymes. A: viruε

RNA target sequenceε; B: Oligonucleotide target sequenceε; C: CMV RNA4 - diphtheria toxin A-chain trans-splicing ribozymes.

Figure 5 is a comparison of cucumber mosaic virus 3/4 sequences.

Figure 6(A) is a diagram of the design of a Gal4- Diphtheria toxin A (DTA) trans-splicing ribozyme; (B) is the complete coding sequence of the Gal4-DTA ribozyme with the isoleucine substitution.

Figure 7 is a diagram of the P-element mediated "enhancer-trapping" method for expression of Gal4 protein. Figure 8 preεentε a partial sequence of wild-type

DTA and DTA 3' exon mutants.

Figure 9 is a map of pGaTB and pGaTN.

Figure 10 is a map of pUAST.

Figure 11 iε a cuticle preparation of a Droεophila embryos expreεεing a Gal4-DTA trans-splicing ribozyme.

Figure 12 preεentε the rationale for "pro- ribozyme" deεign. Arrowε show sites of ribozyme cleavage, "antisenεe" regionε are shown in black, catalytic domains are shown with radial shading, and 3' "exon" εequenceε are εhown with light εhading. In the absence of the target mRNA, trans-splicing ribozymes may transiently base-pair, and react with heterologouε εequenceε (including their own) . In addition, scisεion at the "3' exon" junction will occur. Inactive "pro- ribozymes" are conεtructed to contain extra self- complementary sequenceε which cauεe the catalytic center of the ribozyme to be miε-folded. Active ribozymeε are only formed after base-pairing with the intended target mRNA - and consequent displacement of the interfering secondary structure.

Figure 13 shows the sequence and predicted secondary structure of the CAT-LacZ trans-splicing ribozyme. Ribozyme "core" sequences are shaded (after Cech, Gene 73:259-271 (1988)). Helices P8 are shown for the unmodified ribozyme and pro-ribozymes 1 and 2, with 13 and 18 nucleotides, respectively, of sequence complementary to the "antisense" region (highlighted) . Figure 14 shows (1) active CAT-LacZ ribozyme shown schematically, with "antisense", ribozyme domain with helix P8 and 3' "exon" sequences; (2) (a) inactive CAT-

LacZ pro-ribozyme 2 shown with base-pairing between sequences in the modified helix P8 and the "anti-sense" region; and (b) the active pro-ribozyme, after base- pairing with the CAT mRNA, displacement of the helix P8 - "antisense" pairing, and re-formation of helix P8.

Figure 15 shows stability of CAT-LacZ pro-ribozyme transcripts. Plasmidε containing the CAT-LacZ ribozyme and pro-ribozyme sequences were cleaved with _5coRI and transcribed using T7 or SP6 RNA polymerase and [32- P]UTP. Radiolabeled transcripts were fractionated by

5% polyacrylamide gel electrophoresis in 7M urea and 25% formamide, and autoradiographed. The ribozyme transcripts underwent extensive hydrolysis, primarily at the "3' exon" junction. The pro-ribozyme forms were markedly less reactive.

Figure 16 shows endoribonuclease activity of CAT- LacZ pro-ribozymes. Plasmids containing CAT-LacZ ribozyme and pro-ribozyme sequences were cleaved, with Seal , and transcribed with T7 or SP6 RNA polymerase. Transcriptε were incubated for 30' at 37°c, 45°C and

50°C in 40 mM Tris-HCl pH 7.5, 6 mM MgCl ₂ , 2 mM εpermidine, 10 mM NaCl, 2 mM GTP with radiolabeled CAT RNA, transcribed using T7 RNA polymerase from plasmid cut with PuvII. Products were fractionated by 5% polyacrylamide gel electrophoreεiε in 7M urea and 25% formamide, and autoradiographed. RNA mediated cleavage

of the 173 nt (nucleotides) CAT RNA produces 5' and 3' fragments of 76 nt and 97 nt, respectively.

Figure 17 shows the "wild-type" and modified helices P8 used for pro-ribozyme design with possible base-pairs indicated in schematic form. Those bases which are complementary to the "anti-sense" portion of the corresponding pro-ribozyme, are shown in bold type. The number of complementary bases is listed next to each helix. The helices are ordered by the stability of the corresponding pro-ribozyme transcripts, as measured by the degree of "3' exon" hydrolysiε during in vitro transcription.

Figure 18 shows the stability of GAL4-DTA pro- ribozymes. Plasmids containing ribozyme and pro- ribozyme sequences were linearized with Xhol and transcribed using T7 RNA polymerase. Transcriptε were incubated for 60' at 50 ^β C n 40 mM Tris-HCl pH 7.5, 6 mM MgCl ₂ , 2 mM spermidine, 10 mM NaCl, 1 mM GTP, were fractionated by 5% polyacrylamide gel electrophoresis in 7M urea and 25% formamide, and autoradiographed.

Ribozyme transcriptε are extenεively hydrolysed under these conditions, while pro-ribozyme 1 is less so and pro-ribozyme 2 is stable.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

In the description that follows, a number of termε used in recombinant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Ribozvme. An RNA molecule that inherently posεeεεeε catalytic activity.

.

- 16 -

Trans-splice. A form of genetic manipulation whereby a nucleic acid sequence of a first polynucleotide is co-linearly linked to or inserted into the sequence of a second polynucleotide, in a manner that retains the 3'- 5' phosphodiester linkage between such polynucleotides. By "directed" trans- splicing or "substrate-specific" trans-splicing is meant a trans-splicing reaction that requires a specific specie of RNA as a substrate for the trans- splicing reaction (that is, a specific specie of RNA in which to splice the transpoεed sequence) . Directed trans-splicing may target more than one RNA specie if the ribozyme or pro-ribozyme is designed to be directed against a target sequence present in a related set of RNAs.

Target RNA. An RNA molecule that is a substrate for the catalytic activity of a ribozyme or pro- ribozyme of the invention.

Expression Casεette. A genetic sequence that provides sequences neceεεary for the expression of a ribozyme or pro-ribozyme of the invention.

Stably. By "stably" inserting a sequence into a genome is intended insertion in a manner that results in inheritance of such sequence in copies of such genome.

Operable linkage. An "operable linkage" is a linkage in which a sequence is connected to another sequence (or sequenceε) in εuch a way as to be capable of altering the functioning of the sequence (or sequenceε) . For example, by operably linking a ribozyme or pro-ribozyme encoding εequence to a promoter, expreεεion of the ribozyme or pro-ribozyme encoding εequence is placed under the influence or control of that promoter. Two nucleic acid sequenceε (such as a ribozyme or pro-ribozyme encoding sequence and a promoter region sequence at the 5' end of the

encoding sequence) are said to be operably linked if induction of promoter function results in the transcription of the ribozyme or pro-ribozyme encoding sequence and if the nature of the linkage between the two sequenceε doeε not (1) reεult in the introduction of a frame-εhift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the ribozyme. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter were capable of effecting the synthesis of that nucleic acid sequence.

II. Engineering of the Ribozyme of the Invention

The trans-splicing ribozymes, pro-ribozymes and methods of the invention provide, for the first time, a ribozyme capable of directed trans-splicing into any RNA εequence, and especially into mature (non-intron- containing) mRNA. The trans-splicing ribozyme as described herein, with its extended complementarity to the target, greatly differs from T. thermophila derived endoribonuclease activities described in the art. The additional complementarity of the ribozymes of the invention confers increased affinity and specificity for the target and the complementarity is not an integral part of the catalytic activity. In addition, cleavage occurs efficiently and precisely in the absence of denaturantε and at high concentrations of Mg ⁺⁺ . The guidelines described herein for the design of trans-splicing ribozymes are conεervative, baεed on the well characterized propertieε of group I self-splicing introns and are meant to provide a general scheme for the design of any directed trans-splicing ribozyme. Accordingly, the guidelines preεented herein are not limited to the group I intron of the T . thermophil a

pre-mRNA and may be used by one of skill in the art to design a ribozyme of the invention with other group I introns using such guidelines and knowledge in the art. The native T. thermophila ribozyme (the intron sequence) is located from base 53 to base 465 in the εequence below of the T. thermophila extrachromosomal rDNA: TGACGCAATT CAACCAAGCG CGGGTAAACG GCGGGAGTAA CTATGACTCT CTAAATAGCA ATATTTACCT TTGGAGGGAA AAGTTATCAG GCATGCACCT

CCTAGCTAGT CTTTAAACCA ATAGATTGCA TCGGTTTAAA AGGCAAGACC

GTCAAATTGC GGGAAAGGGG TCAACAGCCG TTCAGTACCA AGTCTCAGGG

GAAACTTTGA CATGGCCTTG CAAAGGGTAT GGTAATAAGC TGACGGACAT

GGTCCTAACC ACGCAGCCAA GTCCTAAGTC AACAGATCTT CTGTTGATAT GGATGCAGTT CACAGACTAA ATGTCGGTCG GGGAAGATGT ATTCTTCTCA

TAAGATATAG TCGGACCTCT CCTTAATGGG AGGTAGCGGA TGAATGGATG

CAACACTGGA GCCGCTGGGA ACTAATTTGT ATGCGAAAGT ATATTGATTA

GTTTTGGAGT ACTCGTAAGG TAGCCAAATG CCTCGTCATC TAATTAGTGA

CGCGCATGAA TGGATTA [SEQ ID NO.l] (Kan, N.C. et al . , Nucl. Acids Reε. 10:2809-2822

(1982) ) .

As described herein, the directed trans-splicing ribozymes of the invention are engineered using the catalytic core of this intron. The intron, and its catalytic core can be isolated by methods known in the art. The catalytic core of the intron, that is, the truncated intron, differs form the full-length intron only in that it is truncated at the S al site, thus removing the last five nucleotideε of the intron. The truncated intron RNA may be prepared by techniques known in the art or may be purchased commercially in kit form from commercial εourceε εuch aε, for example, product #72000 from US Biochemical, Cleveland, OH

(RNAzyme™ Tet 1.0 Kit). Thiε US Biochemical kit provideε ribozyme and the protocol for the use of the ribozyme. Transcribed Tet.l cDNA may be used as the substrate for polymerase chain reaction (PCR) mutagenesis as described below, to produce a synthetic trans-splicing enzyme.

Substrate specificity of the ribozyme of the invention, that is, the ability of the ribozyme to "target" a specific RNA as a subεtrate, is conferred by fusing complementary sequences specific to the target

(subεtrate) RNA to the 5' terminuε of the ribozyme.

Directed trans-splicing specificity of the ribozyme of the invention, that is, specificity in trans-splicing a desired foreign sequence of interest with the sequence of a target RNA, is conferred by providing a new 3' exon at the 3' terminuε of the ribozyme. Detailε of the deεign are further provided below.

To alter the εtructural and catalytic propertieε of the Group I intronε, exon sequences replace the flanking sequence of such introns so that only the catalytic core of the intron, the ribozyme, remainε. The reεulting modified ribozyme can interact with εubεtrate RNAε in tranε . When truncated forms of the intron (i.e., the catalytic "core," i.e. truncated at the Seal site, removing the laεt five nucleotideε of the intron) are incubated with sequences corresponding to the 5' εplice junction of the native ribozyme, the εite undergoeε guanoεine-dependent cleavage in mimicry of the first step in εplicing.

Engineering of the ribozymeε of the invention requireε conεideration of the four guidelineε that follow.

First, a εplice εite must be chosen within the target RNA. In the final trans-εplicing complex, only the 5' portion of the PI duplex iε contributed by the

target RNA. Only a single conserved residue, uracil, is required immediately 5' of the intended splice site. Thiε iε the sole sequence requirement in the target RNA. There iε no inate structure required of the target RNA. Mature mRNA may be targeted and the trans- εplicing reaction performed in the cell's cytoplasm rather than in the nucleus against pre-mRNA. This obviates the need for high concentrations of ribozyme in a cell's nucleus. Second, having chosen a particular target sequence, compensating sequence changes must be added to the 5' section of the ribozyme in order to allow the formation of a suitable helix Pi between the target and ribozyme RNAs. It is highly desired is that the helix PI should contain a U:G base-pair at the intended 5' splice site, and should be positioned at the 4th, 5th (preferred) or 6th position from the baεe of the helix (Doudna, J.A. , et al . , "RNA Structure, Not Sequence Deter ineε The 5 ¹ Splice-Site Specificity of a Group I Intron," Proc . Natl . Acad . Sci . USA 86:7402-7406

(1989) , incorporated herein by reference) . For the native T. thermophila intron, Pi extendε for an additional 3 baεe pairs past the intended 5' splice εite, and, in a preferred embodiment, this is maintained in the trans-splicing ribozyme of the invention. For trans-splicing to be efficient, the substrate and endoribonucleolytic intron RNAs roust base-pair to form helix Pi, with a resulting wobble U:G base-pair. Cleavage of the target RNA occurs at the phosphodiester bond immediately 3' to (after the) U:G base-pair. Phylogenetic comparisons and mutational analyses indicate that the nature of the sequenceε immediately adjacent the conserved uracil residue at the 5' εplice site are unimportant for catalysis, provided the base-pairing of helix Pi iε maintained.

Third, the exon sequenceε flanking the 3' splice εite must be chosen, and adjustments made in the 5' section of the ribozyme, if necessary, to allow the formation of a stable PIO helix. While the PIO helix may be dispenseεd with if neceεεary, itε preεence enhances εplicing and preferred embodiments of the ribozyme of the invention retain the PIO helix (Suh, E.R. et al . , "Baεe Pairing Between The 3' Exon And An Internal Guide Sequence Increaεeε 3' Splice Site Specificity in the Tetrahymena Self-Splicing rRNA

Intron," Mol . Cell . Biol . 10:2960-2965 (1990)). The helices PI and P10 overlap along the T. thermophila intron IGS, and the 2nd and 3rd residueε following both the 5' and 3' εplice sites are complementary to the same residueε in the IGS (Figure 2). While there may be εome advantage in following thiε, many natural group I intronε do not εhare thiε conεtraint, so the choice of 3' exon sequences may be determined primarily by experimental considerationε. Such considerations reflect the wide flexibility in choice of splice sites.

For example, if it is deεired to join two sequenceε at a given point, the εequence at such point cannot be mutated or otherwise altered by the trans-splicing event. Either PI or P10 can be made shorter if the overlapping sequences don't otherwise accomodate for the deεired εplice εite.

The sequence requirements for 3 ¹ splice-site selection appear to lie mainly within the structure of the intron (the ribozyme) itself, including helix P9.0 and the adjoining 3' guanosine residue which delineates the 3' intron boundary. P9.0 iε wholly contained within the intron εequenceε and helpε define the adjacent 3' εplice εite. For the trans-εplicing deεign, the P9.0 helix and the reεt of the functional RNA elementε within the intron are not altered. The εtructural characteristicε of the P9.0 helix are known (Michel, F.

et al. , "The Guanosine Binding Site of the Tetrahymena Ribozyme," Nature 342:391-395 (1989)). However, flanking sequences within the 3' exon are required for the formation of helix PIO and efficient splicing, as shown by mutational analysis.

Fourth, a region of complementary εequence is placed at the 5' terminus of the trans-splicing ribozyme in order to increase its affinity and specificity for the target RΝA. As shown herein, an arbitrary length of around 40 residueε haε been uεed.

Other lengths may be used provided they are not detrimental to the desired effect.

For example, εtarting with the T. thermophila self-splicing intron (diagrammed below) :

1 5 ¹ PI I U A G C A A C U C U C U A A A U

1 * 1 1 1 - 1 1 1 A G G G A G G U U U C C A U U U riboz Iyme core G U A I A I G 1 G1 U I A I . . . 3'

I PIO

2 (The "1" and "2" in the above diagram (and in other ribozyme diagrams throughout the application) note the first and second splice siteε, reεpectively. )

(1) a "5"' site iε choεen adjacent to a uracil reεidue within a choεen target RΝA. The sequences involved in complementarity do not immediately abut sequences involved in PI helix formation but are separated, for example, by five nucleotides also involved in P10 formation;

(2) sequences complementary to the choεen RΝA are fuεed to the 5 ¹ portion of the self-splicing Group I intron. Base-pairing between ribozyme and target RΝA allow formation the of the helix PI;

(3) the chosen "3' exon" sequenceε are fuεed to the 3' portion of the ribozyme, maintaining the conserved helix P10; and

(4) to increase affinity for the target RNA, if desired, a section of extended sequence complementarity is fused to the 5' portion of the ribozyme to allow the formation of 30-40 base-pairs. The alignment of the resulting trans-εplicing ribozyme with itε target RNA may be diagrammed aε εhown immediately below. The target RNA sequence represents the top line. The ribozyme sequence is aligned below it, a continuous sequence wrapping around the lower two lineε wherein the hybridization of the nucleotideε at the 5' and 3' ends and PI and PIO of the ribozyme may be seen.

Alignment of the Ribozyme of the Invention with a Target RNA

1 5' PI I

. . . A U G N N N N N N N U N n I n I n I n I n I *G n I

I ribozyme core G N I PIO

2

According to the invention, trans-splicing ribozymeε can be deεigned that will trans-splice esεentially any RNA sequence onto any RNA target. It is not necessary that the target contain an intron sequence or that the ribozyme be an intron in the target sequence. For example, a strategy for εuch deεign may include (1) the identification of the desired target RNA (2) cloning and/or εequencing of the desired target RNA or portion thereof (3) selection of a desired coding sequence to trans-εplice into the target RNA, (4) the construction of a ribozyme of the invention capable of hybridizing to such target using the guidelines herein and (5) confirmation that the ribozyme of the invention will utilize the target as a substrate for the specific trans-splicing reaction that iε deεired and (6) the inεertion of the ribozyme into the deεired host cell.

Choice of a target RNA will reflect the desired purpose of the trans-splicing reaction. If the purpose of the reaction iε to inactivate a specific RNA, then such RNA must be trans-spliced at a position that deεtroyε all functional peptide domains encoded by such RNA and at a position that does not reεult in continued expression of the undesired genetic sequences. If more than one allele of the gene encoding such RNA exists, the ribozyme should preferably be designed to inactivate the target RNA at a site common to all expresεed formε. Alternatively, more than one ribozyme may be provided to the cell, each deεigned to inactivate a εpecific allelic form of the target RNA.

When only inactivation of the target RNA iε deεired, and not the expreεεion of a new, deεired RNA εequence, it iε not neceεεary that the foreign RNA donated by the ribozyme provide a εequence capable of

being translated by the host cell, and a sequence containing translational stop codons may be used as a truncated intron, for example, the intron ribozyme truncated at the Seal site. If the purpose of the trans-εplicing reaction is to provide a genetic trait to a host cell, then the choice of target RNA will reflect the desired expression pattern of the genetic trait. If it is desired that the genetic trait be continuously expressed by the host, then the target RNA should also to be continuously expressed. If it is desired that the genetic trait be εelectively expreεεed only under a deεired growth, hormonal, or environmental condition, then the target RNA should also be selectively expressed under such conditions.

It iε not neceεεary that expresεion of the ribozyme itεelf be selectively limited to a deεired growth, hormonal, or environmental condition if the substrate for such ribozyme is not otherwise present in the host as the ribozyme itself iε not translated by the host. Thus, εequenceε encoded by the RNA donated by the ribozyme of the invention are not translated in a hoεt until the trans-splicing event occurs and such event may be controlled by the expression of the ribozyme substrate in the host.

If desired, expression of the ribozyme may be engineered to occur in response to the same factors that induce expression of a regulated target, or, expression of the ribozyme may be engineered to provide an additional level of regulation so as to limit the occurrence of the trans-εplicing event to thoεe conditionε under which both the ribozyme and target are εelectively induced in the cell, but by different factorε, the combination of thoεe factors being the undesired event. Such regulation would allow the hoεt cell to expreεε the ribozyme'ε target under thoεe

conditionε in which the ribozyme itεelf waε not co- expressed.

The sequence of the ribozyme domain that hybridizes to the target RNA is determined by the εequence of the target RNA. The sequence of the target

RNA is determined after cloning sequences encoding such RNA or after sequencing a peptide encoded by such target and deducing an RNA sequence that would encode such a peptide. Cloning techniques known in the art may be used for the cloning of a sequence encoding a target RNA.

The selection of a desired sequence to be trans- spliced into the target RNA (herein termed the "trans- spliced sequence") will reflect the purpose of the trans-splicing. If a trans-splicing event is desired that does not result in the expreεεion of a new genetic εequence, then the trans-spliced sequence need not encode a translatable protein sequence. If a trans- splicing event iε deεired that doeε result in the expreεsion of a new genetic sequence, and especially a new peptide or protein εequence, then the trans-spliced sequence may further provide translational stop codons, and other information neceεεary for the correct tranεlational processing of the RNA in the host cell. If a specific protein product is desired as a result of the trans-splicing event then it would be necesεary to maintain the amino acid reading frame in the reεulting fuεion.

The identification and confirmation of the εpecificity of a ribozyme of the invention is made by testing a putative ribozyme'ε ability to catalyze the desired trans-splicing reaction only in the presence of the deεired target εequence. The trans-splicing reaction should not occur if the only RNA sequences present are non-target εequenceε to which such ribozyme should not be responsive (or less responεive) . Such

characterization may be performed with the asεiεtance of a marker such that correct (or incorrect) ribozyme activity may be more easily monitored. In most caseε it is sufficient to test the ribozyme against its intended target in vitro and then transform a host cell with it for study of its in vivo effects.

When it is desired to eliminate a host'ε RNA, such elimination should be as complete as possible. When it is desired to provide a new genetic sequence to a host cell, the trans-splicing reaction of the invention need not be complete. It is an advantage of the invention that, depending upon the biological activity of the peptide that is translated from such genetic sequence, the trans-splicing event may in fact be quite inefficient, as long aε sufficient trans-splicing occurs to provide sufficient mRNA and thus encoded polypeptide to the host for the desired purpose.

Transcription of the ribozyme of the invention in a hoεt cell occurε after introduction of the ribozyme gene into the hoεt cell. If the εtable retention of the ribozyme by the hoεt cell iε not desired, such ribozyme may be chemically or enzymatically synthesized and provided to the hoεt cell by mechanical methods, such as microinjection, lipoεome-mediated tranεfection, electroporation, or calcium phosphate precipitation.

Alternatively, when stable retention of the gene encoding the ribozyme is deεired, such retention may be achieved by stably inserting at least one DNA copy of the ribozyme into the hoεt'ε chromoεome, or by providing a DNA copy of the ribozyme on a plasmid that iε stably retained by the hoεt cell.

Preferably the ribozyme of the invention is inserted into the hoεt'ε chromoεome aε part of an expreεεion caεεette, εuch caεεette providing tranεcriptional regulatory ele entε that will control the tranεcription of the ribozyme in the host cell.

Such elementε may include, but not necesεarily be limited to, a promoter element, an enhancer or UAS element, and a transcriptional terminator εignal. Polyadenylation iε not neceεεary as the ribozyme is not translated. However, such polyadenylation signalε may be provided in connection with the sequence encoding the element to be trans-spliced.

Expression of a ribozyme whose coding sequence haε been εtably inserted into a hoεt'ε chromoεome iε controlled by the promoter εequence that iε operably linked to the ribozyme coding sequences. The promoter that directε expreεεion of the ribozyme may be any promoter functional in the hoεt cell, prokaryotic promoterε being deεired in prokaryotic cellε and eukaryotic promoterε in eukaryotic cells. A promoter is composed of discrete moduleε that direct the tranεcriptional activation and/or repression of the promoter in the host cell. Such moduleε may be mixed and matched in the ribozyme'ε promoter so as to provide for the proper expresεion of the ribozyme in the host.

A eukaryotic promoter may be any promoter functional in eukaryotic cellε, and eεpecially may be any of an RNA poly eraεe I, II or III specificity. If it is desired to expreεε the ribozyme in a wide variety of eukaryotic hoεt cells, a promoter functional in most eukaryotic host cellε should be selected, such as a rRNA or a tRNA promoter, or the promoter for a widely expreεεed mRNA εuch as the promoter for an actin gene, or a glycolytic gene. If it iε deεired to expreεs the ribozyme only in a certain cell or tisεue type, a cell- εpecific (or tiεεue-specific) promoter elementε functional only in that cell or tiεεue type should be εelected.

The trans-splicing reaction iε chemically the εa e

whether it is performed in vitro or in vivo. However, in vivo, since cofactors are usually already present in the host cell, the presence of the target and the ribozyme will suffice to result in trans-splicing. The trans-splicing ribozymes and methods of the invention are usful in producing a gene activity useful for the genetic modification, and/or cell death, of targeted cells. For example, the trans-splicing reaction of the invention is useful to introduce a protein with toxic properties into a desired cell. The susceptibility of cells will be determined by the choice of the target RNA and the regulatory controls that dictate expresεion of the ribozyme. For example, a ribozyme that tranεpoεeε an RNA sequence encoding a toxic protein may be engineered so that expression of the ribozyme will depend upon the characteristics of an operably-linked promoter. In a highly preferred embodiment, diptheria toxin peptide A iε encoded by that part of the ribozyme that iε tranεpoεed into a deεired target in the hoεt. Conditional expreεεion of the ribozyme and diphtheria toxin peptide A chain reεultε in the death of the hoεt cell. Other uεeful peptide toxinε include ricin, exotonin A, and herpeε thymidine kinase (Evans, G.A., Geneε & Dev . 3:259-263 (1989)). In addition, various lytic enzymes have the potential for disrupting cellular metabolism. For example, a fungal ribonuclease may be used to cauεe male sterility in plants (Mariani, C. et al . , Nature 347 : 121-141 (1990)). Particular tiεεueε might be deεtroyed due to limited expreεsion of the target RNA.

Further, if a viral RNA iε uεed aε target, new formε of viruε reεiεtance, or therapieε may be engineered.

A binary system for control of tiεεue-εpecific gene expreεεion and/or for ectopic ablation may be designed using the ribozymes of the invention. For example, lines of Droεophila that expresε the yeaεt

tranεcription activator GAL4 in a tiεεue and εpatial- εpecific pattern using P-element enhancer-trap vectors may be used. Any tranεcriptional activator may be used in place of GAL4 and the invention is not intended to be limited to GAL4. A gene encoding a fusion ribozyme that is capable of trans-splicing the DTA sequence may be placed under the control of the GAL4-UAS promoter and inserted into Drosophila in a genetically stable manner. Such ribozymes will not be expresεed in Drosophila in the abεence of GAL4. Accordingly, crossing Drosophila hosts genetically carrying this ribozyme construct with Drosophila hosts that expresε GAL4 in a tiεεue-εpecific manner result in progeny hat, when GAL4 expression is induced, exhibit a pattern of cell death similar to the pattern of GAL4 expression.

In addition, by targetting the ribozyme to trans- splice with the GAL4 mRNA, the splicing activity of the ribozyme inactivates GAL4 expresεion and ribozyme expreεεion may be εelf-regulated.

Pro-ribozymes

A trans-splicing ribozyme, aε deεcribed above, conεiεts of three fused sequence elementε - a 5' "anti- sense" region which is complementary to the target RNA, the catalytic region which is based on a self-εplicing Group I intron, and 3' "exon" εequenceε. The 5' region can base pair with the chosen target RNA, to bring it into proximity with the catalytic εequenceε of the Group I intron. The εtructure of the Group I intron provideε a chemical environment εuitable to catalyze the preciεe εplicing of the target RNA with the 3' "exon" sequences. However, in the absence of the appropriate target RNA, the ribozyme sequences can still catalyze εciεεion at the 3' "exon" junction

(εi ilar hydrolyεiε iε εeen for Group I εelf-εplicing

- 32 - intons (Zaug et al ., Science 231:470-475 (1986)), and may be able to catalyze illegitimate splicing events through transient base-pairing of the ribozyme with heterologous RNA εequences (which may include their own) . Such side-reactionε and illegitimate splicing eventε are unwanted, and may be deleterious. For example, if trans-splicing is to be used for conditional delivery of a toxin in vivo, illegitimate trans-splicing might result in unexpected expresεion of the toxic activity. Spontaneouε cleavage at the 3'

"exon" junction would lower the efficiency of trans- εplicing.

To help avoid these problems, "pro-ribozyme" forms of the trans-εplicing RNAε have been conεtructed wherein for example, helix P8 is disrupted. The pro- ribozymes are conεtructed to contain extra self- complementary sequenceε which cauεe the catalytic center of the ribozyme to be miε-folded. The pro- ribozy eε are inactive in the abεence of the intended target RNA; active forms are only formed after base- pairing of the ribozyme and target RNAs - with consequent displacement of the interfering secondary structure within the ribozyme. Pro-ribozymes are intended to be catalytically inert species in the abεence of the target RNA, to eliminate unwanted εelf- cleavage, self-splicing and illegitimate trans-splicing reactions in vitro and in vivo (Figure 12) .

The pro-ribozymes deεcribed here are conformation- ally diεrupted and therefore inactive formε of the trans-splicing activities. Thuε the pro-ribozymeε posεeεε little εelf-cleavage activity. They are only re-activated by εpecific interaction with the target RNA, and thuε are εubεtrate-activated ribozymes which are less likely to catalyze trans-splicing to an unintended target RNA. Trans-splicing ribozymes are intended to be used for the delivery of new gene

activitieε in vivo, and any reduction in the extent of unwanted εide reactions or illegitimate splicing is desirable, and may be necesεary.

While the disruption of helix P8 haε been exemplified here for the trans-splicing pro-ribozymes, other helices which are required for catalytic activity could also have been used.

The εame approach, of diεrupting the conformation of a catalytically important εtructure in εuch a way that only baεe-pairing with the intended εubεtrate RNA will allow the formation of an active ribozyme, could be applied to other ribozyme deεignε. For example, the loop εequence of a "hammerhead" type endoribonucleaεe (Haseloff et al . , Nature 334:585-591 (1988)) could be extended and made complementary to one of the "anti¬ sense" arms of the ribozyme - εimilar to the above modification of helix P8. Endoribonuclease activity would only be exhibited after base-pairing with the choεen target RNA, displacement of the disrupting εecondary εtructure, and reformation of the εte -loop εtructure required for catalyεiε. Thiε would effectively increaεe the εpecificity of the ribozyme of its target.

In addition, the activation of a pro-ribozyme need not rely on baεe-pairing with the εubεtrate itεelf.

Inεtead, a choεen third RNA or εεDNA or even protein might be required for activity. An additional baεe- pairing or RNA-protein interaction would be required for the formation of an active ribozyme complex. The availability of εuch additional componentε would determine ribozyme activity, and could be uεed to alter ribozyme εelectivity.

The ribozyme or pro-ribozyme of the invention may be introduced into any hoεt cell, prokaryotic or eukaryotic and eεpecially into a plant or mammalian hoεt cell, and eεpecially a human cell, either in

culture or in vivo , using techniques known in the art appropriate to such hosts. The ribozymes of the invention may also be engineered to destroy viruseε. In one embodiment, the ribozyme or pro-ribozyme of the invention is provided in a genetically εtable manner to a host cell prior to a viral attack. Infection by the appropriate virus, or expression of the latent virus in such host cell, (resulting in the appearance of the ribozyme's or pro-ribozyme target RNA in the host cell) , would stimulate the catalytic activity of the ribozyme and destruction of the viral RNA target and/or production of a- toxin via trans-splicing resulting in death of the virus infected cells. In another embodiment, the ribozyme or pro-ribozyme may be engineered and packaged into the virus itself. Such embodiments would be especially useful in the design of viruεeε for inveεtigative purpoεeε, wherein the ribozyme or pro-ribozyme may be designed to deεtroy the function of a specific viral RNA and thus allow the study of viral function in the absence of εuch RNA.

Viruεeε carrying ribozymeε may alεo be uεed aε carrierε to tranεfect hoεt cellε with a deεired ribozyme or pro- ribozyme activity.

Male or female sterility may be engineered in agronomically important species using the ribozymeε or pro-ribozymeε of the invention. For example, male sterility in tobacco may be engineered by targetting TA29 or TA13 mRNA (tobacco anther-specific genes; Seurinck, J. et al . , Nucl . Acids Res . 18:3403 (1990) with a ribozyme or pro-ribozyme of the invention that trans-spliceε the DTA 3 ^« exon into thoεe targets.

The form of crop plants may be manipulated by εelective deεtruction or modification of tiεsueε uεing the ribozymes or pro-ribozymeε of the invention. For example, εeedleεε fruits may be made by targetting the seed εtorage protein mRNA with a ribozyme or pro-

ribozyme of the invention that trans-splices the DTA 3' exon into the target.

Transgenic plantε may be protected againεt infection by expreεsion of virus-specific ribozymes or pro-ribozyme to kill infected cells. This would be an artificial form the "hypersensitive responεe." For example, cucumber moεaic virus coat protein mRNA may be targeted with a ribozyme or pro-ribozyme of the invention that trans-splices the DTA 3' exon into the target.

Populations of micro-organismε may be made reεistant to specific pathogens by introduction of trans-splicing ribozymeε or pro-ribozymeε. For example, cheeεe-making bacteria may be made resistant to phage infection by targetting the phage RNA with a bacterial toxin gene or lytic enzyme encoded by the 3' exon provided by the ribozyme or pro-ribozyme of the invention, for example, which would interfere with phage replication by causing premature lysiε after phage infection.

Viruε pathogenε could be conεtructed to deliver toxic activities via trans-εplicing. In thiε way, εpecific cell typeε could be targeted for ablation, εuch aε for cancer or viral therapy. For example, HIV mRNA may be targeted by a ribozyme or pro-ribozyme of the invention that carrieε the DTA 3' exon, for either viruε or liposome delivery.

The examples below are for illustrative purposes only and are not deemed to limit the scope of the invention.

EXAMPLES

Example 1

Construction and Characterization of a CAT-LacZ Trans-Splicing Ribozyme

I. PCR Amplification and Cloning of the Ribozyme of the Invention

Following the guidelines outlined above, a trans- splicing fusion ribozyme was deεigned that will splice a portion of the amino-terminal coding sequence of E . col i /3-galactosidase (LacZ) mRNA to a site in the chloramphenicol acetyl tranεferaεe (CAT) mRNA (Figure 3). The sections of new sequence flanking the T. thermophila ribozyme core and the 3' exon were εyntheεized as oligonucleotides. The intact ribozyme sequence waε then assembled by succeεεive polymeraεe chain reactions, using the synthetic adaptor oligonucleotides aε primerε with ribozyme and β- galactoεidaεe DNA templateε (while there are other methodε available, thiε method iε moεt convenient) .

For the conεtruction of a ribozyme capable of εplicing 3-galactosidase (LacZ) α-peptide coding εequence to a εite in the 5' coding εequence of the chloramphenicol acetyl tranεferaεe (CAT) , three oligonucleotideε were synthesized.

Oligonucleotide 1

5'-GGCCA AGCTT CTTTA CGATG CCATT GGGAT ATATC AACGG TGGTA TAAAC CCGTG GTTTT TAAAA GTTAT CAGGC ATGCA CC-3 ' [SEQ ID NO. 2]

Oligonucleotide 2

5'-GATTA GTTTT GGAGT ACTCG TACGG ATTCA CGGCC GTCGT TTTAC AA-3' [SEQ ID NO. 3]

Oligonucleotide 3

5 ^, -GGCCG AATTC TTACA ATTTC CATTC AGGCT GCGCA ACTGT TGG-

2 ' [SEQ ID NO. 4]

^• 5

Oligonucleotides 2 and 3 (200 pmoles each) were combined with 0.1 μg PvuII-cut pGEM4 DNA (which contained the LacZ α-peptide εequence) , and subjected to PCR amplification in a volume of 100 μl containing:

10

50 mM KC1,

10 mM Tris-HCl pH 8.3, 1.5 mM MgCl ₂ , 0.4 mM dNTPs, 15 0.1% gelatin, and

5 U TaqI DNA polymerase, and incubated for 30 cycles, 1 min § 94°C, 2 mins € 50 ^β C, 2 mins § 72°C. 0

Plaεmid pGEM4 iε commercially available from Promega Corporation, Madison WI, USA.

The amplified product of 210 base-pairs was 5 purified using low-gelling temperature agarose electrophoresiε, and was used as primer in a second round of PCR amplification.

Following the second round of PCR amplification, 2.0 μg of 210 base-pair amplified product, 200 pmoles 0 oligonucleotide 1 and 0.1 μg 450 base-pair fragment containing the T. thermophila IVS were mixed and subjected to PCR amplification using the conditionε shown above. The resulting 660 base-pair product was digested with the restriction endonucleases .EcoRI and 5 Hindlll, and cloned into the plasmid vector pGEM4. The complete sequence of the CAT-LacZ α-peptide ribozyme DNA sequence is preεented as SEQ ID NO. 5 and Figure 3B.

The cloning vector containing the cloned εequenceε 0 waε transformed into, and propagated in, the bacterial hoεt XLl/Blue (Strategene, La Jolla, California), using techniques known in the art (Maniatis, Molecular

Cloning, A Laboratory Guide , 2nd edition, 1989, Cold Spring Harbor Laboratory, Publishers) . However, any bacterial host capable of stably maintaining the vector may be used, for example the JM109.

The plasmid may be extracted from the host cell for further analysiε using techniques commonly known in the art (Maniatis, Molecular Cloning, A Laboratory Guide , 2nd edition, 1989, Cold Spring Harbor Laboratory, Publishers) .

II. In vitro Transcription of Cloned Ribozyme and Tarσet RNAs

Using standard procedures, cloned sequences were purified from the bacterial hoεt and the plasmid linearized using a restriction endonuclease that does not cut the ^" ribozyme sequence, (for example, EcoRI ) , and transcribed uεing T7 RNA polymeraεe in a volume of 100 μl, containing:

5 μg linearized plasmid DNA, 40 M Tris-HC pH 7.5,

6 mM MgCl ₂ , 2 mM spermidine, lOmM NaCl, lOmM DTT, lmM NTPs (containing 20 μCi [α- ³² P]UTP, if labelled RNA tranεcriptε were deεired) , 100 U RNaεin, and

50 U T7 RNA polymeraεe, and the reaction waε incubated at 37°C for 2 hours.

•RNA tranεcriptε were purified by 5% polyacrylamide gel electrophoreεiε before uεe (TBE, 7M urea gel) . RNAs containing active T. thermophila IVA sequences undergo some spontaneouε scisεion at the 3' intron-exon junction during tranεcription. Frag entε are removed by electrophoretic purification for clarity of analyεiε during εubεequent tranε-splicing aεεayε.

III. In Vitro Trans-εplicing Reaction Conditionε

Target and/or tranε-splicing ribozymeε are incubated under the following conditions: 0.1-0.5 μg RNA component (amount depends on type of experiment, uεually ribozyme in 5-fold exceεε of target) ,

30 mM Tris-HCl pH 7.5, 100 mM NaCl, 2mM GTP,

5 mM MgCl ₂ , in a volume of 5 μl at 42°C, 60 mins.

The reaction is diluted with 95 μl 0.1 mM Na ₂ EDTA,

200 mM NaCl, and ethanol precipated. The RNAs are then analyεed on 5% polyacrylamide gelε containing TBE buffer, 7M urea and 25% formamide, and autoradiographed.

IV. Aεεav of Endonucleolytic Activity

After baεe-pairing of the ribozyme and target, the firεt εtep in trans-εplicing is the guanoεine mediated cleavage of the target RNA at the intended 5' splice site. Annealing and trans-splicing may be performed in a buffer εuch as 30 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl ₂ , 2 mM GTP at 42°C. As the 3' εplice εite iε diεpenεable for thiε reaction, truncated trans-splicing ribozymes should behave as highly-specific endoribonucleases. To test this activity, shortened in vitro transcriptε of the CAT-LacZ α-peptide trans- splicing ribozyme described above (SEQ ID NO. 5 and Figure 3) were incubated with CAT mRNA sequenceε. The CAT-LacZ ribozyme caεεette iε on a Hindlll-EcoRI fragment. The S al cleavage εite markε a poεition 5 baεeε upstream of the 3' splice εite. The ribozyme εpecifically cleaved the target RNA at the expected single site to produce the expected size fragments.

V. The Trans-splicing Reaction

To confirm the ability of the CAT-LacZ α-peptide ribozyme to catalyze the ligation of 3' exon sequences at the 5' splice site, variouε formε were incubated with radiolabelled CAT RNA. Ribozyme tranεcripts were synthesized from DNA templates which had been 3' truncated at one of several positions, ranging from the end of the ribozyme core through the exon sequence. Incubation with labelled CAT led to the formation of the expected spliced products, which differed in length depending on the extent of 3* exon sequence.

In addition, a certain proportion of the CAT-LacZ αpeptide ribozyme molecules underwent spontaneous cleavage at the 3' splice site during in vitro transcription, similar to the intact T. thermophila intron. These cleaved forms, terminated at the guanosine residue adjacent the 3' splice εite, were also incubated with CAT RNA. In this case, the ribozyme itself is ligated to a 3' portion of the CAT RNA, to produce a product of about 550 nucleotides in size. This reaction is similar to the self-circularization of the intact intron, and the same ligation product is found in the other trans-splicing reactions.

VI. Accuracy of the Trans-splicing

The products from a CAT-LacZ α-peptide trans- splicing reaction were reverse-transcribed, and amplified by polymerase chain reaction using two oligonucleotides complementary to εequenceε on either εide of the predicted εplice εiteε. Amplified εequenceε were cloned and εequenced. Individual recombinantε εhowed no variation from the expected εequence of the spliced products. Aε found in studies

with the intact intron, εplicing appearε to be highly accurate.

Accordingly, the εtudies above show that a trans- splicing ribozyme designed according to the guidelineε of the invention is capable of accurate, effective trans-splicing in vitro.

Example 2

Design of a Trans-Splicing Ribozyme that Provides Plant Virus Reεiεtance

Cucumber mosaic virus (CMV) is a pandemic virus with a large number of known strains. Nine sequence strains are shown in the region of the start of their coat protein cistron encoded in RNA 3 and the subgeno ic mRNA 4 (SEQ ID NOS. 7-25; Figures 4(A) and 5) . Two sites have been chosen which are conserved in sequence and downstream from the AUG start codon of the coat protein. Oligonucleotides for the conεtruction of ribozymes capable of trans-splicing the ile-mutant form of DTA into the CMV coat protein mRNA are shown in Figure 4B and is discuεsed below.

The trans-splicing ribozymes shown in Figure 4C and D are targetted to the CMV virus sequenceε εhown in

Figure 4B and will result not only in the cleavage of the CMV RNA molecules but in the expresεion of diphtheria toxin A-chain in the infected cell. The trans-splicing cassettes shown in Figure 4 may be transformed into any CMV-susceptible plant species uεing techniqueε known in the art, and transgenic progeny challenged by CMV infection. The deεign of the ribozyme iε εuch that virus infection is necesεary to initiate toxin production via RNA trans-splicing because the ribozyme itself is not translated. The localized death of the infected cellε that reεultε from expreεεion of the toxin could limit replication and

εpread of the viruε within the plant giving an artificial hyperεenεitive reεponεe.

Example 3

Conεtruction and Characterization of a Gal4- Diphtheria Toxin A Chain Trans-Splicing Ribozvme

According to the invention and the methods described in Example l, a fusion ribozyme haε been designed that is a Gal4-Diphtheria toxin A chain trans- splicing ribozyme (Figure 6) . The sequence of this ribozyme is εhown aε SEQ ID NO. 6. The GAL4-DTA ribozyme caεεette is a Sall-Xhol fragment. The Seal εite markε a poεition 5 baεeε upεtream of the 3' splice εite. Thiε ribozyme iε capable of splicing the coding εequence for the A chain of the diphtheria toxin to a εite in the 5' region of the GAL4 mRNA. This tranε- εplicing activity is active both in vitro (as above) and in vivo (below) . The major criteria for succesεful deεign of the GAL4-DTA ribozyme, and any trans-splicing ribozyme that trans-εpliceε a sequence encoding a toxic product, are not only the efficient and precise catalysiε of trans-splicing, but also that expreεεion of the toxic product, for example, DTA doeε not occur in the abεence of trans-splicing.

The catalytic portion of the ribozyme is constructed according to the deεign outlined above, and 5' and 3' splice εiteε choεen within the 5 ¹ coding regionε of GAL4 and DTA, reεpectively. The 3' exon εequence correεpondε to that of a DTA gene already uεed for expreεεion in eukaryotes, except for the removal of the firεt AUG codon and several proximal amino acids. The original C. diphtheriae form of DTA also differs in this 5' region, utilizing a CUG codon for translation

initiation. The original DTA sequence also contains a signal peptide leader sequence which is absent.

These ribozyme molecules can undergo spontaneous sciεsion at the 3' splice site. Given the extreme toxicity of DTA, it is important that any liberated 3 ¹ exon sequenceε not give rise to toxic translation products. The 3' exon contained an in-frame methionine at position 13, which could conceivably give rise to a truncated but toxic polypeptide. To eliminate this posεibility, the wild-type εequence (Rz-DTA,,^) waε altered from methionine at this poεition to isoleucine (Rz-DTA _ile ) or leucine (Rz-DTA _leu ) in two separate ribozyme conεtructionε (Figure 6) .

Example 4

In Vivo Activity of the Ribozv eε of the Invention

I. Introduction

The in vivo activity of a ribozyme deεigned according to the guidelineε provided herein, and the ability of εuch a ribozyme to deliver new gene ac ivitieε to host cellε, waε demonstrated using the Gal4-Diphtheria toxin A chain trans-splicing ribozyme described (Example 3 and in Figure 6) to deliver the highly toxic diphtheria toxin A product to a host cell.

In thiε system, Drosophila waε the chosen host and it was desired to control expreεεion of the ribozyme of the invention in a tiεsue-εpecif c manner within the Droεophila host. Diphtheria toxin iε εecreted by CorynebacteriUJΠ diphtheriae lyεogenic for B phage. The toxin iε produced aε a single polypeptide which undergoeε proteolyεiε to produce A and B chainε. The A chain (DTA) contains a potent ADP riboεylaεe activity which is εpecific for the eukaryote translation elongation factor EF-2. The presence of even a few molecules of

thiε enzyme is enough to cause cessation of translation and eventual death in a variety of eukaryote cells. The B chain allows intracellular delivery by attachment of the toxin to cell surface receptors by binding mannose reεidues, is endocytosed and enters the cytoplasm by vesicular fusion.

In the absence of the B-chain, the A-chain is much lesε toxic when preεent extracellularly. Thiε property, and itε extreme toxicity, have εuggeεted its use for ectopic ablation experiments. For example, sequences encoding DTA have been expresεed in transgenic mice, using an opsin promoter to drive expreεεion in developing eyeε. The reεulting mice are blind, with deformed eyeε (Breitman, M.L., Science 238:1563-1565 (1987)). In other εtudies, ablation of the mouse pancreas was performed (Palmiter, R.D. et al .. Cell 50:435-443 (1987)) and Wert, S.E. et al . , Am. Rev. Reεpir. Diε . 141 fno. 4, part 2) :A695 (1990) described ablation of alveolar cells by use of a chimeric gene conεiεting of the promoter and 5' flanking sequence of the human surfactant protein C gene (expresεed in type II alveolar cellε) and the DTA gene.

However, uεing thiε type of approach, it is not possible to maintain or propagate transformed organismε which might have more εevere, or lethal phenotypes. In addition, transformation of certain specieε, εuch as Droεophila , with intact DTA sequenceε has not been reported to date. Leaky expreεεion of the DTA gene during εuch transformations leads to immediate death.

II. The Drosphila System

A general method for targeting gene expreεεion in Droεophila haε been developed. Firεt, the εyεtem allowε the rapid generation of individual εtrainε in

which ectopic gene expreεεion can be directed to different tissues or cell types: the enhancer detector technique iε utilized (O'Kane, C.J. and Gehring, W.J. , Proc. Natl . Acad . Sci . USA:9123-9127 (1987); Bellen et al . , Genes and Development 3:1288-1300 (1989); Bier et al . , Genes and Development 3:1273-1287 (1989)) to expresε a tranεcriptional activator protein in a wide variety of patterns in embryos, in larvae and in adults. Second, the method separates the activator from its target gene in distinct lines, to ensure that the individual parent lines are viable: in one line the activator protein is present but haε no target gene to activate, in the εecond line the target gene iε silent. When the two lineε are croεsed, the target gene iε turned on only in the progeny of the crosε, allowing dominant phenotypeε (including lethality) to be conveniently εtudied.

To ectopically expreεε only the gene of intereεt, a tranεcriptional activator that haε no endogenouε targetε in flieε iε required. An activator from yeaεt,

Gal4, can activate tranεcription in flieε but only from promoterε that bear Gal4 binding sites (Fischer et al . , Nature 332:853-865 (1988)). To target gene expresεion, Gal4 iε restricted to particular cells in two ways: either Gal4 tranεcription iε driven by characterized fly promoterε, or an enhancerless Gal4 gene is randomly integrated in the Droεophila genome, bringing it under the control of a diverse array of genomic enhancers. To assay transactivation by Gal4, flies that expresε Gal4 are croεεed to thoεe bearing a lacZ gene whoεe tranεcription iε driven by Gal4 binding εiteε (Fiεcher et al . , Nature 332:853-865 (1988)). β-galactosidaεe iε expreεεed only in thoεe cellε in which Gal4 iε firεt expreεεed. Tissue- and cell-specific transactivation of l acZ has been demonstrated in strainε in which Gal4

is expressed and in which a variety of patterns are established.

With this system, it iε now posεible: 1) to place Gal4 binding sites upstream of any coding sequence; 2) to activate that gene only within cellε where Gal4 is expresεed and 3) to observe the effect of this aberrant expression on development. In cases where ectopic expression is lethal, this method allows the two parent lines (one expressing Gal4, the other carrying a silent gene bearing Gal4 binding sites in its promoter) to be stably propagated. Phenotypeε can then be studied in the progeny of a croεε.

Ill. Vectorε The vectorε utilized as starting materials in these studieε include:

1) pGATB and pGATN (figure 9) : Theεe vectorε are uεed for cloning promoterε and enhancerε upεtream of a promoterleεε Gal4 gene. Vectorε were conεtructed in which either a unique

NotI or BamHI εite iε inεerted upεtream of the Gal4 coding region. Once a promoter haε been linked to the Gal4 coding sequence, the gene can be excised from the pHSREM vector backbone (Knipple and Marsella-Herrick, Nucl . Acids Res . 16:7748 (1988)) and moved into a P- element vector. The Rh2 promoter has been cloned (Mismer et al. , Genetics 120:173-180 (1988)) into thiε vector and flieε have been generated in which Gal4 iε expreεεed only in the ocelli. 2) pGa B: Thiε is a Gal4 vector for use in enhancer detection.

An enhancerlesε Gal4 gene waε εubcloned into the vector plwB (Wilεon et al . , Genes and Development 3:1301-1313 (1989)) to create pGawB. plwB was first digested with Hindlll to remove the lacZ gene and the

N-terminuε of the P-tranεpoεaεe gene. Theεe were

replaced with the entire Gal4 coding region behind the TATA box of the P-transpoεaεe gene.

3) pUAST (Figure 10) : This plasmid was uεed for cloning coding sequences downstream of the Gal UAS. A vector into which genes can be subcloned behind the Gal4 UAS (Upεtream Activation Sequence) was constructed in the P-element vector, pCaSpeR3 (C. Thu mel, Univ. of Utah Medical Center, Salt Lake City, Utah, personal communication) . Five Gal4 binding sites were inserted, followed by the hsp70 TATA box and transcriptional start, a polylinker, and the SV40 intron and polyadenylation site. Unique sites into which genes, or cDNAs, can be inserted include: EcoRI , Bgl ll , NotI , Xhol , Kpnl and Xbal .

IV. Droεophila Strains

The genetic techniques described herein used to characterize the strains of Droεophila utilized in theεe studieε are well known in the art ("Genetic Variationε of Droεophila melanogaεter, " D. Lindsley and

E.H. Grell, edε) .

The P-element tranεpoεonε are mobilized uεing the "ju pεtarter" strain that carries Δ2-3, a defective P-element on the third chromosome that expresses high levels of a constitutively active tranεpoεaεe

(Robertεon et al . , Geneticε 118:451-470 (1988)). The three εtockε currently uεed to generate and map the inεertion lines were deposited in the Drosophila Stock Center, Indiana University Department of Biology, Jordan Hall A 503, Bloomington, Indiana 47405:

1: y w; +/+; Sb P[ry ⁺ , Δ2-3]/TM6, Ubx 2: w; +/+; TM3, Sb/CxD (deposit no. 3665) 3: w; CyO/Sco; +/+ (deposit no. 3666) where the genetic characteriεtics of the three chrorooso eε are εeparated by εemicolonε. Thuε, for example, in strain 1, the firεt chromoεome (the X

chromosome) is homozygous for yellow and white ("y w") , the second chromosome iε wild-type ("+/+") , and the third chromosome carries the stubble gene ("Sb") , and the P element transpoεon rosy gene ("ry ⁺ ") and Δ2-3, while the second third chromosome carries balancer inversions ("/TM6, Ubx") .

V. Strategy for Generating Gal4 Expresεion Patternε

A. Scheme used to isolate transformants

Constructε are injected into embryoε derived from the stock;

99 y w/y w ; Δ2-3,Sb/TM6,Ubx X 66 y w/Y ; Δ2-3,Sb/TM6,Ubx FO; Establiεh εingle lineε

9 y w/y w ; Δ2-3,Sb/TM6,Ubx X 6 y w/Y; +/+ or

6 y w/Y ; Δ2-3,Sb/TM6,Ubx X 9 y w/y w; +/+

Fl Select Tw±l and fSb± progeny and eεtablish stocks 9 y w/y w ; +/TM6,Ubx X 6 y w/Y; +/+ or

6 y w/Y ; +/TM6,Ubx X 9 y w/y w; +/+

B. Schemes used to jump the enhancerlesε Gal4 insert

1. Jumps from the X-chromoεome

99 FM3/FM7,w; +/+ X _^ 66 y w/Y; Δ2-3,Sb/TM6,Ubx

99 FM7,W/ P[Gal4,W ⁺⁾ X 66 FM7/Y ; Δ2-3,Sb/+

99 FM7,w/ P[Gal4,w ⁺ ]; Δ2-3,Sb/+ X 66 y w/Y; +/+

9 FM7,w/ y w ; Δ2-3,Sb/+ X 6 y w/Y; +/+ Select [w ⁺ ] and [B] progeny and establish stocks

2. Jumps from the Δ2-3-chromoεome

99 y w/y w X 66 y w/Y ; P [Gal4 , w ⁺ ] , Δ2-3 , Sb/+

Select [w ⁺ ] and [Sb ⁺ ] progeny and eεtabliεh stocks.

C. Chromosomal εegregation

To analyze the segregation of the insertions two stocks are used: w;+/+; TM3, Sb/CxD and w; CyO/Sco;+/+.

Method To create a large number of strains that expresε

Gal4 in a cell- or tiεεue-εpecific manner enhancer detection vectorε have been built that carry different verεionε of the Gal4 gene. Two geneε, encoding either the full-length protein or a truncated protein, have been cloned into roεy (ry ⁺ ) and white (w ⁺ ) P-element vectorε (modified verεionε of plArB and plwB; Wilεon et al . , Geneε and Development 3:1301-1313 (1989)). Uεing ry ^"1, or w ⁺ aε a screen, these vectors have been mobilized by introduction of the Δ2-3 gene (Robertεon et al . , Genetics 118:461-470 (1988)). To visualize the expreεsion pattern of Gal4, the Gal4 inεertion lineε are croεsed to a strain that carries the lacZ gene under the control of the Gal4 UAS (Fiεcher et al . Nature 332:853-865 1988). Embryoε, larvae and adultε derived from theεe crosses are screened for β- galactoεidaεe expreεsion either by an enzyme assay, with X-gal as a εubεtrate, or by staining with monoclonal antibodies against 3-galactoεidaεe. β- galactoεidaεe encoded by the UAS-J acZ construct is localized in the cytoplasm.

Approximately 500 Gal4-insertion strainε have been εcreened and many that can be used to activate geneε in εpecific tiεεueε have, been identified εuch aε, for example, epidermal εtripeε, meεoderm, the central nervous εyεtem and the peripheral nervous εyεte . Many of the lineε expreεε jS-galactoεidaεe in the salivary

glandε aε well aε in other tiεεueε. It is posεible that in conεtructing the enhancerleεε-Gal4 tranεpoεon a poεition-dependent salivary gland enhancer was fortuitously generated.

VI. Sample Screen

To activate a gene (Gene X) in a specific pattern, a Gal4 insertion line is selected and croεεed to a εtrain that carrieε Gene X cloned behind the GAL UAS.

VII. Summary of the GAL4/UAS Syεte without the Ribozyme

The Gal4/UAS εyεtem iε a two-part εyεtem for controlling gene activatiion. The method iε versatile, can be tisεue-specific and does not appear to exhibit a basal level of expreεsion except perhaps, aε deεcribed herein, for a UAS-DTA conεtruct. It can be uεed to ectopically expreεε characterized geneε, to expreεε modified genes that would otherwise be lethal to the organism and to expresε geneε from other species to study their effect on Droεophila development. Since the method makes it poεεible to produce dominant, gain- of-function mutationε, epiεtaεiε teεtε and εcreenε for enhancerε or εuppreεεorε of visible or lethal phenotypeε can be carried out. The Gal4 εyεtem alεo allowε the expreεεion of toxic productε to εtudy the conεequenceε of cell- and tisεue-εpecific ablation.

VIII. Use of Gal4-Exoreεεing Droεophila with the DTA Ribozyme of the Invention

Expresεion of the fusion ribozyme carrying the sequences encoding the DTA protein was placed under the control of a the GAL4 UAS (upstream activator sequence) in pUAST (Figure 10). As stated supra , using modified P-element enhancer-trap vectors described above, a large number of stable lines of Drosophila were constructed which each express the yeast transcriptional activator GAL4 in specific spatial and temporal patterns in the developing flies. Any gene under the control of the GAL4 upstream activator sequence (UAS) can be transformed and maintained singly, then induced in particular Droεophila tisεueε by genetic croεεing to lineε which expreεε GAL4 (Figure 7) . However, it waε not poεεible to take advantage of the Gal4 εyεtem for expreεεion of DTA per se without further modification, due to the difficulty in producing UAS-DTA transfor antε through leaky expreεεion of the DTA.

It waε found that uεe of thiε two-element syεtem aε a meanε of conditionally expreεεing DTA via a tranε- εplicing ribozyme (Figure 6) overcame theεe problemε.

In those cells expresεing GAL , the GAL4 protein provideε the activity neceεεary for ribozyme tranεcription, and the GAL4 mRNA provideε the target for trans-εplicing neceεεary for DTA production. Droεophila embryos may be injected with ribozyme sequenceε placed under the control of a UAS promoter aε described above, using techniques known in the art. Embryoε injected with the Rz-DTA _met conεtruction will not εurvive, whereas normal tranεfor ed flieε were obtained from embryoε injected with both Rz-DTA _lle and

Rz-DTA _leu . Thiε reεult εuggeεted that the internal AUG codon waε indeed acting as an initiation codon for the

tranεlation of a toxic product after injection. The codon iε adjacent to propoεed NAD+ binding εite in the DTA εequence, and to εequenceε conεerved in the diεtantly related exotoxin A, another EF-2 εpecific ADP-ribosylase from Pεeudomonaε aeruginosa .

Transgenic flies containing the Rz-DTA _ile and Rz- DTA _leu sequences under control of the GAL4 UAS were crossed to flies producing GAL4 in particular patterns of expression. For example, in one characterized line, line 1J3, the GAL4 gene was been inserted near the hairy gene, and mirrored its pattern of expresεion. The hairy gene product iε produced in epidermal εtripeε in the even-numbered abdominal εegmentε during e bryogeneεiε. When a UAS-driven LacZ gene waε introduced into 1J3 in which GAL4 iε expreεεed in the same pattern as the hairy gene product, S-galactosidaεe waε found localized within the even-numbered εtripeε. When flieε containing the Rz-DTA _leu gene were croεεed to thiε GAL4-expreεεing line, normal progeny reεulted. However, when flieε containing Rz-DTA _ile were croεεed to the GAL4-expreεεing line, development of the progeny waε arrested in e bryogenesis. Darker colored bandε were evident on the cuticleε of the embryoε, conεiεtent with the death of underlying cellε. When cuticle preparationε were examined, the even-numbered denticle bandε were diεrupted or miεεing, particularly thoεe of the 4th, 6th and 8th εtripeε (Figure 11) . Other specific patterns of cell death were observed when the containing Rz-DTA _ile flieε are croεεed to different GAL4 expreεεing geneε.

Example 5 Design of Pro-ribozv eε

As a test for the design of pro-ribozymes, the

CAT-LacZ trans-splicing ribozyme which described earlier was modified (Figure 2) . Phylogenetic comparisons and mutational analysis (for review, see Cech, Ann Rev. Biochem . 59:543-568 (1990)) have indicated that a core region of the group I self- splicing introns is highly conserved and important for activity (Figure 8) . For the construction of trans- splicing pro-ribozymeε a helix immediately adjacent to this region, P8, was disrupted. In the first experiments, 13 or 18 nucleotides of new sequence were introduced into the 5' strand and loop of helix P8, to produce pro-ribozyme 1 and 2 , respectively. The extra nucleotides were complementary to the 5' "anti-sense" portion of the ribozyme, while the flanking sequences were adjusted to conserve (l) the actual sequences at the base of P8, and (2) the extent of base-pairing possible within P8 (Figure 13) . The extent of self- complementarity between the sequenceε inserted into helix P8 and the 5' "anti-senεe" region of the pro- ribozyme iε εuch that thiε new helix would be expected to form in nascent transcriptε, in preference to helix P8. The formation of thiε alternative helix would also be expected to disrupt flanking secondary and perhaps tertiary interactions within the catalytic core of the ribozyme. Thus, mis-folding of the pro-ribozyme would render it catalytically inactive (Figure 14). However, base-pairing of the pro-ribozyme with the intended target RNA would displace the P8-"anti-senεe" baεe- pairing, sequester the "anti-senεe" εequences and allow re-formation of the P8 helix and an active catalytic

domain. Diεplacement of the P8-"anti-εenεe" helix resultε in a greater sum of base-pairs and allows proper folding of the catalytic domain, so should be energetically favored.

CAT-LacZ pro-ribozymes

Cloned sequences corresponding to the two CAT-LacZ pro-ribozymes were conεtructed using PCR-mutagenesiε as discussed above, and RNAs were produced by in vitro transcription. The CAT-LacZ trans-splicing ribozyme waε obεerved to undergo scisεion during transcription at the 3' εplice junction, aε a reεult of hydrolysiε catalyzed by the intron εequenceε. Similar hydrolysiε iε εeen in in vitro tranεcriptε of the unmodified

Tetrahymena thermophila intron. In contraεt, tranεcriptε of the different CAT-LacZ pro-ribozymeε are more εtable, with little cleavage evident under the εa e conditionε (Figure 15) . Thiε indicateε that the pro-ribozymes are inactive, which would be expected if the catalytic sequenceε were miε-folded. Truncated formε of the pro-ribozymeε were teεted for specific endoribonuclease activity directed against the CAT RNA. CAT-LacZ pro-ribozyme RNAs were tranεcribed from templateε truncated at the Seal εite, to remove the 3' εplice junction and LacZ εequenceε. Both ribozyme and pro-ribozyme RNAε are εtable after removal of the 3' splice εite. Incubation of the truncated pro-ribozymeε with CAT RNA led to εpecific cleavage of the target RNA to give fragments of the expected εizeε (Figure 16) .

Specific cleavage activity waε εeen at 37, 45 and 50 degreeε.

Pro-ribozyme forms of the GAL4-DTA trans-splicing ribozyme were also conεtructed (Figure 17) . Regionε of 20 nucleotides (complementary to the "anti-senεe" region) were inεerted into the 5' εtrand and loop of

helix P8. The two pro-ribozymeε differed in the extent of baεe-pairing poεεible in the modified heliceε P8, and GAL4-DTA pro-ribozyme 1 poεεeεεing both a longer stem and fewer (3) acceεεible bases in the loop. The helix P8 of GAL4-DTA pro-ribozyme 2 more closely rese bleε that of the CAT-LacZ pro-ribozyme 2, with a larger loop (14 baεeε) containing sequenceε complementary to the "anti-sense" region. Transcripts of the GAL4-DTA pro-ribozymes are more stable than those of the unmodified ribozyme. In particular, pro- ribozyme 2 iε mainly intact after incubation in conditionε that reεult in eεsentially complete self- cleavage of the ribozyme form (30'§ 50°C, 10 mM MgCl ₂ , 2 mM GTP, see Figure 18) .

Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditionε, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Haseloff, James

Goodman, Howard M. Brand, Andrea Perrimon, Norbert

(ii) TITLE OF INVENTION: Cell Ablation Using Trans-Splicing Ribozymes

(iii) NUMBER OF SEQUENCES: 56

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox

(B) STREET: 1225 Connecticut Avenue, N.W., Suite 300

(C) CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20036

(v) 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 #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT

(B) FILING DATE: herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/642,330

(B) FILING DATE: 17-JAN-1991

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Goldstein, Jorge A

(B) REGISTRATION NUMBER: 29,021

(C) REFERENCE/DOCKET NUMBER: 0609.3496604

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (202)833-7533

(B) TELEFAX: (202)833-8716

(2) INFORMATION FOR SEQ ID NO :1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 517 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : both

(D) TOPOLOGY : linear (ii ) MOLECULAR TYPE : DNA

(xi ) SEQUENCE DESCRIPTION : SEQ ID N0 : 1 :

TGACGCAATT CAACCAAGCG CGGGTAAACG GCGGGAGTAA CTATGACTCT 50

CTAAATAGCA ATATTTACCT TTGGAGGGAA AAGTTATCAG GCATGCACCT 100

CCTAGCTAGT CTTTAAACCA ATAGATTGCA TCGGTTTAAA AGGCAAGACC 150

GTCAAATTGC GGGAAAGGGG TCAACAGCCG TTCAGTACCA AGTCTCAGGG 200

GAAACTTTGA CATGGCCTTG CAAAGGGTAT GGTAATAAGC TGACGGACAT 250

GGTCCTAACC ACGCAGCCAA GTCCTAAGTC AACAGATCTT CTGTTGATAT 300

GGATGCAGTT CACAGACTAA ATGTCGGTCG GGGAAGATGT ATTCTTCTCA 350

TAAGATATAG TCGGACCTCT CCTTAATGGG AGGTAGCGGA TGAATGGATG 400

CAACACTGGA GCCGCTGGGA ACTAATTTGT ATGCGAAAGT ATATTGATTA 450

GTTTTGGAGT ACTCGTAAGG TAGCCAAATG CCTCGTCATC TAATTAGTGA 500

CGCGCATGAA TGGATTA 517

(2) INFORMATION FOR SEQ ID N0: 2 :

( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 82 bases

(B) TYPE : nucleic acid

( C) STRANDEDNESS : both

(D) TOPOLOGY : linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

GGCCAAGCTT CTTTACGATG CCATTGGGAT ATATCAACCG TGGTATAAAC 50

CCGTGGTTTT TAAAAGTTAT CAGGCATGCA CC 82

(2) INFORMATION FOR SEQ IQ N0:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

GATTAGTTTT GGAGTACTCG TACGGATTCA CGGCCGTCGT TTTACAA 47

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

GGCCGAATTC TTACAATTTC CATTCAGGCT GCGCAACTGT TGG 43

(2) INFORMATION FOR SEQ ID N0:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 623 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

GGGAGACCGG AAGCTTCTTT ACGATGCCAT TGGGATATAT CAACGGTGGT 50

ATAAAGCCGT GGTTTTTAAA AGTTATCAGG CATGCACCTG GTAGCTAGTC 100

TTTAAACCAA TAGATTGCAT CGGTTTAAAA GGCAAGACCG TCAAATTGCG 150

GGAAAGGGGT CAACAGCCGT TCAGTACCAA GTCTCAGGGG AAACTTTGAG 200

ATGGCCTTGC AAAGGGTATG GTAATAAGCT GACGGACATG GTCCTAACCA 250

CGCAGCCAAG TCCTAAGTCA ACAGATCTTC TGTTGATATG GATGCAGTTC 300

ACAGACTAAA TGTCGGTCGG GGAAGATGTA TTCTTCTCAT AAGATATAGT 350

CGGACCTCTC CTTAATGGGA GCTAGCGGAT 6AAGTGATGC AACACTGGAG 400

CCGCTGGGAA CTAATTTGTA TGCGAAAGTA TATTGATTAG TTTTGGAGTA 450

CTCGTACGGA TTCACTGGCC GTCGTTTTAC AACGTCGTGA CTGGGAAAAC 500

CCTGGCGTTA CCCAACTTAA TCGCCTTGCA GCACATCCCC CTTTCGCCAG 550

CTGGCGTAAT AGCGAAGAGG CCCGCACCGA TCGCCCTTCC CAACAGTTGC 600

GCAGCCTGAA TGGAAATTGT AAG 623

(2) INFORMATION FOR SEQ ID NO: 6 :

( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 1038 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : both

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

GTCGACCTTT TTAAGTCGGC AAATATCGCA TGTTTGTTCG ATAGACATCG AGTGGCTTCA 60

AAAGTTATCA GGCATGCACC TGGTAGCTAG TCTTTAAACC AATAGATTGC ATCGGTTTAA 120

AAGGCAAGAC CGTCAAATTG CGGGAAAGGG GTCAACAGCC GTTCAGTACC AAGTCTCAGG 180

GGAAACTTTG AGATGGCCTT GCAAAGGGTA TGGTAATAAG CTGACGGACA TGGTCCTAAC 240

CACGCAGCCA AGTCCTAAGT CAACAGATCT TCTGTTGATA TGGATGCAGT TCACAGACTA 300

AATGTCGGTC GGGGAAGATG TATTCTTCTC ATAAGATATA GTCGGACCTC TCCTTAATGG 360

GAGCTAGCGG ATGAAGTGAT GCAACACTGG AGCCGCTGGG AACTAATTTG TATGCGAAAG 420

TATATTGATT AGTTTTGGAG TACTCGTCTC GATGATGTTG TTGATTCTTC TAAATCTTTT 480

GTGATTGAAA ACTTTTCTTC GTACCACGGG ACTAAACCTG GTTATGTAGA TTCCATTCAA 540

AAAGGTATAC AAAAGCCAAA ATCTGGTACA CAAGGAAATT ATGACGATGA TTGGAAAGGG 600

TTTTATAGTA CCGACAATAA ATACGACGCT GCGGGATACT CTGTAGATAA TGAAAACCCG 660

CTCTCTGGAA AAGCTGGAGG CGTGGTCAAA GTGACGTATC CAGGACTGAC GAAGGTTCTC 720

GCACTAAAAG TGGATAATGC CGAAACTATT AAGAAAGAGT TAGGTTTAAG TCTCACTGAA 780

CCGTTGATGG AGCAAGTCGG AACGGAAGAG TTTATCAAAA GGTTCGGTGA TGGTGCTTCG 840

CGTGTAGTGC TCAGCCTTCC CTTCGCTGAG GGGAGTTCTA GCGTTGAATA TATTAATAAC 900

TGGGAACAGG CGAAAGCGTT AAGCGTAGAA CTTQAGATTA ATTTTGAAAC CCGTGGAAAA 960

C6TGGCCAAG ATGCGATGTA TGAGTATATG GCTCAAGCCT GTGCAGGAAA TCGTGTCAGG 1020

CGATCTTTGT GACTCGAG 1038

(2) INFORMATION FOR SEQ ID N0:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 134 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

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

GTTTAGTTGT TCACCTGAGT CGTGTGTTTT GTATTTTGCG TCTTAGTGTG 50

CCTATGGACA AATCTGGATC TCCCAATGCT AGTAGAACCT CCCGGCGTCG 100

TCGCCCGCGT AGAGGTTCTC GGTCCGCTTC TGGT 134

(2) INFORMATION FOR SEQ ID N0:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 134 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

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

GTTTAGTTGT TCACCTGAGT CGTGTTTTCT TTGTTTTGCG TCTCAGTGTG 50

CCTATGGACA AATCTGGATC TCCCAATGCT AGTAGAACCT CCCGGCGTCG 100

TCGCCCGCGT AGAGGTTCTC GGTCCGCTTC TGGT 134

(2) INFORMATION FOR SEQ ID N0:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 152 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

GTTATTGTCT ACTGACTATA TAGAGAGTGT TTGTGCTGTG TTTTCTCTTT 50

TGTGTCGTAG AATTGAGTCG AGTCATGGAC AAATCTGAAT CAACCAGTGC 100

TGGTCGTAAC CGTCGACGTC GTCCGCGTCG TGGTTCCCGC TCCGCCCCCT 150

CC 152

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 152 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GTTATTGTCT ACTGACTATA TAGAGAGTGT GTGTGCTGTG TTTTCTCTTT 50

TGTGTCGTAG AATTGAGTCG AGTCATGGAT AAATCTGAAT CAACCAGTGC 100

TGGTCGTAAC CGTCGACGTC GTCCGCGTCG TGGTTCCCGC TCCGCCTCCT 150

CC 152

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 131 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

AGAGAGTGTG TGTGCTGTGT TTTCTCTTTT GTGTCGTAGA ATTGAGTCGA 50

GTCATGGACA AATCTGAATC AACCAGTGCT GGTCGTAACC GTCGACGTCG 100

TCCGCGTCGT GGTTCCCGCT CCGCCCCCTC C 131

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 154 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GTTATTGTCT ACTGATTGTA TAAAGAGTGT GTGTGTGCTG TGTTTTCTCT 50

TTTACGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 100

GCTGGTCGCA ACCGTCGACG TCGTCCGCGT CGTGGTTCCC GCTCCGCCCC 150

CTCC 154

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 154 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

GTTATTGTCT ACTGACTATA TAGAGAGTGT GTGTGTGCTG TGTTTTCTCT 50

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 100

GCTGGTCGTA ACCGTCGACG TCGTTTGCGT CGTGGTTCCC GCTCCGCCTC 150

CTCC 154

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 130 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

GAGTGTGTAT GTGCTGTGTT TTCTCTTTTG TGTCGTAGAA TTGAGTCGAG 50

TCATGGACAA ATCTGAATCA ACCAGTGCTG GTCGTAACCG TCGACGTCGT 100

CCGCGTCGTG GTTCCCGCTC CGCCCCCTCC 130

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 152 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

GTTATTGTCT ACTGACTATA TAGAGAGTGT GTGTGCTGTG TTTTCTCTTT 50

TGTGTCGTAG AATTGAGTCG AGTCATGGAC AAATCTGAAT CAACCAGTGC 100

TGGTCGTAAC CATCGACGTC GTCCGCGTCG TGGTTCCCGC TCCGCCCCCT 150

CC 152

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

GGAGGGGGCG GAGCGGGAAC CACGACGCGG ACGACGTCGA CGGTTACGAC 50 CAGCCCTGGT AGATTCAGAT TTGTCCAT 78

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

TTTGCGTCTT AGTGTGCCTA TGGACAAATC TGGATCTCCC AATGCTAGT 49

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:

TTTGCGTCTC AGTGTGCCTA TGGACAAATC TGGATCTCCC AATGCTAGT 49

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID N0:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ATAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID N0:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

( ii) MOLECULAR TYPE : nucleic acid

(xi ) SEQUENCE DESCRIPTION : SEQ ID 0: 21 :

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID N0:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:

TTTACGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID N0:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: . 56 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 bases

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULAR TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

TTTGTGTCGT AGAATTGAGT CGAGTCATGG ACAAATCTGA ATCAACCAGT 50

GCTGGT 56

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

AATTTTGTGT CGTAGAATTG AGTCGAGTCA TGGACAAATC TGAATCAACC 50

AGTGCTGCA 59

(2) INFORMATION FOR SEQ ID N0:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

GCACTGGTTG ATTCAGATTT GTCCATGACT CGACTCAATT CTACGACACA 50

A 51

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

AATTTTGTGT CGTAGAATTG AGTCGAGTCA TGGACAAATC TGAATCAACC 50

AGTGCTGCA 59

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

AGCATTGGTA TCATCAGGTT TGT 23

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:

GTTGATGATG TTGTTGATTC T 21

(2) INFORMATION FOR SEQ ID N0:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: Amino Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:

MET ASP LYS PHE ASP ASP VAL VAL ASP SER 5 10

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

ATGGACAAAT TTGATGATGT TGTTGATTCT 30

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

AATTTTGTGT CGTAGAATTG AGTCGAGTCA TGGACAAATC TGAATCAACC 50

AGTGCTGCA 59

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:

AGCCATCCTT GGTTCAG 17

(2) INFORMATION FOR SEQ ID N0:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:

GTAAGGGTGG ATGTT 15

(2) INFORMATION FOR SEQ ID N0:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: Amino Acid (D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

MET ASP LYS SER GLU LEU ARG VAL ASP VAL 1 5 10

(2) INFORMATION FOR SEQ ID N0:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:

ATGGACAAAT CTGAATTAAG GGTGGATGTT 30

(2) INFORMATION FOR SEQ ID N0:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:

TCTCGATGAT GTTGTTGATT CTTCTAAATC TTTTGTGATG GAAAACTTTT 50

CTTCGTACCA CGGGACTAAA 70

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: Amino Acid (D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

MET GLU ASN PHE SER SER TYR HIS GLY THR LYS 1 5 10

(2) INFORMATION FOR SEQ ID N0:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

TCTCGATGAT GTTGTTGATT CTTCTAAATC TTTTGTGATT GAAAACTTTT 50

CTTCGTACCA CGGGACTAAA 70

(2) INFORMATION FOR SEQ ID N0:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41 :

TCTCGATGAT GTTGTTGATT CTTCTAAATC TTTTGTGTTG GAAAACTTTT 50

CTTCGTACCA CGGGACTAAA 70

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

ATGAAGCTTC TCGATGATGT TGTTGATTCT TCTAAATCTT TTGTGATGGA 50

AAACTTTTCT TCGTACCACG GGACTAAA 78

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: Amino Acid (D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:

MET LYS LEU LEU ASP ASP VAL VAL ASP SER SER LYS SER PHE VAL 1 5 10 15

MET GLU ASN PHE SER SER TYR HIS GLY THR LYS 20 25

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

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

ATGGAGAAAA AAATCACTGG ATATACCACC GTTGATATAT C 41

(2) INFORMATION FOR SEQ ID N0:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: Amino Acid (D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:

MET GLU LYS LYS ILE THR ASP SER LEU ALA VAL VAL LEU GLN ARG 1 5 10 15

ARG ASP 17

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:

ATGGAGAAAA AAATTACGGA TTCACTGGCC GTCGTTTTAC AACGTCGTGA 50

51

(2) INFORMATION FOR SEQ ID N0:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:

ATGAAGCTAC TGTCTTCTAT CGAACAAGCA TGCGATATTT 40

(2) INFORMATION FOR SEQ ID N0:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: Amino Acid (D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:

MET LYS LEU LEU ASP ASP VAL VAL ASP SER SER LYS SER PHE VAL 1 5 10 15

MET GLU ASN PHE SER 20

(2) INFORMATION FOR SEQ ID N0:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 bases

(B) TYPE: Nucleic Acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: Linear

(ii) MOLECULAR TYPE: Nucleic Acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:

ATGAAGCTTC TCGATGATGT TGTTGATTCT TCTAAATCTT TTGTGATGGA 50

AAACTTTTCT 60

(2) INFORMATION FOR SEQ ID N0:50:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 72 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:

AUGGAGAAAA AAAUCACUGG AUAUACCACC GUUGAUAUAU CCCAAUGGCA UCGUAAAGAA 60

CAUUUUGAGG CA 72

(2) INFORMATION FOR SEQ ID N0:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 479 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:

AAGCUUCUUU ACGAUGCCAU UGGGAUAUAU CAACGGUGGU AUAAAGCCGU GGUUUUUAAA 60

AGUUAUCAGG CAUGCACCUG GUAGCUAGUC UUUAAACCAA UAGAUUGCAU CGGUUUAAAA 120

GGCAAGACCG UCAAAUUGCG GGAAAGGGGU CAACAGCCGU UCAGUACCAA GUCUCAGGGG 180

AAACUUUGAG AUGGCCUUGC AAAGGGUAUG GUAAUAAGCU GACGGACAUG GUCCUAACCA 240

CGCAGCCAAG UCCUAAGUCA ACAGAUCUUC UGUUGAUAUG GAUGCAGUAC AGACUAAAUG 300

UCGGUCGGGG AAGAUGUAUU CUUCUCAUAA CAUAUAGUCG GACCUCUCCU UAAUGGGAGC 360

UAGCGGAUGA AGUGAUGCAA CACUGGAGCC GCUGGGAACU AAUUUGUAUG CGAAAGUAUA 420

UUGAUUAGUU UUGGAGUACU CGUACGGAUU CACUGGCCGU CCUGUUACAA CGUCGUGAC 479

(2) INFORMATION FOR SEQ ID N0:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 479 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:

AAGCUUCUUU ACGAUGCCAU UGGGAUAUAU CAACGGUGGU AUAAAGCCGU GGUUUUUAAA 60

AGUUAUCAGG CAUGCACCUG GUAGCUAGUC UUUAAACCAA UAGAUUGCAU CGGUUUAAAA 120

GGCAAGACCG UCAAAUUGCG GGAAAGGGGU CAACAGCCGU UCAGUACCAA GUCUCAGGGG 180

AAACUUUGAG AUGGCCUUGC AAAGGGUAUG GUAAUAAGCU GACGGACAUG GUCCUAACCA 240

CGCAGCCAAG UCCUAAGUCA ACAGAUCUUC UGUUGAUAUG GAUGCAGUAC AGACUAAAUG 300

UCGGUCGGGG AAGAUGUAUU CUUCUCAUAA CAUAUAGUCG GACCUCUCCU UAAUGGGAGC 360

UAGCGGAUGA AGUGAUGCAA CACUGGAGCC GCUGGGAACU AAUUUGUAUG CGAAAGUAUA 420

UUGAUUAGUU UUGGAGUACU CGUACGGAUU CACUGGCCGU CCUGUUACAA CGUCGUGAC 479

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 480 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: RNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

AAGCUUCUUU ACGAUGCCAU UGGGAUAUAU CAACGGUGGU AUAAAGCCGU GGUUUUUAAA 60

AGUUAUCAGG CAUGCACCUG GUAGCUAGUC UUUAAACCAA UAGAUUGCAU CGGUUUAAAA 120

GGCAAGACCG UCAAAUUGCG GGAAAGGGGU CAACAGCCGU UCAGUACCAA GUCUCAGGGG 180

AAACUUUGAG AUGGCCUUGC AAAGGGUAUG GUAAUAAGCU GACGGACAUG GUCCUAACCA 240

CGCAGCCAAG UCCUAAGUCA ACAGAUCUUC UGUUGAUAUG GAUGCAGUAC AGACUAAAUG 300

UCGGUCGGGA CCGUUGAUAU AUGGUUCAUA ACAUAUAGUC GGACCUCUCC UUAAUGGGAG 360

CUAGCGGAUG AAGUGAUGCA ACACUGGAGC CGCUGGGAAC UAAUUUGUAU GCGAAAGUAU 420

AUUGAUUAGU UUUGGAGUAC UCGUACGGAU UCACUGGCCG UCCUGUUACA ACGUCGUGAC 480

(2) INFORMATION FOR SEQ ID NO: 54 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 487 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

AAGCUUCUUU ACGAUGCCAU UGGGAUAUAU CAACGGUGGU AUAAAGCCGU GGUUUUUAAA 60

AGUUAUCAGG CAUGCACCUG GUAGCUAGUC UUUAAACCAA UAGAUUGCAU CGGUUUAAAA 120

GGCAAGACCG UCAAAUUGCG GGAAAGGGGU CAACAGCCGU UCAGUACCAA GUCUCAGGGG 180

AAACUUUGAG AUGGCCUUGC AAAGGGUAUG GUAAUAAGCU GACGGACAUG GUCCUAACCA 240

CGCAGCCAAG UCCUAAGUCA ACAGAUCUUC UGUUGAUAUG GAUGCAGUAC AGACUAAAUG 300

UCGGUCGGGA CCGUUGAUAU AUCCCAAACG GUUCAUAACA UAUAGUCGGA CCUCUCCUUA 360

AUGGGAGCUA GCGGAU6AAG UGAUGCAACA CUGGAGCCGC UGGGAACUAA UUUGUAUGCG 420

AAAGUAUAUU GAUUAGUUUU GGAGUACUCG UACGGAUUCA CUGGCCGUCC UGUUACAACG 480

UCGUGAC 487

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1044 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 55 :

GTCGACCTTT TTAAGTCGGC AAATATCGCA TGTTTGTTCG ATAGACATCG AGTGGCTTCA 60

AAAGTTATCA GGCATGCACC TGGTAGCTAG TCTTTAAACC AATAGATTGC ATCGGTTTAA 120

AAGGCAAGAC CGTCAAATTG CGGGAAAGGG GTCAACAGCC GTTCAGTACC AAGTCTCAGG 180

GGAAACTTTG AGATGGCCTT GCAAAGGGTA TGGTAATAAG CTGACGGACA TGGTCCTAAC 240

CACGCAGCCA AGTCCTAAGT CAACAGATCT TCTGTTGATA TGGATGCAGT TCACAGACTA 300

AATGTCGGTC GGGGAACAAC ATGCGATATT GTTCTCATAA GATATAGTCG GACCTCTCCT 360

TAATGGGAGC TAGCGGATGA AGTGATGCAA CACTGGAGCC GCTGGGAACT AATTTGTATG 420

CGAAAGTATA TTGATTAGTT TTGGAGTACT CGTCTCGATG ATGTTGTTGA TTCTTCTAAA 480

TCTTTTGTGA TTGAAAACTT TTCTTCGTAC CACGGGACTA AACCTGGTTA TGTAGATTCC 540

ATTCAAAAAG GTATACAAAA GCCAAAATCT GGTACACAAG GAAATTATGA CGATGATTGG 600

AAAGGGTTTT ATAGTACCGA CAATAAATAC GACGCTGCGG GATACTCTGT AGATAATGAA 660

AACCCGCTCT CTGGAAAAGC TGGAGGCGTG GTCAAAGTGA CGTATCCAGG ACTGACGAAG 720

GTTCTCGCAC TAAAAGTGGA TAATGCCGAA ACTATTAAGA AAGAGTTAGG TTTAAGTCTC 780

ACTGAACCGT TGATGGAGCA AGTCGGAACG GAAGAGTTTA TCAAAAGGTT CGGTGATGGT 840

GCTTCGCGTG TAGTGCTCAG CCTTCCCTTC GCTGAGGGGA GTTCTAGCGT TGAATATATT 900

AATAACTGGG AACAGGCGAA AGCGTTAAGC GTAGAACTTG AGATTAATTT TGAAACCCGT 960

GGAAAACGTG GCCAAGATGC GATGTATGAG TATATGGCTC AAGCCTGTGC AGGAAATCGT 1020

GTCAGGCGAT CTTTGTGACT CGAG 1044

( 2 ) INFORMATION FOR SEQ ID NO : 56 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 1047 base pairs

(B) TYPE : nucleic acid

(C ) STRANDEDNESS : both

( D) TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:

GTCGACCTTT TTAAGTCGGC AAATATCGCA TGTTTGTTCG ATAGACATCG AGTGGCTTCA 60

AAAGTTATCA GGCATGCACC TGGTAGCTAG TCTTTAAACC AATAGATTGC ATCGGTTTAA 120

AAGGCAAGAC CGTCAAATTG CGGGAAAGGG GTCAACAGCC GTTCAGTACC AAGTCTCAGG 180

GGAAACTTTG AGATGGCCTT GCAAAGGGTA TGGTAATAAG CTGACGGACA TGGTCCTAAC 240

CACGCAGCCA AGTCCTAAGT CAACAGATCT TCTGTTGATA TGGATGCAGT TCACAGACTA 300

AATGTCGGTC GGGCAAACAT GCGATATTTG CCGTTTGTCA TAAGATATAG TCGGACCTCT 360

CCTTAATGGG AGCTAGCGGA TGAAGTGATG CAACACTGGA GCCGCTGGGA ACTAATTTGT 420

ATGCGAAAGT ATATTGATTA GTTTTGGAGT ACTCGTCTCG ATGATGTTGT TGATTCTTCT 480

AAATCTTTTG TGATTGAAAA CTTTTCTTCG TACCACGGGA CTAAACCTGG TTATGTAGAT 540

TCCATTCAAA AAGGTATACA AAAGCCAAAA TCTGGTACAC AAGGAAATTA TGACGATGAT 600

TGGAAAGGGT TTTATAGTAC CGACAATAAA TACGACGCTG CG6GATACTC TGTAGATAAT 660

GAAAACCCGC TCTCTGGAAA AGCTGGAGGC GTGGTCAAAG TGACGTATCC AGGACTGACG 720

AAGGTTCTCG CACTAAAAGT GGATAATGCC GAAACTATTA AGAAAGAGTT AGGTTTAAGT 780

CTCACTGAAC CGTTGATGGA GCAAGTCGGA ACGGAAGAGT TTATCAAAAG GTTCGGTGAT 840

GGTGCTTCGC GTGTAGTGCT CAGCCTTCCC TTCGCTGAGG GGAGTTCTAG CGTTGAATAT 900

ATTAATAACT GGGAACAGGC GAAAGCGTTA AGCGTAGAAC TTGAGATTAA TTTTGAAACC 960

CGTGGAAAAC GTGGCCAAGA TGCGATGTAT GAGTATATGG CTCAAGCCTG TGCAGGAAAT 1020

CGTGTCAGGC GATCTTTGTG ACTCGAG 1047