MXPA98002771A - Proteins involved in the direction of the peptidilo transfer center, therapeutic agents and methods corresponding - Google Patents

Abstract

The present invention relates to the degradation of mRNA is an important point of control of gene expression and has been shown to bind the translation process, a clear example of this binding is the observation that nonsense mutations accelerate the degradation of MRNA, this report demonstrates that a subset of mof alleles (framework maintenance) in yeast, which is isolated as chromosomal mutations that increase the efficiency of frame change at the site of the LA virus frame change and caused the loss of LA satellite of the M1 virus, also affects the mRNA degradation pathway mediated by nonsense mutations, the mRNA levels containing nonsense mutations rose in the cells that reach the mof4-1 alleles, in addition mof4-1 is allelic UPF1, which has been shown to be involved in the degradation pathway of mRNA mediated by nonsense mutations, although the cells that reach the mof4-1 allele lose the M1 virus more alleles f (for example upf1, upf2, upf3) involved in the maintenance of mRNA degradation mediated by nonsense mutations maintains M1, the alleles ifs1 and ifs2 previously identified as mutations that improve the frame change in the frame change signal Ribosomal 1 from mutations originating in the mouse mammary tumor virus was shown to be allelic to the UPF2 and UPF1 genes, respectively, and both ifs strains maintained M1; the mof4-1 strain is more sensitive to paromomycin aminoglycoside a strain upf1g (D) and increases the efficiency of frame change in a strain mof4-a that grows in the presence of paromomycin, these results indicate that the upf1-p has a dual function in translation and rotation of AR

Description

• r , # PROTEINS INVOLVED IN THE DIRECTION OF PEPTIDILO TRANSFER CENTERS, THERAPEUTIC AGENTS AND CORRESPONDING METHODS FIELD OF THE INVENTION The present invention relates to the identification __j2D of proteins that are involved in the frame shift ^ of mRNA and on the degradation path of nonsense mRNA » As well as the RNAs encoding recombinant genes with increased stability in vivo. The identification of these proteins and these stabilized RNAs provides in vitro analysis systems to identify agents that affect the functional activity of mRNAs by altering the frequency of displacement of frame. These agents will be antiviral drugs and useful antimicrobials. The analysis systems herein additionally provide for the identification of agents that affect the stability of RNA. These agents will be useful - x * to treat diseases associated with nonsent mutations. 2C also provide opposite-sense RNAs 'stablished, and peptides encoding stabilized mRNA' for use in two-hybrid ligand analysis systems.

BACKGROUND OF THE INVENTION INVERSION OF RNA AND THE DISPLACEMENT OF RIBOSOMICAL FRAMEWORK Many studies have indicated that the procedures investment and translation of mRNA are linked directly CPeltz co-authors »Proq. Nucí Acid Res. & Mol. Biol. »47» 271-298 (1994) 3. A clear example of the relationship between the inversion of translational mRNA is the observation that the nonsense mutations in a gene can decrease the stability of transcripts that do not contain CPeltz meaning and coauthors »Prog. Nuci. Acid Res. & Mol. Biol. »47» 271-298 (1994) 3. The yeast Saccharomyces cerevisiae has been used to identify and characterize the transaction factors involved in the path of decomposition of mRNA »5 mediated by the lack of sense. Mutations in the UPF1 »UPF2 and UPF3 genes raise the concentration of mRNAs that do not make sense in cells» by increasing their half-lives without affecting the breakdown of most wild-type transcripts CLeeds and co-authors »Genes S Dev. »5: 2303-2314 0 (1991) 3» Leeds and co-authors Mol. Cel 1. Biol. »12» 2165-2177 (1992); Peltz and co-authors »Genes & Dev. «7: 1737-1754 (1993); Do and co-authors »Mol. Cell. Biol. 15. "809-823 (1995): Cui and coauthors, Genes &Dev., 9" 437-454 (1995), He and coauthors, Genes &Dev., 9, 437-454 (1995) 3. Cu and co-authors, Genes &Dev Dev., Pp. 437-454 (1995), He and coauthors »Genes &Dev.» 9 »437-454 (1995) 3. Upflp not only works on trajectory 9 of decomposition of mRNA mediated by nonsense, but also has separable functions involved in the modulation of nonsense and in the suppression of frame shift "which suggests that these procedures are directly linked to the fact that they can share similar components. of the translational apparatus that is able to stabilize nonsense transcripts and suppress J n nonsense and frame shift mutations »mechanisms have been developed that regulate the expression of genes. inducing elongation of ribosomes to displace the reading frame in response to ribosomal frame shift signals »CChandler-specific and co-authors» Mol. Microbiol .. 7 »497-503 (1993); Farabaugh and coauthors. J. Biol. Bhe. 270: 103S1-103S4 (1995); Hayashi and co-authors. Biochem.

J ^. 306: 1-10 (1995) 3. Frame shift events produce fusion proteins in which the N-terminal and C-terminal domains are encoded in two distinct overlapping reading frames. The displacement of the ribosomal frame is different from the displacement suppression of the framework since these events are directed to sequences and specific structures of mRNA »instead of being a consequence of mutations in gene host products.

THE DISPLACEMENT OF VIRAL FRAMEWORK Most of the examples of ribosomal frame shift in viruses have been identified, all of which use their RNA strands (+) as: 1) multiple protein products »that encode mRNA; 2) the species of RNA that is packed in nascent viral particles and 3) the template for reproduction of the viral genetic material. The production of multiple protein products could be achieved by splicing or editing the mRNA. However, these mechanisms impose the consequence of producing altered (+) NA filaments, which results in the production of mutant viral genomes, unless an RNA site necessary for packaging or reproduction is removed by splicing or editing. of genomic RNA. Perhaps for that reason it is not known that the (+) ssRNA and dsRNA viruses use splicing or mRNA editing; and retroviruses remove their packing site (PSD when splicing their CMann RNAs and co-authors) Cel 1 33: 153-159 (1983), Antanabe and Temin »Proc. Nati. Acad. Se. USA 79: 5986-5990 (1982) 3. By using the ribosomal frame shift and / or the reading trough of the termination codons to form fusion proteins, the RNA (+) filament templates are not altered and "in such a way" the production and the packaging of the viral genomes CIcho and Wickner »J. Biol. Chem., 264: 6716-6723, (1989) 3. For example» the killer virus system in the yeast consists of the LA and M RNA double viruses filament »and used ribosomal frame-shifted -1 for the expression of the appropriate gene CWickner» RB »J. Biol. Chem., 268: 3797-3899 (1993) 3. The 5 dsARN AI virus has two open reading frames The 5 'gag encodes the Gag protein and the 3' pol gene encodes a multifunctional protein domain required for viral RNA packaging.

J & CWickner, R.B.J. Biol. Chem., 26B: 3797-3800 (1993) 3. A ribosomal frame shift event -1 is responsible of the production of the Gag-Pol fusion protein. M ,. »a satellite dsARN virus of L-A encodes a secreted killer toxin CWickner. R. B. »J. Biol. Chem.» 268: 3797-3800 (1993) 3 »is encapsulated and reproduced using the gene products synthesized by the L-A virus. The previous results have shown that mutations that alter the efficiency of ribosomal frame shift 1 of L-A virus change the ratio of Gag to Gag-Pol synthesized and cause the loss of satellite virus ^ CDinman and co-authors »J. Virol. , 66: 3669-3676 (1992). Dimitans and coauthors »Genetics» 136: 75-86 (1994) 3.

THE FRAMEWORK DISPLACEMENT MECHANISMS The displacement of ribosomal frame in the direction -1 in retroviruses. in viruses (+) ssRNA and erx viruses dsARN. requires a special sequence »X XXY YYZ (the frame 0 is indicated by the spaces) called "slippery site" Jacks and Varmus »Science 230: 1237-1242 (1985) 3. The simultaneous slippage of site A and P »tRNAs bound to the ribosome, at a base in the 5 'direction, still leaves their non-wobbly bases paired correctly in the new reading frame. A second promoter element Jacks and coauthors »Cel 1 55: 447-458 (1988) 3, usually pseudonymous RNA, is localized medically 3 'to the site of A ^ Sliding CBrown and Geiduschek »J. Biol. Chem., 262: 13953-13958 (1987); Dinman and co-authors, Proc. Nati Acad. Sci. USA, 88: 174-178 (1991); TenDam and coauthors Biochemistry »31: 11665- 11676 (1992) 3. The pseudonym of mRNA makes the ribosome pause over the slip site "and is believed to increase the likelihood of ribosomal movement in 5T (Somogyi et al., Mol. Cel. Biol., 13: 6931-6940 (1993); Your and coauthors, Proc. Nati Acad. Sci. USA, 89: 8636-8640 (1992) 3. The efficiency of the ribosomal frame shift -1 can be affected by the ability of the tRNAs bound to the ribosome to disappear from the frame O »the ability of those tRNAs to re-pair in the frame -1, the position Relative of the RNA pseudoknots from the slip site and its thermodynamic stability CBrierly and coauthors »J. Mol. Biol. , 227: 463-479 (1992); Brierl and coauthors, J. Mo1. Bio1. 227: 463-479 (1991); Bro and coauthors, (19B7), supra; Dinman and coauthors (1991) supra; Dinman and Wickner, J: Virology »Gß 36G9-3676 (1992); Jacks and coauthors (1988) supra; Morikawa and Bishop. rology 186: 389- * 397 (1992) 3. In vitro mutagenesis, directed to the site, has been used to examine the slip site and pseudonymous mRNA. The pseudonymous structure of mRNA is required for efficient efficient ribosomal frame shifting "5 in yeast L-A virus CDinman and co-authors" (1991), supra; Dinman and coauthors »(1992), supra. A screen for mutations that increased the _ ^ g? efficiencies in ribosomal frame shift -1, programmed, in cells, identified nine chromosomal mutants that were named mof (by Ma ntenance Of Frame (maintenance of frame) CDinman and coauthors »J. Virol., 66: 3669-3676 (1992), Dinman and coauthors, Genetics, 136: 75-86 (1994) 3. The screen originally used to identify the mof mutants used a construct in which the lacZ gene was inserted downstream of the ribosomal frame shift signal L-1 and in the reading frame -1 with respect to an initial translation site. The analysis for mof mutants was based on the identification of cells with higher beta-galactosidase activities. as a consequence of the increased efficiency in ribosomal frame shift -1. The mRNA reporter used on this screen has a region encoding protein »5 'of the short frame shift site (approximately 100 nt)» followed by approximately 3.1 kb of lacZ mRNA »which is out of frame with the open 5T reading frame. Thus, it is conceivable that the translation apparatus can see the B * transcript reported as a mRNA containing aberrant nonsense. Seen in this way »the activity of beta-gal actsidase observed in the mof strains can be the result of mutations that stabilize the transcripts that contain nonsense. Thus, the experiments presented here investigate the phenotypes of mRNA-mediated degradation of nonsense mutants and relate this phenotype to the ability to maintain the killer phenotype. The citation of any reference in the present No. 0 should be considered as the admission that said reference is available as "prior art" for the present solitude.

BRIEF DESCRIPTION OF THE INVENTION In its first aspect, the present invention relates to the identification of genes and proteins encoded by them, which are involved in the mRNA frame shift, and can also play a role in the path of 0 degradation. of mnn sinsent do. In a "mode" exemplified in the examples that are incorporated herein, the allele mof4-1 has been identified as UPF1 of Saccharomyces cerevisiae and it has been found that the UPF1 protein complements the 5 m or 4-1 mutation. Thus, UPF1 is identified as a protein associated with the mRNA frame shift. The mof4-l * mutation is associated with an increase in ribosomal frame-shifting efficiency -1 of the viral mRNA. In another modality "mof2-l has been identified as an allele of the gene suil" that encodes the SUI1 protein (Castilho-Valavicius and co-authors »1990» Genetics 124: 124: 4B3-495 »Yoon and Donahue» 1992, Mol. Cell. Biol. 12: 248-260). This protein has a human counterpart (Fields and Adams, 1994, Biochem, Biophys, Research Commun., 198: 288-291, Kasperaitis and co-authors, 1995, FEBS Let., 365: 47-50). It has been discovered that The human homolog of SUI1 can complement the mutation in mof2-l of S. cerevisiae, thereby confirming its role for both yeast and human proteins in the ribosomal frame shift of the viral mRNA. In another embodiment, the present invention relates to the identification of mof5-l as an allele of the PRP19 gene (also known as CDC40) (Jones and co-authors, 1995 »Proc. Nati, Acad. Sci. USA, Vaisman and co-authors» Mol. Gen. Genetics 247: 123- 136; Vijayraghavan and co-authors, 1989 »Genes &Dev.» 3: 1206-1216). 20 The identification of these genes gives the selection and testing of the agents that affect the efficiency of the ribosomal frame shift. In a specific modality »the agents that interfere with the ATPase activity. helicase activity or motif configuration zinc index »can be selected for testing. These agents can be useful drugs to treat IO viral infections »since many retroviruses» mainly HIV »coronaviruses and other RNA viruses are associated with medical and veterinary pathologies. By providing the identity of the proteins that modulate the framework displacement events, an initial screen for agents can include agglutination analysis of these proteins. The ability of a binding agent to affect the efficiency of frame travel can be measured in vitro »po? example, using a killer analysis such as the one described in the example included here (Dinman and Wickner »1922» J. Virol., 66: 3669-3676). This analysis can be used to test the effectiveness of the agents on the activity of the proteins associated with the frame displacement, coming from human source as well as from yeasts or other non-human source, including, but not limited to, those of animals. Agents that increase or decrease the efficiency of frame shift alter the ratio of Gag to Gag-pol proteins, expressed by viral genes. An increase of this proportion out of a narrow scale does not coincide with the process of assembling the viral particles, because too much Gag-pol is available. A decrease in this ratio results in the suppression of viral reproduction, because the level of expression of pol is too much. low. In any case, the final result is an interference in the production of viral particles. In a specific * modality, antibiotics that increase or decrease the frame shift are effective against HIV. The present invention additionally relates to the identification of genes associated with the path of degradation of nonsense mRNA. In a specific modality "it is found that UPFl is involved in the mRNA degradation procedure" and that the UPF! found in mof4-, stabilizes the mRNA nonsense. Thus, the present invention is directed to a discriminatory analysis for the identification of agents that affect the function of the proteins involved in the path of decomposition of the mRNA of sinsent do. Such agents can be tested for their ability to stabilize nonsense or short mRNA transcripts. The The identification of a protein involved in this trajectory allows the rational selection of such agents. Many analyzes are known in the art for nonsense-mediated decomposition (Zhang and co-authors »1995» Mol. Cell. Biol. »15: 2231-2244; Hagan and co-authors» 1995, Mol. Cel 1. Biol., 15: 809 - 20 823; Peltz and coauthors, 1994, in Progress in Nucleic Acid Research and Molecular Biology 47: 271-298). In a specific modality "agents that interfere with ATPase activity" can be selected for testing for helicase activity or the zinc finger motif configuration. Another discovery of the present invention is that recombinant genes for mRNA expression »particularly * for frame shift analysis» can produce current arti results because the site of the reporter transcript 5 * of the frame shift site can being too short and »in such a way» is recognized as an aberrant transcript by the degradation machinery. In that case, the increased activity of the gene at the site of frame shift may be the result of mutations that stabilize the transcripts that contain the nonsense in * place of those that increase the displacement efficiency of O frame. The present invention has advantageously identified this problem for the first time and "in such a manner" provides strategies to overcome the rapid degradation of short transcripts that are recognized by the translation apparatus as a mRNA that contains aberrant nonsense. 5 This last result has critical implications for many different technologies. For example, the identification of agents that inhibit the path of decomposition or stabilize nonsense transcripts can be critical to the success of RNA technology in the opposite or opposite direction. Opposite-sense RNAs are small, diffuse, non-transiated, and highly structured transcripts that pair with specific target RNAs, in regions of complementarity, thereby controlling the function or expression of the target RNA. However, the attempts to apply opposite-direction RNA technology have had limited success. The limiting factor seems to be to obtain sufficient concentrations of the opposite-sense RNA in a cell to inhibit or reduce the expression of the target gene. It is likely that an impediment to obtaining sufficient concentration is the path of decomposition nonsense »since short opposite 'RNA transcripts', which are not intended to encode a gene product, will probably lead to the rapid termination of the ^ É-translation "in case it occurs and» consequently, due to the rapid degradation and low abundance of sense RNA opposite in the cell. Thus, the agents of the invention that stabilize the aberrant mRNA transcripts can also stabilize the opposite sense RNAs. The ability to stabilize nonsense mRNA has important implications for treating diseases associated with nonsense mutations, such as thalassemia. As with any biological system »there will be a small amount of deletion of a nonsense mutation» which results in the expression of a full-length protein (which may or may not include substitution or omission of 2? amino acids). In the natural state, such low amounts of full-length protein are produced that a pathology results. However, by stabilizing the mRNA of nonsense, it dramatically increases the likelihood of "full reading" transcripts and may allow the sufficient expression of the protein to overcome the pathological phenotype.

The ability to stabilize short RNA transcripts will also help develop two-hybrid systems to genetically identify interacting proteins. For example. it is possible to analyze various peptides that interact with protein destinations "if the mRNAs encoding said peptides can be stabilized to allow translation. The RNAs encoding small peptide libraries will be unstable and will be degraded by the degradation path of mRNA mediated by nonsense. in the absence of any agent that stabilizes them. Alternatively, strains that inactivate the path of decomposition and stabilize the aberrant RNA can be useful discrimination systems for two-hybrid systems, since the natural tendency of such strains is to stabilize the mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE: The "integrated model" of ribosomal frame shift -1. (A) illustrates how the ribosomal frame shift -1 is related to the translational elongation stages. (B) illustrates how the ribosomal frame displacement +1 mediated by Tyl is related to the translation elongation. FIGURE 2: Characterization of the mRNA abundance of mRNAs containing nonsense in the mof strains. A) The abundance of mRNA for the CYH2 precursor and mRNA of CYH2 was determined by RNA staining analysis of the RNAs of strains harboring the MQF- * (WT), UPFl - * - (WT), upfl t and the eight different alleles of mof. RNA staining containing 20 μg of RNA per channel was hybridized with radiolabeled CYH2 probe. A schematic representation of the CYH2 precursor and its detached products is shown below the autogram. B) We determined the abundance of the mRNA for the wild type PGKl transcript and the mini-PGKl mRNA that O contains the nonsense »by RNA stain analysis of the RNAs of strains that host the HQF * (WT), mof4- 1 and upf -2. The RNA stain was hybridized with a radiolabeled DNA fragment of the PGK1 gene. The schematic representations of the mini-PGKl allele containing the nonsense and the wild-type PKG1 5 gene are shown to the right of the autogramm. OR The mof4-l strain was transformed with centromere plasmids containing just the vector that hosts either the UPF1 gene or the UPF2 gene and the mof4-1 diploid cells crossed with an upfl / strain. The abundance of 0 mRNA of the CYH2 precursor was determined by RNA spot analysis as described in (A). FIGURE 3: The killer phenotype and the maintenance of the co-segregated M with mof4-1. The tetrada analysis was carried out from a cross (JD830 crossing) between a strain mof4-1 5 (JD474-3D) that does not maintain the killer phenotype (K-) nor the double-filament RNA j_ (M _) with a strain of type s lvestre * (JD742-2D; M + K +). Both parental strains contained the framework displacement construction of the integrated LacZ gene osomically (1 eu2-l:: pJD85; CDinman and Wickner »Genetics 136: 73-86 (1994) 3). The spores of each tetrad were analyzed for their beta-galactosidase activity »their killer phenotype and their ability to propagate the M double-stranded RNA» as described in "Materials and Methods". Beta-galactosidase activity (Y axis) for each series of * tetradas (X axis) is shown as well as the capacity of each one of the spores to maintain the killer phenotype Kfl + / K-) or the A double-stranded RNA (1. ^ ^ '). FIGURE 4: Identification and characterization of the lesion in the mof4-1 allele. Hybrid genes between the wild type UPF1 and the mo-1 alleles represented schematically in panel A were constructed »transformed to an upfl / \ strain and the abundance of the CYH2 precursor was determined by RNA staining analysis» as described in figure 1. An auto-radiogram of this analysis is shown in panel B. The rectangle in the Panel A represents the sequences of the wild-type gene UPF1, while the rectangle that is shaded represents sequences of the mof4-1 allele. The cysteine-rich region of the UPF1 gene is represented by the gray rectangle in the wild-type UPF1 gene. The dark vertical line represents the location of the mutation within the mof4-1 allele. The sequence of the mof4-l allele was determined and the change in sequence is shown. For each hybrid allele shown in panel A, two identical constructs were prepared from different PCR reactions, and designated with subscript 1 or 2 in panel B. The restriction endonucleases depicted in panel A are: EKEcorRI ), Bst (BstXI), Asp (Asp718) »BKBa HI). FIGURE 5: A) Cloning strategy to clone roof2-1. B) Sequence analysis to identify the mutation in the mo allele 2-1. FIGURE 6: Cloning strategy to clone mof5- 0 1 ^. FIGURE 7: Neither mof2- nor suil- repress the expression of GCN4. Sógenas strains l / \ »transformed with pSUIl» pmof2-l »psuil-1 or PHUIS0SUI1, with the following plasmids in ormadores: pGCN4 = contains the PCN4 upstream of the controlling region» plus the first 10 amino codons of the structural gene GCN4 »fused with the lacZ gene. pORFl only contains the first upstream ORF (uORF) of GCN4 and the AUGs of the other uORF have been omitted. In pFG-lacZ »uORFl is fused in frame with the GCN4-lacZ fusion. The 0 cells were developed overnight in selective media, diluted to the logarithm-medium phase and developed for two more hours. The cultures were divided and 3-aroinotriazole was added »by de-repressing GCN4 expression. After six hours the beta-galactosidase activities were determined. The Relationships of Beta-galactosidase Activities from Depressed to Suppressed Cells are shown. There are no significant differences in these relationships between wild type s cells and mof2-1 cells, their 1-1 or HUIS0SUI1. FIGURE 8: Selective medium (H-trp) containing the indicated concentrations of anisomycin (A »C) or sparso icine (B. D) was inoculated to a D.0.β of 0.200 JD88 cells harboring the pT125 plasmids ( frame control O) »pF8 (ribosomal frame displacement tester derived from -1 LA) Jiménez and coauthors. Biochem. Biophys. Acta. 383: 427 (1975) 3 or pJD104 (ribosomal frame displacement tester derived from +1 Tyl) CBalasundara and co-authors. Proc. Nati Acad. Sci. USA 91: 172 (1994) 3, and incubated at 30 ° C for 5 hours "after which the activity of galactosidase (-gal) was determined. (A. B) The activities of _-gal produced from pT125 »as a percentage of the controls without drug. ((C, D) The multiples of change in the ribosomal efficiencies -1 or +1, in comparison with the controls without drug (-1 = l.B9_ »+1 = 5.5J4) FIGURE 9: 1906 cells (MATa leu2 mak8-2 K ~ MKT-) harboring pJD136.0 or pJD136.-l (LEU2 CEN vectors in which the HindIII fragments of pT125 or pFB were cloned which contain ribosomal framework shift fragments of frame control 0 or -1) were inoculated to an ODßBO of 0.2 in H-Leu medium containing the indicated concentrations of anisomycin or sparsomine, and incubated at 30 ° C for 5 hours, after which the activities of _-Galactosidase (_-gal). (A, B) As described above for Figure 8A and BB. (C, D) The ordinate illustrates the actual efficiencies of ribosomal frame shift -1. described in figure S, JDBB cells were used in the H-trp medium containing the indicated concentrations of drugs and the Ribosomal frame displacement sequences -1 after 5 hours of incubation at 30 ° C. - # FIGURE 11. JD88 cells were cultured in rich medium that contained the indicated concentrations of anisomycin (A) or of sparsomici (B). Aliquots of the cells were removed after 24, 48, 72, 96 or 120 hours, washed twice with sterile water, dried on rich media and grown in individual colonies at 30 ° X. These were extended as a replica on indicator plates and marked in terms of their killer phenotypes. The loss of killer phenotype was measured by dividing the number of colonies without killer (K-) by the total number of colonies. Each data series corresponds to a total of more than 100 colonies. 20 FIGURE 12. (A) A single colony without killer (K-) of each drug concentration was collected in the 72-hour data series, randomly, and the total nucleic acid (TAN) CDinman and Wickner were extracted » Viology 66: 3669 (1992) 3. TAN amounts of approximately equal to through a non-denaturing TAE-agarose gel and stained with ethidium bromide. The * 4.6 kb bands of L-A and L-BC and the 1.8 kb bands of M ^ dsARN are indicated. (B) The RNA was denatured in the gel shown in (A) »it was transferred to ni-rocellulose and hybridized with filament RNA probes L-A and M. (+)» Labeled with C32P3CTP. as described in CDinman 5 and Wickner. Genetics 136: 75 (1994) 3. FIGURE 13: Ribosomal displacement -1 in vitro.

A luciferase-based system and trainer was constructed with ^^ base in pT7-LUC minus 3tUTR-Aßo CGallie and co-authors »Mol. Gen.

Genet .. 288: 258 (1991) 3 Cal that is referenced here as pLUCO). The construction of the ribosomal frame shift test plasmid -1 (pJD120, referred to herein as pLUC-1) contains (ds 5 'to 3'). an AUG start codon »a ribosomal frame shift signal L-A -1 (from pFB). followed by the luciferase cDNA that is in the reading frame -1 with respect to the start codon. The transcripts of polyadenylated mRNA were made, crowned with meth T'pppG. using pLUGO and pLUC-1. li eal zados with Dra I, T7-RNA-polymerase and an in vitro transcription kit mMessage mMachine (A bion). The extracts of yeast competent for translation »from yeast strain JD696 (MAT_ura3-52 CL-A-O M- or L-BC-? 3) which was described in Cllizuka and co-authors, Mol. Ce11. Biol. , 14: 7322 (1994) 3. 20 ng of LUCO or LUC-1 mRNA was incubated and the indicated concentrations of anisomycin or sparsomycin were incubated. 24 ° C for one hour »with 15 μl of the yeast extracts, in triplicate, and the luciferase activities of each sample were determined using a luminometer (Turner Designs Model /20). (A, B) Luciferase activities generated using the LUCO reporter mRNA as a percentage of the controls without drug. (C, D) Efficiency of frame displacement -ribosomal which was calculated by dividing the luciferase activity of the LUC-1 reporter mRNA between that generated from the LUCO control mRNA. _ ^? FIGURE 14.- The LUCO and LUC-1 mRNA were used to ^ measure the effects of anisomycin and sparsomycin on the translation and the displacement of ribosomal framework -1 in a transliteration system of rabbit reticulocytes in vitro (Retic Lysated IVT equipment »Ambion). As in Figure 13, 20 ng of the LUCO or LUC-1 mRNA was used for these analyzes. FIGURE 15: Anisomycin and sparsomycin HIV degrees decrease. Virus producing cells (HIVgpt # 69) were incubated in DMEM containing 10% fetal calf serum "with the indicated concentrations of anisomycin or sparsomycin, and incubated at 37 ° C. After 4 days, the supernatants containing virus were harvested. made a series of dilutions and incubated the virus reporter cells (HELA T4) with 0.3 ml of diluted supernatant solutions for three hours, after which they were aspirated and replaced with 3 ml of DMEM / 105. of fetal calf serum. Each dilution was tested in duplicate.

After 24 hours the growth medium was replaced with DMEM / 1054 fetal calf serum containing 7 μg / ml gpt to select against uninfected cells. Subsequently, half was charged every three days. After 14 days »the colonies were counted for each dilution and the number of colony forming units was determined» multiplying the dilution factor by 3.33. (A) HIV titers of human cells treated with anisomycin (blank squares) or esparsomine (blank diamonds). (B) Effects of anisomycin (blank bars) and of sparsomycin (black bars) on HIV titers "as a percentage of infected cells" not treated. FIGURE 16: (A) Effects of anisomycin on the ribosomal frame shift -1. (B) Effects of sparsomycin on the ribosomal frame shift -1. The wild-type mutant mof cells were treated with the indicated amounts of each drug and the relative ribosomal shift was evaluated and normalized to cells without drug treatment. FIGURE 17: Effects of drugs at a concentration of 6 μg / ml on the suppression of nonsense in UPF + and UPF- strains. (A) Development of the strain. (B) Markers.

DETAILED DESCRIPTION OF THE INVENTION The displacement of ribosomal framework is a critical aspect of gene expression in many retroviruses and RNA viruses. Furthermore, the degradation of mRNA is an important control point in the regulation of gene expression and has been shown to be linked to the translation process. A clear example of this link is the observation that nonsense mutations accelerate the degradation of mRNAs. The present invention represents the first identification of proteins that mediate any of these activities. Understanding the way in which ribosomes maintain the translational reading frame is a great challenge for the fields of translational control and virology. In the last ten years it has been shown that many eukaryotic viruses induce ribosomes to shift the reading frame in order to regulate the expression of gene products that have enzymatic functions. Historically. Many of the definitive experiments in molecular biology and virology amphos have exploited amphionic cell systems / genetically malleable viruses. The present invention is based »in part. in studies on the displacement of ribosomal framework in one of these systems. the L-A virus of the yeast Saccharomyces cerevisiae. These studies have shown that: 1) viral mRNA sequences and secondary structures can modulate the efficiency of the ribosomal frame shift -1 »2) the efficiency of the ribosomal frame shift -1 is critical for the propagation of the satellite virus? from L-A »and 3) mutants can be obtained from the chromosomal gene products of the host yeast» involved in the maintenance of the translational reading frame CMaintenance p_f translational reading Frame (mof) 3, many of which have interesting secondary phenotypes. Figure 1 shows a model that integrates the translational elongation cycle, describing the current model the ribosomal displacement -1. Examination of this integrated model reveals that, since the ribosomal frame shift -1 occurs during the translational lengthening »f when both ribosomal sites A and P are occupied, this event must occur after the aminoacyl-tRNA delivery by EF-lalfa to site A, and before EF-2 mediates the translocation; Peptide ligation formation and peptidyl transfer occur within these parameters. Excess expression of a fragment of the yeast ribosomal L3 protein, which is involved in the formation of the center of pepti di 1-transferasa CFried and Warner, Proc. Nati Acad. Sci. USA 78: 238 (1981), Schultz and Friesen, J. Bacteriol.

- ^ W- 155: 8 (1983); Schultze and Nierhaus, EMBO J. 5: 609 (1982) 3 increases the efficiency of the ribosomal frame shift -1 and promotes the loss of MA in the wild-type cells.

Thus, the pharmacological agents that affect the peptidi 1-transferase center affect the efficiency of ribosomal frame shift -1 and interfere with viral propagation. Figure IB shows that, in contrast, since the ribosomal frame displacement +1, directed by Tyl requires the sliding of a specific P site tRNA »this procedure should not be performed by this class of drugs. Rather, one could predict the conditions that would serve to change the translation parameters during the time when only the P site is occupied by the l-tRNA peptide. would affect the efficiency of ribosomal displacement +1. Current models describing the ribosomal frame shift -1 and +1 have been integrated within the context of the translational elongation cycle. The resulting "integrated model" has tremendous predictive value with respect to the identification of the agents that can change the efficiencies of the ribosomal frame shift in any direction that "in turn" is predicted to oscillate the capacity of the cells to propagate a wide range of viruses that are based on ribosomal framework displacement strategies to ensure correct morphogenesis. Human pathogens would include most of the retroviruses "including HIV" as well as numerous viral pathogens dsRNA and filament ssRNA (+). This strategy also has applications with respect to viral diseases of veterinary and agricultural importance. The model predicts that agents that change the translation parameters "after the insertion and selection of aa-tRNA by EF-1" through the peptidyl transfer step "and before the EF2-mediated translocation" should alter the efficiency of the ribosomal frame shift - 1. Therefore, although this has been proven using only two antibiotics that specifically "determine" the function of peptide l-transferase of eukaryotic ribosomes, the model predicts that other antibiotics that act in the same step (for example, the sesqui terpene antibiotics of the trichodermin group) should give results if they are. In particular »the present invention refers» in part »to the discovery that a subset of mof alleles in the yeast (Dinman and Wickner» 1994 »Genetics 136: 75-86) were isolated as chromosomal mutations that increased the efficiency of the displacement of frame at the site of displacement and frame of the virus L-A »and caused loss of the virus Ma satellite of L-A» also affected the path of decomposition of mRNA mediated by nonsense. The mRNA levels containing nonsense were elevated in cells harboring the mof4-l allele and to a lesser extent. in cells containing the mof2-1 alleles. mof5-1 and mof8-1. The invention further relates to the discovery that mof4-1 is allelic with respect to UPF1, which has been shown to be involved in the decomposition path of mRNA mediated by nonsense. Additionally "it has been found that mof2-l is allelic to SUI1" having a human homologue "and it has been found that mof5-l is allelic to PRP17 / CDC40. The cloning of mof4-lestá described in the example that follows. Example 2 presents the cloning of mof2-1 and identifies the mutation of this allele; Figure 6 presents the cloning of mof5-1. A genetic and biochemical study of the UPF1 gene was carried out in order to understand the functioning mechanism of Upflp in the degradation path of mRNA mediated by nonsense. Our analysis suggests that Upflp is a multifunctional protein with "separable activities" that can affect the change of mRNA and the suppression of nonsense. Mutations have been identified in conserved Upflp helicase motifs that inactivate its mRNA decomposition function "while not allowing deletion of the nonsense alleles 1eu2-2 and tyr7-1. In particular »a mutation located at the ATP binding and the hydrolysis motif of Upflp that changed aspartic acid and glutamic acid to alanine residues (DE572AA)» lacked ATPase and helicase activity and formed an Upflp: RNA complex in the absence of ATP. However »surprisingly» the Upflp: RNA complex dissociated as a consequence of ATP binding. This result suggests that an ATP binding »independently of its hydrolysis, can modulate the formation of the Upflp: RNA complex for this mutant protein. Furthermore, mutations in the "cysteine / histidine rich" amino terminal region of Upflp have been identified, and they have been chemically characterized as having normal activities of mRNA degradation mediated by nonsense but they are capable of suppressing alleles. nonsense leu2-2 and ryr7-l. The biochemical characterization of these mutant proteins showed that they fear altered RNA binding properties. Additional entity. using the two-hybrid system »Upflp-Upf2p was characterized and the interactions of Upf2p-Upf3p were demonstrated. Mutations in the cysteine / histidine rich region of Upflp abolish the Upflp-Upf2p interaction. Based on these results, the role of the Upf complex in the degradation of mRNA mediated by nonsense and the suppression of nonsense seems to be mediated by separate domains in the protein. This has obvious implications for the determination or labeling of drugs, since one or the other domains can be determined for the development of drugs, for example, through the use of combinatorial banking techniques or rational drug design techniques. In another aspect "a clone has been obtained as described here" which shows that one of said mutants »mof2-1» is urx single allele of the yeast SUI1 gene. Although mutants suil-1 have somewhat elevated efficiencies of ribosomal frame shift -1 »mof2-l is unique in that it is the only known allele of SUI1 that promotes the loss of satellite virus ^ dsRNA. Additionally »the ribosomal frame shift -1 is specifically affected by these mutants» since they have no effects on the ribosomal displacement in the +1 direction. The mof2-l mutation also affects the degradation path of mRNA mediated by nonsense. In this aspect »the present application shows that the mutation of mof2-1 has a mRNA degradation phenotype mediated by nonsense, intermediate» since it requires a minimum of two elements downstream »in order to activate this trajectory. The ability to suppress the his4"" * ° mutation demonstrates that the mof2-l mutants have a Sui phenotype. However, unlike the sui2 and SUI3 mutants, the mof2-1 mutants are not able to repress the expression of the GCN4 gene. The expression of the human homolog of this gene can correct the mutant phenotypes in the yeast. Based on these new data, the present invention teaches that the Suil protein (Suilp) plays a role in the monitoring of translational fidelity during all stages of translation. Furthermore, when determining the role of the Suil / Mof2 protein in the translation, the present invention indicates that this protein helps the reading function of the ribosome test in all the translational steps, including the beginning, the elongation and the termination. . Recently »reversal analysis of the mutants suil has identified five suppressor sites called ssu (supreeoree of suil)» that enhance the development of a mutant suil at the restriction temperatures. Ssul encodes the ribosomal protein S4 RPS4 and ssu4 encodes RPS26. RPS4 corresponds to E. coli S5, a ram mutant Cribosomal ambigúity »ribosomal ambiguity) 3. Ram proteins have been implicated in the editing of the ribosomal P site during # translational lengthening. Although it is not intended to be limited in that manner, the present invention is further based on the hypothesis that, by interacting with the yeast equivalent of the bacterial Ram proteins, mof2-5-1 acts as a yeast ram mutant. The yeast equivalent of bacterial ram proteins are: bacterial S5 = yeast RPS4 = SUP44, and bacterial S4 = yeast RPS13A ^ - = sup46. The invention additionally allows the examination of: 1) if SUP44 and / or SUP46 are synthetically lethal with mof2-1; 2) if the excess expression of sup44 and sup46 of wild types is capable of suppressing the phenotypes mof2-l and 3) determines whether wild-type Suilp is associated with the ribosome »and if there are qualitative differences between the different forms of Suilp (Suil-lp, Mot2-lp and the human Suilp) to agglutinate the ribosomes. The present invention additionally launches the hypothesis that the Mof2 (Mof2p) protein is a general regulator of translational fidelity »and acts as a factor stimulator on the hydrolysis of the nucleotide triphosphate (NTP). Suilp physically interacts with eIF5, stimulating the hydrolysis of GTP during the start step. In addition, two of the mutants ssu, ssu2 and ssu3 encode eIF5 and eIF2gamma, respectively. The suppressor mutations in these Two maps of G proteins, to regions that contain homology with G proteins, suggest that these mutants # suppressors alter the fidelity of the translation by changing the GTP hydrolysis activity in these translational initiation factors. Accordingly, the present invention proposes: 1) to examine the effects of wild-type Mof2-lp, Suil-lp and hISOSUI1p on the hydrolysis of GTP with purified G proteins which are known to be involved in the elongation phase of the translation »that is to say» EFlalfa and EF-2 »2) examine the effects of these forms of Suilp on the # ATP hydrolysis »using Upflp and its mutants» 3) examine the synthetic lethality of mof2-l with TEF2 alleles (encoding EF-lalfa) »EF-2 and UPFl. The gene dosage experiments indicate whether the expression process of EF-lalfa. EF-2 or Upflp can suppress mof2-1 waves mutations of their l-l. The invention also relates to the discovery that the ifsl and ifs2 alleles. that were previously identified as mutations that increase the frame shift in the ribosomal frame shift signal -1 »from the mammary tumor virus in mice »are all related to the UPF2 and UPF1 genes, respectively, although both ifs strains maintained M. In addition, the expression of the N-terminal 100 amino acids of the TCM1 / MAK8 gene also increases the efficiency of the ribosomal frame shift -1 and interferes with the reproduction of the virus M., dsARN of L-A. Additionally, "other non-antibiotic agents that affect elongation within the specific window" should also not affect the efficiency of the ribosomal frame shift -1. To determine an appropriate scale of concentrations, the effects of anisomycin and sparsomycin on cell development were analyzed. It was determined that anisomycin concentrations ranging between 0.786 and 3. 8 μM »and the concentrations of sparsomycin varying between 0.52 and 2.6 μM» inhibited the overall cell growth rates in less than 3054 (data not shown). These drug concentration scales were selected for additional in vivo investigations.

THE DISPLACEMENT OF THE RIBOSOMIC FRAMEWORK AND THE SINSENTIDO IN THE DESCO POSITIONED RNA The strategy employed in the identification of the mof mutants is based on finding cells that express increased amounts of beta-gal. Increased ribosomal frame shift efficiencies -1 would result in increased expression of beta-gal when the lacZ gene is downstream of a La ribosomal frame shift signal -1 and LA reading frame -1"with with respect to the initial translation site. However, the same result could be observed as a result of chromosomal mutations different from those that affect efficiencies in the displacement of the ribosomal framework -1. For example, "mutations that increase the stability of the lacZ gene product" or mutations that increase its transcription rate "could also produce the desired result. The most interesting possibility of all 5 would be that of mutations that increase the half-life of the lacZ reporter mRNA. It tested all the mof mutants in terms of their ^ w phenotypes (11) of mRNA degradation specific for Upf. Since it is known that the half-life of the endogenous CYH2 0 precursor mRNA increases in the upf mutants, the abundance of the CYH2 precursor was determined in wild-type and mof mutants. The abundance of the precursor RNA of CYH2 was slightly elevated in the mutants mof2-1, mof5-1 and mof8-1 »and increased greatly in the mof -1 cells. The phenotypes of degradation of mRNA mediated by nonsense »mutants, of mof2-1, mof4-1. mof5-1, mof7-1 and mofB-1 are increased by using a mini-PGKl reporter construct, which contains * only a DSE. Increasing the number of DSE decreases this mutant phenotype, especially in the mof2-1 mutants. 20 The complementation test revealed that mof2-1, mof5- 1 and mofß-l do not correspond to any known mutation in the degradation path of mRNA mediated by nonsense; we have identified mo 4-1 as an allele of the UPFl gene. The diploid cells that are the result of a cell cross mof4-l and upf1-2 »had beta-gal activities that were not distinguishable from that of participant mo 4-1. and the abundance of the CYH2 precursor in these cells remained high. The introduction of a single-copy centromere-based plasmid, containing the UPF1 gene in the mof4-1 cells, was able to correct both mutant phenotypes; while the introduction of similar urx plasmid containing the UPF2 gene or the vector alone, had no effect on the mutant phenotypes of the mof4-1 cells. ^ Although the half-lives of the ribosomal frame-shifting mRNA reporters -1 are increased in the 0 upf mutants, the ribosomes that the transirads continue to move the frame with the same efficiency. Thus, although the upf mutants must be indistinguishable from the mof mutants by the beta-gal analysis, the upf mutants must be able to maintain the virus ^ because the ratio of Gag to Gag-pol 5 would remain unaffected. However, the true mof mutants, by virtue of their effect on the efficiency of the ribosomal frame shift -1, should not be able to propagate M, ... Three of the four mof mutants also have the Upf phenotype »ie» mof2-1 »mof4-1» mof5-1 are unable to propagate M ,. and »therefore» are true mutants mof.

Only mofB-1 »which has a weak mutant phenotype of mRNA degradation nonsense of Upf» is able to maintain M. The cloning and characterization of mof4-1 is a model for understanding the procedures of translational lengthening "and how the perspectives of the present can be applied to the rational development of antiviral agents that specifically indicate or determine the displacement of the ribosomal framework. The mof -1 allele of UPF1 is interesting because it is the only known allele of UPF1 that is unable to maintain the M satellite virus. With its increased efficiency of ribosomal frame shift-1 and the high abundance of nonsense m-RNA, the mof4-l allele of UPF1 defines a new class of mutant. The sequence of the t L allele mof4-1 was formed and determined to consist of a missense mutation of cystine (Cys) by tyrosine at amino acid 0 62. the first Cys residue in the putative zinc index. The present data demonstrate that there is a connection between the phenomena defined by the mof and upf mutants, which illuminates the continuity in the translation process »from the stability of mRNA through the synthesis of the complete protein product» mof4-l it is also sensitive to paronomici a »which is a translational inhibitor, and the ribosomal frame 1 shift can be increased with increasing concentrations of paronomycin. This represents the first demonstration that the efficiency of displacement of 0 ribosomal framework -1 can be modulated by a specific drug and, as such, has wide pharmacological implications. In addition to examining the effects of the mof mutants on the accumulation of the mRNA of the CYH2 precursor, the abundance 5 of the other mRNA containing nonsense »was also determined. These mRNAs are encoded by alleles of PGI that contain different high codons (UUA, UAG, UGA), the HIS4 gene, with a high codon inserted in the Nhel site (HIS4-UGA (Nhe)) and the PKGl gene. full length containing nonsense codons at different positions within the coding region (PGKl-n-UAG-AU) ((25 »57) and see Figures 2A-2D for constructions). It was previously demonstrated that the stability of these mRNAs was z ^ dependent on the gene products Upf. The mRNA level of the mini-PGKl and HIS4-10 UAG (Nhe) alleles, in the mof2-1, mof5-1 and roofB-1 cells were almost as high as in mof4-l and in the Upf »cells and were not dependent on the type of stop codon. Interestingly, the location of the high codon within the PGK1 gene affects the degradation phenotype of mRNA mediated by nonsense, 5 especially in the case of mof2-1. In the H2 mutation (3). the UAG terminator occurs before all the downstream elements »and there are only two downstream (known) 5 'elements of the H2 (2) mutation. The levels of mRNAs and PGK-n-UAG-AU (where nonsense codons occur after the downstream elements have been moved) in the mof2-1 cells equal those seen in the wild-type cells. This is an interesting discovery and the probable hypotheses for these different effects will be described later. In view of the foregoing, "it becomes evident that the present invention provides numerous routes to effect the ribosomal frame shift" which has important implications for antiviral therapy and for the suppression of nonsense pathological mutations. It is very important that two antibiotics and an approximately 100 amino acid N-terminal segment of a ribosome binding protein »L3» interrupt the normal trajectories of frame shift and nonsense decay. Thus, the present invention provides drugs for use as an viral compounds or for altering the ribosomal decomposition. The term "drugs" is used herein to refer to a compound "such as an antibiotic or a protein" that can affect the function of the peptidyl transferase center. Such compounds can increase or decrease the frame displacement efficiency -i; in either case »the result is the interruption of the expression of the protein that has antiviral consequences» or it can suppress the nonsense mutations.

GENES THAT CODIFY THE DISPLACEMENT OF MARC DECOMPOSITION PROTEIN FRAMEWORK In accordance with the present invention, molecular biology and microbiology techniques can be used.

Recombinant DNA »conventional» within the experience of the technique. Esae techniques are fully described in the literature. see »for example, Sambrook, Fritsch & Maniatis »Molecular Cloninq: A Laboratory Manual» 2a. edition (19B9), Cold Spring Harbor Laboratory Press »Cold Spring Harbor» New York (in the present "Sambrook and others» 1989"), DNA cloninq: A Practical Approach» volumes I and II (D. N. Glover ed. »1985); 5 01 Igonucleotide Synthesis (M. J. Gait ed. »1984); Nucleic Acid Hybridization CB. D. Hames & S. J. Higgins eds. (1985) 3, Transcription and Translation CB. D. Hames and S. J. Higgins eds. gj (1984) 3; Animal Cell Culture CR. i: Freshney, ed. (1986) 3; Immobili ed Celis And Enzymes CIRL Press »(1986) 3; B. Perbal. A 0 Practical Guide To Molecular Cloning (1984); F. M. Ausebel et al. (Eds.). Current Protocols n Molecular Biology, John Wiley & Sons, Inc. »(1994). Therefore "as is evident here" the terms will have the definitions given below. 15 A "vector" is a replicon »such as a plasmid. phage or cosmid »to which another segment of DNA can be attached. in order to reproduce the attached segment. A "replicon1" is a rx genetic element (eg, plasmid »chromosome» virus) that functions as an autonomous unit of in vivo reproduction of DNA »that is, capable of reproducing under its own control. A "caset" refers to a segment of DNA that can be inserted into a urx vector "at specific restriction sites. The DNA segment encodes a polypeptide of interest "and the cassette and restriction sites are designed to ensure the insertion of the cassette into the appropriate reading frame for transcription and translation.

A cell has been "transfected" by exogenous or heterologous DNA when said DNA has been introduced into the cell. A cell has been "transformed" by exogenous or heterologous DNA when the transfected DNA makes a phenotypic change. Preferably, the transformation DNA must be integrated (covalently linked) into the chromosomal DNA, which constitutes the genome of the cell. "Heterologous" DNA refers to DNA not naturally located in the cell "or a chromosomal site of the IO cell. Preferably the heterologous DNA includes a gene foreign to the cell. A "nucleic acid molecule" refers to the polymeric form phosphate ester of the ribonucleosides (adenosine »guanosine» uridine and citid na: "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine: "DNA molecules") or any phosphoester analogs thereof such as phosphorothioates and thioesters either in the form of a single filament or double filament They are possible the double-strand helices DNA-DNA »DNA-RNA and RNA-RNA. The term "nucleic acid molecule" and "in particular" DNA or RNA molecule only refers to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. So, this term includes the Double-stranded DNA found, among others, in linear or circular DNA molecules (eg, # restriction fragments), plasmids and chromosomes. By discussing the structure of the "particular" double-stranded DNA molecules, the sequences can be described here according to the normal convention, giving only the sequence in the 5 to 3 direction along the non-transcribed strand of DNA (that is, the formula that has a homologous sequence to the mRNA). A "DNA molecule ^^ recombinant "is a DNA molecule that has undergone a ^ molecular biological manipulation. A nucleic acid molecule is "hybridizable" to another nucleic acid molecule »such as a cDNA» a genomic DNA or an RNA »when a single-filament form of the nucleic acid molecule is attached to another nucleic acid molecule under the appropriate conditions of temperature and ionic concentration of the solution (see Sambrook et al., Supra). The conditions of temperature and ionic concentration determine the "severity" of the hybridization. For the preliminary purification of homologous nucleic acids, low-stringency hybridization conditions can be used. that correspond to a Tm of 55 ° C, for example 5x SSC, 0.154 SDS, 0.2554 milk, s n formamide; or 30J4 of for a da, 5x SSC, 0.5JÍ of SDS). Hybridization conditions of moderate severity correspond to a higher Tm, for example, 4054 of formamide, with 5x or 5x of SCC. Hybridization conditions of severe severity correspond to the maximum Tm »for example» 505Í of formamide »5x or 6x of SCC. Hybridization requires that the two nucleic acids contain complementary sequences "although depending on the severity of the hybridization" the inequalities between the aces are possible. The appropriate severity for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation which are variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids and nucleic acids having these sequences. The relative stability O (corresponding to higher Tm) of the nucleic acid hybridizations decreases in the following order: RNA: RNA »AD: RNA» DNA: DNA. For hybrids of more than 100 nucleotides long, equations have been derived to calculate Tm (see Sambrook and others »supra» 9.50-0.51). For hybridization with 5 shorter nucleic acids »that is,» oligonucleotides »the position of the inequalities becomes more important» and the length of the oligonucleotide determines its specificity (see Sambrook et al. »Supra» 11.7-11.8). Preferably the minimum length for urx hybridizable nucleic acid is at least about 10 nucleotides; preferably »at least about 15 nucleotides; and better still »the length is at least 20 nucleotides. In a specific embodiment, the term "normal hybridization conditions" refers to a Tm of 55 ° C and uses the conditions indicated above. In a preferred embodiment »the Tm is 60 ° C and» in the most preferred embodiment »the Tm is 65 ° C. "Homologous recombination" refers to the insertion of a foreign DNA sequence of a vector into a chromosome. Preferably, the vector signals or determines a chromosomal site 5 specific for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology of the chromosome to permit complementary binding and incorporation of the vector into the chromosome. The longest homology regions and the greater degrees of sequence similarity may increase the efficiency of homologous recombination. A "coding sequence" of DNA is a double-stranded DNA sequence that is transcribed and translated to a polypeptide in a cell in vitro or in vivo, when placed under the control of appropriate regulatory sequences. The limits of the coding sequence are determined by the initial codon at the 5"end (amino) and the translation stop codon at the 3T (carboxyl) end.A coding sequence may include, but is not limited to them» Prokaryotic sequences »cDNA from eukaryotic mRNA» sequence of genomic DNA from eukaryotic (eg mammalian) DNA and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, usually one will be located The polyadenylation signal and the transcription termination sequence »3» with respect to the coding sequence.

The transcriptional and translational control sequences are 'regulatory DNA' sequences such as promoters, termino-enhancer and the like 'that provide for the expression of a coding sequence in an amphiphile cell. In eukaryotic cells, pol adenylation signals are control sequences. A "promoter sequence" is a "DNA regulatory region" capable of binding the RNA-polymerase in a cell and initiating the transcription of a coding sequence downstream. (address 3). For purposes of defining the present invention, the promoter sequence is located at its 3 'end by the transcription initiation site and extends upstream (in the 5"direction) to include the minimum number of bases or elements necessary to initiate the transcription to levels detectable above the background. Within the promoter sequence will be found a transcription start site (conveniently defined for example) by mapping with the endonuclease SI). as well as the protein binding domains (consensual sequences) »that are responsible for the binding of the RNA-pol merase. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell "when the RNA polymerase transcribes the coding sequence in the mRNA" which is then divided from the Trans-RNA is transferred to the protein encoded by the coding sequence.

As used herein, the term "homologous" in all its grammatical forms and its orthographic variations refers to the relationship between proteins possessing a "common evolution origin", which include the proteins of 5 superfa ilias (for example, example »immunoglobulin super amyla) and homologous proteins of different species (for example» light chain myosin »etc.). (Reeck f and coauthors »19B7» Cell 50: 667). These proteins (and their coding genes) have sequence homology »as reflected by its high degree of sequence similarity. Consequently, the term "sequence similarity", in all its grammatical forms, refers to the degree of identity or correspondence between the nucleic acid or amino acid sequences of the proteins which may or may not share a common evolution origin (cf. Reeck and co-authors, supra). However, in common usage and in the present application, the term "homologous", when modified with an adverb, such as "strongly", may refer to sequence similarity and not to a common evolution origin. In a specific embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (preferably at least about 7554 and, better still, at least about 25%) 90 or 9554) of the nucleotides coincide in the defined length of the DNA sequences. Substances that are substantially homologous can be detected by comparing the sequences by the use of normal application programs., obtainable in the sequence data banks, or in a Southern hybridization experiment, for example under severe conditions as defined for that particular system. The definition of the appropriate hybridization conditions is within the knowledge of those skilled in the art. see »for example» Maniatis et al., supra: DNA Cloning »volumes I and II, supra; Nucleic Acid Hybrization, supra. Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially similar" when more than 3054 of the amino acids are identical, or more than about 6054 are similar (functionally identical). Preferably, similar or homologous sequences are identified by the use using, for example, the GCG application program (Genetics Computer Group, Program Manual for the GCG package, version 7 »Madison» Wisconsin »E. U. A). The term "corresponding to" is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule in which the similarity or homology is measured. Nucleic or amino acid may include spaces. Thus, the term "corresponding to" refers to the similarity of the sequence and not to the numbering of amino acid residues or nucleotide bases. The present invention contemplates the isolation of a gene encoding a frame shift of the protein of Degradation of mRNA of the present invention, which includes a "full-length" or "naturally occurring" form of frame shift or mRNA degradation protein, ^ g »of any eukaryotic origin, such as yeast» but that ^ includes an animal source »particularly of mammal or ave »and better yet, human or vegetable. As used herein, the term "gene" refers to an assembly of nucleotides that encodes a polypeptide and includes cDNA nucleic acids and genomic DNA. A gene encoding a framework or mRNA degradation protein "either genomic DNA or cDNA" can be isolated from any source "in particular from a human cDNA or from a genomic library. Methods for obtaining such genes are well known in the art "as described previously (see, for example, Sambrook et al. 1989" 20 above). A specific example of isolation of said gene is shown in the example included herein. Consequently »any eukaryotic cell can potentially serve as the source of nucleic acid for the molecular cloning of a gene that encodes a protein of frame shift or mRNA degradation. DNA can be obtained by common procedures known in the art from cloned DNA (eg, DNA "bank"). by chemical synthesis, by cloning of cDNA or by cloning genomic DNA or its fragments can be purified from the desired cell (see »for example» Sambrook et al. »1989, supra; Glover, DM (ed.)» 1985 »DNA Cloning: A Practical Approach, MRL Press. Ltd.» Oxford »United Kingdom, volumes I, II). Clones derived from genomic f-DNA may contain regions of regulatory and intron DNA, in addition to the coding regions; loe clones cDNA derivatives will not contain intron sequences. Whatever the source, the gene must be cloned molecularly into a vector suitable for propagating the gene. In a molecular cloning of the gene from DNA genomic, it generates DNA fragments, some of which will encode the desired gene. DNA can be divided into specific sites using various restriction enzymes. Alternatively, DNase can be used in the presence of manganese to fragment the DNA, or it can be physically cut DNA, for example, by sonic treatment. The DNA fragments can then be separated according to size, by common techniques, including but not limited to electrophoresis in agarose and polyacrylamide gel and column chromatography. Once the DNA fragments are generated, the identification of the specific DNA fragment containing the desired gene can be achieved in many different ways. For example, "if a quantity of a portion of the gene or its specific RNA or fragment thereof is available and can be purified and labeled", the generated DNA fragments can be purified by hybridization of the nucleic acid to the labeled probe (Benton and Davis »1977» Science »196: 180» Grunstein and Hogness »1975» Proc. Nati, Acad. Sci. USA »72: 3961). For example »a series of oligonucleotides that correspond to the information of the amino acid sequence The "partial" obtained for the framework shift or mRNA degradation protein can be prepared and can be used as probes for the DNA encoding frame shift protein or mRNA degradation. Preferably "a fragment that is unique with respect to the protection from frame shift or mRNA degradation. Those fragments of DNA that have substantial homology to the probe will hybridize. As noted earlier, "the greater the degree of homology", the more hybridization conditions can be used. severe. In addition, "because frame shift and mRNA degradation proteins are fundamental for translation, they are strongly conserved, for example, from yeast to human. Thus, the identification of said protein in yeast or another eukaryotic cell, leads to the obtaining of Said protein from human cDNA or other animal cDNA libraries. The section »in addition» can be carried out on the basis of the gene properties »for example» if the gene encodes a protein product having an isoelectric »electrophoretic» amino acid composition or a partial amino acid sequence of the protein 5 frame or mRNA degradation "as described here. In such a way »the presence of the gene can be detected by means of analyzes based on the physical or chemical properties ^^ immunological of your expressed product. For example, cDNA clones or DNA clones that are O hybridized-can be selected by selecting the appropriate mRNAs that produce a protein that "for example" has similar or identical electrophoretic migration behavior »isoelectric focus or gel electrophoresis At pH out of equilibrium, proteolytic digestion maps or antigenic properties such as the 5 known for the frame shift protein or mRNA degradation. A gene of the invention can also be identified by selection of mRNA, ie, by nucleic acid hybridization, followed by in vitro translation. In this procedure, nucleotide fragments are used to hybridize the complementary mRNAs. Such DNA fragments may represent purified DNA "available" including DNA from other species »or may be synthetic oligo-nucleotides designated from the partial information of the amino acid sequence. Immunoprecipitation analysis or functional assays of the in vitro translation products of the products of the isolated mRNAs »identifies the mRNA and therefore the" complementary DNA fragments "containing the desired sequences.

Additionally »you can select specific mRNAs by adsorption of polysomes isolated from cells to immobilized antibodies, directed specifically against the frame shift protein or degradation of mRNA. ^^ A radiolabeled cDNA can be synthesized using ^ the selected mRNA (of the adsorbed polysomes) as IO template. The radiolabeled mRNA or cDNA can then be used as a probe to identify the homologous DNA fragments from among other genomic AGN fragments. The present invention also relates to cloning vectors containing genes encoding analogues and derivatives of the invention of frame shift or degradation of mRNA, which have the same homologous functional activity as the frame shift or mRNA degradation protein, and their homologs of other species. The production and use of derivatives and analogues related to frame shift or mRNA degradation protein are within the scope of the present invention. In a specific embodiment, the derivative or analogue is an active entity, that is, it is capable of exhibiting one or more functional activities associated with a protein of the invention. frame shift or degradation of mRNA »full-length, wild-type.

The frame shift or mRNA degradation protein derivatives can be formed by altering the coding nucleic acid sequences »by substitutions» additions or omissions »that provide functionally equivalent molecules. Preferably, derivatives are prepared which have favored or increased functional activity with respect to the frame shift protein or mRNA degradation. Because of the degeneracy of the nucleotide coding sequences O, other DNA sequences that encode substantially the same amino acid sequence as the framework shift or mRNA degradation gene can be used in the practice of the present invention. These include, but not limited to, »the genes allelic »the homologous genes of other species and the nucleotide sequences that comprise all the genes or portions of said genes. that are altered by the substitution of different codons that encode the same amino acid residue within the sequence "producing that way urx silent change. Likewise, "frame shift or mRNA degradation protein derivatives" of the invention "include but are not limited to" those containing as the primary amino acid sequence all or part of the amino acid sequence of a "Frame shift or mRNA degradation protein" which includes altered sequences in which functionally equivalent amino acid residues are substituted by residues within the sequence, which result in a conservative amino acid substitution. For example, one or more amino acid residues may be substituted within the sequence by another amino acid of a similar polarity, which acts as a functional equivalent, which results in a silent alteration. You can select substitutes for ^ fc an amino acid within the sequence of other members of the class to which the amino acid belongs. For example, IO non-polar (hydrophobic) amino acids include: alanine, leucine, isoleucine »valine» proline »phenylalanine» tryptophan and methionine. Polar neutral amino acids include: glycine, serine »threonine, cysteine» tyrosine »asparagine and glutamine. The positively charged (basic) amino acids include: arginine » lysine and histidine. The negatively charged amino acids (acids) include: aspartic acid and glutamic acid. It is not expected that these alterations affect the apparent molecular weight »when determined by polyacrylamide gel electrophoresis» or the isoelectric point. The genes encoding the derivatives and analogs of the frame shift or mRNA degradation protein of the present invention can be produced by various methods known in the art. Manipulations that result in their production may occur at the level of gene of the protein. For example, the gene sequence of the "cloned" mRNA / shift framework protein can be modified by any of numerous strategies known in the art (Sambrook et al. 1989, supra). The sequence can be divided into appropriate sites with one or more restriction endonucleases "followed by additional enzymatic modification" if desired, then isolated and ligated. In the production of the gene that encodes a derivative or analog of the displacement protein ^^ frame or degradation of mRNA »care should be taken in * ^ P_ ensure that the modified gene remains within the same translational reading frame as the frame shift or mRNA degradation protein gene. not interrupted by translational stop signs »in the region of the gene in which the desired activity is coded. Additionally, the nucleic acid sequence encoding the framework shift or mRNA degradation protein can be mutated in vitro or in vivo. to create and / or destroy the translation sequences. start and / 0 * termination »or to create variations in the coding regions and / or to form new endonuclease sites of restricting or destroying previously existing ones. to further facilitate in vitro modification. Preferably, these mutations increase the functional activity of the gene product of the frame shift or degradation protein of mutated mRNA. Any technique Mutagenesis known in the art can be used "including but not limited to site-directed mutagenesis" in vitro (Hutchinson, C., and co-authors, 1978, J. Biol. Chem., 253: 6551; Zoller and Smith, 1984). , DNA, 3: 479-488, Oliphant and coauthors, 1986 »Gene 44: 177» Hutchinson and coauthors »1986, Proc. Nati, Acad. Sci., USA 83: 710), the use of TAB linkers < W > 5 (Pharmacia), etc. Preferred are PCR techniques for site-directed mutagenesis (see Higuchi, 1989, "Using PCR to Eng neer DNA" in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, etc. "Stockton Press" Chapter 6 »pages 61-70). Then the identified and isolated gene can be inserted into the cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include but are not limited to: plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of the vectors include, but are not limited to: E. coli "bacteriophages" such as lambda "or plasmid" derivatives such as pBR322 derivative or pUC plasmid derivatives "eg" pGEX vectors "pmal-c »PFLAG» etc. The Insertion within the cloning vector "for example" can be achieved by ligating the DNA fragment in a cloning vector having complementary coherent ends. However »if the complementary restriction sites used to fragment the DNA are not present in the cloning vector» The ends of the DNA molecules can be enzymatically modified. Alternatively, any desired site can be produced by linking nucleotide sequences (linkers) at the DNA ends; these ligated linkers may comprise chemically »specific» synthesized oligonucleotides that encode restriction endonuclease recognition sequences. Recombinant molecules can be introduced into host cells by transformation, transfection, infection, electroporation, etc., so that many copies of the gene are generated. Preferably, the cloned gene is contained in urx shuttle vector plasmid, which provides dilation in a cloning cell, for example, E. coli, and easy purification by subsequent insertion into an appropriate expression cell line, if so it is desired. For example »a shuttle vector» which is a vector that can be reproduced in more than one type of organism »can be prepared for reproduction in both E. coli and Saccharomyces cerevisiae» by linker sequences of an E. coli plasmid »with sequences that form the 2μ yeast plasmid. In an alternative method "the desired gene can be identified and isolated after insertion into an appropriate cloning vector" in a "shotgun" approach. The enrichment of the desired gene "for example" by fractionation of size "can be carried out before insertion into the cloning vector.

EXPRESSION OF MUNN FRAMING OR DECOMPOSITION PROTEINS The nucleotide sequence encoding the frame shift or mRNA degradation protein »or a functionally active derivative thereof» including a chimeric protein »can be inserted into an appropriate expression vector» that is »a vector containing the * elements necessary for the transcription and translation of IO the protein coding sequence inserted. These elements are referred to here as "promoters". Thus, the nucleic acid encoding the protein of the invention, frame shift or mRNA degradation is operationally associated with a promoter in an expression vector of the invention. Both the cDNA and the genomic sequences can be cloned and expressed under the control of said regulatory sequences. Also an expression vector preferably includes a reproduction origin. You can provide the necessary signal of tranecripeion and translation in a recombinant expression vector "or can be delivered by the natural gene encoding the frame shift or mRNA degradation protein" and / or its flanking region. Potential host / vector systems Include, but are not limited to: mammalian cell systems infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell systems »infected with viruses (eg, baculovirus); microorganisms, such as yeasts containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA or cosmid DNA. The expression elements of vectors vary in their concentrations and in their specificities. Depending on the host-vector system used, it can be used / -_ any of numerous transcription and translation elements suitable. 0 A recombinant protein of the invention can be expressed chemically, frame shift or mRNA degradation, or a fragment »derivative» chimeric construction or functional analogue thereof »after integration of the coding sequence by recombination. In this regard, any of a number of amplification systems can be used to obtain high levels of stable gene expression (see Sambrook et al., 1989, supra). The cell within which the recombinant vector 0 comprising the framework or mRNA degradation protein encoding the nucleic acid is cultured is cultured in an appropriate cell culture medium under conditions that provide for the expression of the framework shift or mRNA degradation protein. for the 5 cell. Any of the previously described methods for the insertion of the DNA fragments can be used. in the cloning vector, to construct any of the expression vectors containing a gene consisting of appropriate transcription / translation control signals and the protein coding sequences. These methods may include in vitro recombinant DNA techniques and synthetic techniques as well as in vivo recombination / ^ -__- (genetic recombination). It is possible to control the expression of the protein of Frame shift or degradation of mRNA by any promoter / enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. The promoters that can be used to control the expression of the gene of the frame shift protein or mRNA degradation include but are not limited to the early promoter region SV40 (Benoist and Chambon »1981» Nature 29 ?: 304-310), the promoter contained in the repeat of the terminal 3r long of the Rous sarcoma virus (Yamamoto and coauthors, 1980 »Ce11 22: 787-797), the herpes thymidine kinase promoter (Wagner and coauthors, 1981, Proc. Nati, Acad. Sci., USA 78: 1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al. coauthors »1982. Nature, 296: 39-42); the prokaryotic expression vectors. such as beta-lactase promoter (Vi lla-Kamaroff and co-authors »1978» Proc. Nati, Acad. Sci. USA 75: 2727-2731) or the tac promoter # (DeBoer and co-authors, 1983 »Proc. Nati. Acad. Sci. USA 8 ?: 21-25); see also Useful proteins from recombinant bacteria "in S entific American 1980" 242: 74-94; the promoter elements of yeasts or other fungi such as the 5 Gal4 promoter »the ADC (alcohol-dehydrogenase) promoter» the P6K promoter (phosphoglycerol kinase) »The alpha! Ina phosphatase promoter and the animal transcription control regions» that exhibit ^ Tissue specificity and that have been used in transgenic animals: the control region of the elastase I gene »that 0 is active in pancreatic acinar cells (Swift and co-authors» 1984 »Cell 38: 639-646; Ornitz and co-authors» 1986, Cold Spring Harbor Sy, Quant. Biol., 50: 399-409, MacDonald, 19B7, Hepatology, 7: 425-515); the controlling region of the insulin gene »that is active in pancreatic beta cells (Hanahan »1985» Nature 315: 115-122); the control region of the immunoglobulin gene »that is active in lymphoid cells (Grosschedl and co-authors» 1984 »Cell 38: 647-658; Ademes and co-authors» 1985 »Nature 318: 533-538; Alexander and coauthors» 19B7, Mol ., Biol. 7: 1436-1444); the region of control of the breast tumor virus in mice, which is active in testicular, breast, lymphoid and mastoid cells (Leder and coauthors, 1986, Cell 45: 486-495), the controlling region of the albumin gene »which is active in the liver (Pinkert and coauthors, 1987 »Genes and Devel.» i: 26B-276); the The controlling region of the α-fetoprotein gene, which is active in the liver (Kru lauf and coauthors »19B5» Mol.

Biol. 5: 1639-1648 »Hammer and coautoree, 1987, Science 235: 53-58)» the controlling region of the alpha-1-antitrypsin gene »that is active in the liver (Kelsey and coauthors, 1987, Genes and Devel ., 1: 161-171); the controlling region of the beta-5 globulin gene, which is active in myeloid cells (Mogram and co-authors, 1985, Nature 315: 338-340; Koll as and co-authors »1986» Cell 46: 89-94); the controlling region of the gene of ^^ basic protein of ina honey »which is active in oligodendrocytic cells, in the brain (Readhead and coauthors, 1987» O Cell 48: 703-712), the controlling region of the light chain-2 gene of the m? osin »that is active in the skeletal muscle (Sani, 1984» Nature »314: 283-286) and the controlling region of the gonadotropic releasing hormone gene» that is active in the hypothalamus (Mason and coauthors, 1986. Science 234: 1372-5787). Expression vectors containing a nucleic acid encoding a frame shift or mRNA degradation protein of the invention can be identified by four general approaches: (a) 0 PCR amplification of the desired plasmid DNA or mRNA specific; b) nucleic acid hybridization; (c) preemption or absence of gene functions selection marker; and (d) expression of the inserted sequences. In the first approach, nucleic acids can be amplified by PCR 5 to give the detection of the amplified product. In the second approach, the presence of a foreign gene inserted into an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene. In the third approach, the recombinant vector system can be identified and selected based on the presence or absence of certain "selectable marker" gene functions (eg, beta-galactosidase activity). activity of ti idine kinase, resistance to antibiotics, the transformation phenotype, the formation of a body of occlusion in baculovirus, etc.) caused by the insertion of foreign genes into the vector. In another example, if the nucleic acid encoding the frame shift or mRNA degradation protein is inserted into the sequence of the vector "selection marker" gene, the recombinants containing the protein insert of the vector can be identified. Frame shift or mRNA degradation »due to the absence of the function of frame shift or mRNA degradation protein gene. In a fourth approach, recombinant expression vectors can be identified by analysis or assay of the activity, biochemical or immunological characteristics of the gene product, expressed by the recombinant "as long as the expressed protein adds up to a conformation of active functionality. . Once a particular recombinant DNA molecule is identified and isolated, various methods known in the art can be used to propagate it. Once an adequate host system and appropriate developmental conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors that can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses, talee like vaccinia virus or adenovirus »insect virus» ^ such as baculovirus; yeast vectors; bacteriophage vectors (eg »lambda) and plasmid DNA vectors 0 and cosmid» to name just a few. In a preferred embodiment "the frame shift protein is expressed in a cell that is virally infected" and the ability of an agent to inhibit or eliminate death by virus is evaluated. In another preferred embodiment, the mRNA degradation protein is co-expressed in a cell with an aberrant mRNA transcript "such as a nonsense transcript" a transcript of opposite sense or a short transcript "and it is possible to evaluate the capacity of an agent to increase the stability of the aberrant 0 transcript. The vectors are introduced into the desired host cells by methods known in the art "for example" media before transfection »electroporation. microinjection »transduction» cell fusion »DEAE dextran» precipitation 5 with calcium phosphate »l pofection (fusion with lysosome a) use of a genetic cannon or a DNA vector transporter (see. # for example »Wuy and coautoree» 1992 »J. Biol. Chem.» 267: 963-967 »Wu and Wu» 1988 »J. Biol. Chem. 263: 14621-14624; Hartmut and co-inventors »Canadian patent application No. 2,012,311» filed on March 15, 1990). 5 DEPURATION FOR AGENTS THAT AFFECT PROTEIN ACTIVITY ^ k Any debugging technique can be used "known in this field" to purge the agents that affect the function of a frame shift protein or a mRNA degradation protein. The present invention contemplates purifications for small molecule ligands. Knowledge of the primary sequence of a frame shift protein or protein degradation of mRNA and the similarity of that sequence with proteins of known function "may provide an initial indication about the agents that are likely to affect the activity of the protein. Identification and debugging of said agents is additionally facilitated when determining the aspects structural proteins »for example» the use of X-ray crystallography »neutron diffraction» nuclear magnetic resonance spectrometry and other techniques for structural determination. These techniques provide rational design or identification of agonists and antagonists. In another aspect "synthetic data banks can be used (Needels and coauthors» 1993 »Generation and screening of an oligonucleotide encoded synthetic peptide library, Proc. Nati. Acad. Sci. USA 90: 10700-4; Lam and co-inventors, US Pat. No. 5,382,513"issued January 17, 1995" Lam and co-inventors "International Patent Publication No. WO 92 / O0252, and Ohimeyer and Co-authors, 1993, Proc. Nati, Acad. Sci. USA 90: 10922-10926 , each of which is incorporated herein by reference in its entirety) and sims, to purge the agents according to the present invention. Purification can be carried out with recombinant cells expressing the frame shift protein or the mRNA degradation protein or, alternatively, with the purified protein. For example, the ability of the labeled protein to bind to a molecule in a combinatorial bank can be used as a "clearance assay" as described in the references cited above.

OPPOSITE SENSE RNA AND RIBOZYMES The present invention extends to the preparation of nucleotides of opposite or opposite direction and ribozymes that incorporate the strategies indicated herein or that take advantage of the discovery that the degradation path of aberrant mRNA can shorten the half-life of opposite-sense RNA or ribozymes »in order to render them ineffective for the intended purpose, that is, to interfere with the expression of a gene at the translational level. This approach uses the opposite-sense nucleic acid and ribozymes to block the translation of a specific mRNA, either by masking the mRNA with a nucleic acid in the opposite direction or by dividing it with a ribozyme. Nucleic acids of opposite direction are DNA or RNA molecules that are complementary to at least one ^ t portion of a specific mRNA molecule (see Marcus-Sekura, 1988, Anal. Biochem 172: 29B). In the cell »they hybridize to the mRNA» forming a double-filament molecule The cell does not transfer a mRNA in this double-filament form. Therefore, nucleic acids of opposite direction interfere with the expression of mRNA to protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the start codon of AUG »will be particularly efficient» since they are easy to synthesize and probably involve fewer problems than larger molecules when introduced into organ cells. Opposite methods have been used to inhibit the expression of many in v tro genes (Marcus-Sekura »1988. supra; Habor and co-authors, 1988. J. Exp. Med. 1689: 1237). Ribozymes are RNA molecules that have the ability to specifically separate other RNA molecules from a single strand »in a manner somewhat analogous to DNA restriction endonucleases. The ribozymes were discovered from the observation that certain mRNAs have the ability to remove their own introns. By modifying the nucleotide sequence of these RNAs, the researchers have been able to engineered molecules that recognize specific nucleotide sequences in an RNA molecule and divide it (Cech, 19B8, J. Am. Med. Assoc. 260-3030 ). Because they are specific for the sequence, only mRNAs with particular sequences are inactivated. Researchers have identified two types of ribozymes. The Tetrahi ena type and the "hammer head" type. The ribozymes of the tetrahimena type recognize sequences of four bases »while those of the type" martillo head "recognize sequences of eleven to eighteen bases. The longer the recognition sequence, the more likely it is to occur exclusively in target mRNA species. Accordingly, ribozymes of the hammerhead type are preferred to ribozymes of the tetrahimena type to inactivate a specific mRNA species, and 18-base recognition sequences are preferable to shorter recognition sequences.

ANTIVIRAL THERAPY In yet another embodiment, the present invention provides a means to treat viral infections by providing agents that modulate frame shift efficiency and thereby "directly affect viral or assembly viral particles. The efficiency of the ribosomal frame shift -1 in the naturally occurring L-A slip site is 1,854 to 2,054 (16). The change in efficiency of the displacement of the ribosomal framework -1 using the drugs or the molecular biological reagents of the present invention "changes the ratio of Gag to Gag-pol" that is synthesized. This, in turn, "affects the assembly of the viral particle and the RNA packaging" that affects the ability of the cell to propagate the virus. In particular »the change in the sequence of the slip site affects the efficiency of the ribosomal frame shift -1. The efficiency of the ribosomal frame shift -1 can also be affected by introducing the mutated cellular gene products that interact with the translational apparatus »in particular the peptide transfer center lo. Molecular and genetic biological methods have been used to demonstrate that the efficiency at 1.954 of the displacement of the ribosomal framework produces an optimal ratio of structural to enzymatic proteins. The change in frame shift efficiencies greater than 2-3 times results in the loss of the Mp- satellite virus, whether the virus is supported by LA cDNA clones containing altered slip sites, or by the wild type L-A virus in mutant host cells. Even slight changes in ribosomal frame shift efficiencies -1 signifi- cantly decrease the number of copies of Mx. The present invention advantageously provides drugs and methods for identifying drugs for use in antiviral therapy (or nonsense suppression) of viruses that utilize the basic-1-ribosomal frame-shifting mechanism that includes four large families of animal viruses and three large families of plant viruses. For example, almost all retroviruses use ^ St- ribosomal frame shift -1, which includes lentivirus (immunodeficiency virus), talee as HIV-1 and HIV-2 »VIS» VIF »VIB» V sna virus »arthritis-encephalitis virus and the equine infectious anemia virus: the foamviruses (loe sparkling viruses) ), such as the human foamy virus and other mammalian foamy viruses »viruses li T cell vectors »such as HTLV-I» HTLV-II, STLV and BLV; poultry leukosis viruses »such as leukemia and sarcoma viruses in many birds» including commercial poultry; Type B retroviruses »including mouse mammary tumor virus and D-type retroviruses» such as Mason-Pfizer monkey virus and ovine pulmonary adenocarcinoma viruses. In addition »many coronaviruses use the -1 frame shift» including human coronaviruses. such as 229-E »0C43; the animal coronaviruses »such as the calf coronavirus» the virus of the transmissible gastroenteritis of the cerco, the encephalomyelitis virus hemagl utinante of the pig and the virus of the porcine epidemic diarrhea; canine coronaviruses, feline infectious peritonitis viruses and feline enteric coronaviruses; the infectious poultry bronchitis virus and the blue turkey crest virus; the mouse hepatitis virus, the rat coronavirue and the rabbit coronavirus. Yep.? larovirus (a type of coronavirus) »is involved as well as human toroviruses associated with enteric and respiratory diseases; the breda virus in W * calves and the bovine respiratory virus; the berne virus of the horses »porcine torovirus» feline torovirus. Another coronavirus is the arterivirus, which includes simian hemorrhagic fever virus, equine arteritis virus, Leystad virus (in pigs), virus VR2332 (in pigs) and lactate-dehydrogenase elevator virus (in rodents).

Other viruses and paramyx irus animals »such as the human-1 ribosomal frame shift» reported in measles, and astroviruses »such as human astrov rus 1-6 and the bovine, ovine, porcine, canine and duck astroviruses. Plant viruses that involve a mechanism of displacement of frame -1 include tetraviruses »such as the so-called virus (for example» the southern bean mosaic virus »the houndstooth fire virus)» the leuteovi us (for example »the virue of the yellow chip of barley, the yellowing beet southern virus and the potato leafroller virus). enamoviruses (for example, pea mosaic virus) and u braviruses (for example, carrot mottle virus) to busviruses such as the busvirus (for example, the tomato foliage achaping virus) ), carmoviruses (for example, mottled carnation virus), necroviruses (for example, tobacco necrosis virus), diantoviruses (for example, necrotic mosaic virus of red cloves) and maquiomoviruses (for example, corn chlorotic mottle virus). Additionally. the toti virus, such as L-A and L-BC (yeast) and other fungal viruses »the Giradia lambia virus (intestinal parasite). the virus Triconella vaginalis (human parasite) »the virus Leishmania brasil iensis (human parasite) and other protozoan viruses» are viruses of frame displacement -i.

SUPPRESSION OF PATHOLOGICAL SINSENTIDO MUTATIONS The path of decomposition of mRNA mediated by nonsense regulates the decomposition of transcripts that have acquired nonsense codons by means of a mutagenic event. As such »this trajectory is clearly involved in the regulation of gene expression. More important »modulate this trajectory can solve the lack of gene expression due to a nonsense mutation. Also the opposite-sense RNAs can be substrates for the decomposition path of mRNA mediated by nonsense. which leads to »a decrease in its cellular concentration and to the reduction of its capacity to inhibit the expression of the gene. Mutations or drugs that inactivate this pathway can increase the abundance of "sineentido" RNA. which results in an increase in the efficiency with which the opposite sense RNAs inhibit the expression of the gene. The modulation of the decomposition path of the mRNA also has significant potential to treat certain % terms. It finds nonsense mutations in many genes that result in diseases. A list of such diseases (and the allele that contains nonsense) is given below: non-spherocytic hemolytic anemia (TPI). beta thalasse ia (ß-GLOBINA) hypercholesterolemia (LDL RECEPTOR Lebanese aliele). pulmonary emphysema (alpha-1 ANTITRYPSINE) » adrenal hyperplasia (STEROID-21 HIDROLASE (CYP21)). deficiency of apolipoprotein C-II (APOLIPOPROTEIN C- ^ p. ov.1 'hemophilia B (FACTOR IX OI.TI_.AND' 'Bernard-Soulier syndrome (GLICOPROTEIN IBalpha) »fructose intolerance (ALDOLASE B)» resistance to ineulin (RECEPTOR E INSULIN) »urine disease such as maple syrup (alpha-CETOACIDO DEHYDROGENASE). thrombosis (PROTEIN S), goiter and hypothyroidism (THYLOGLOBULINE), chronic granulomatous (CITOCHROME B558). Sanhoff's disease (HEBX). von W disease type III (vonVILLEBRAND FACTOR) »spin atrophy (ORNITHINE) AMINOTRANSFERASE) »rickets resistant to 1.25- dihydroxy tamine D3 (VITAMIN D RECEPTOR). is erocystosis (ERYTHROCITE BAND 3). cystic fibrosis (CFTR) and spherocytosis (ERITROIDE ANKYRIN).

THERAPY WITH GENES AND TRANSGENIC VECTORS In a "modality" a gene encoding a ribosomal frame shift protein or "mutant" mRNA degradation protein or a "polypeptide domain fragment thereof" or a gene encoding an antisense or a ribozyme specific for a protein ribosomal frame shift or "wild-type" mRNA degradation is introduced in vivo into a viral vector. Such vectors include DNA viruses attenuated or defective, such as but not limited to: herpes simplex virus (HSV). the papilloma virus »the Epstein Barr virus (EBV)» the adenovirus »the adeno-associated virus (AAV) and the like. Defective viruses "that totally or almost totally lack viral genes" are preferred. The defective virus is not ineffective after its introduction into a cell. The use of defective viral vectors allows for administration to cells in a specific, localized area without concern as to whether the vector can infect other cells. Thus »adipose tissue can be fixed as a specific target. Examples of particular vectors include but are not limited to: a defective herpes virus 1 vector (VHS1) CKaplin and co-authors, Molec. Cell. Neurosci., 2: 320-330 (1991) 3, an attenuated adenovirus vector, such as the vector described by Stratford Perr audet and co-authors CJ. Clin. Invest. 0: 626-630, (1992) 3, and a defective vector of the adeno-associated virus CSamulski and co-authors. J. Virol. 61: 3096-3101 (1987); 5 Samulski and co-authors, J. Virol. »63: 3822-3828 (1989) 3. Preferably "for in vivo administration" an appropriate immunosuppressive treatment is used »together , with the viral vector »for example» the adenovirus vector »to avoid the immuno-deactivation of the viral vector and the transfected O-cells. For example, »immunosuppressive cytokines» such as interleukin-12 (IL-12) »interferon-gamma (IFN-gamma) or anti-CD4 antibody» can be administered to block humoral or cellular immune responses »to vectors viral Cvéase »for example» Wilson » Nature Medicine (1995) 3. Furthermore, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens. In another embodiment, the gene can be introduced into a retroviral vector, for example, as described in Anderson and co-inventors, U.S. Patent 4,650,764; Temin and coinventores »US Patent No. 4» 980 »289; Markowitz and coauthors »1988» J. Virol. »62: 112 ?; Temin and co-inventors »U.S. Patent No. 5,124-263; International Patent Publication No. WO 95/07358 »published on March 16, 1995 by Dougherty and co-inventors; and K? o and coauthors »1993» Blood 82: 845.

The supply from gene to destination is described in the international patent publication WO 95/28494. published in October 1995. Alternatively, the vector can be introduced in vivo by lipofection. During the last decade, there has been an increasing use of liposomes to encapsulate and transfect nucleic acids in vitro. The synthetic cationic molecules designated to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for the in vivo transfection of a gene coding for a CFelgner marker and co-authors. Proc. Nati Acad. HE . U.S.A. »84: 7413-7417 (1987); see Mackey and coauthors. Proc. Nati. »Acad. Sci. U.S.A. »85: 8027-8031 (1988) 3. The use of cationic lipids can promote the encapsulation of negatively charged nucleic acids and also promotes fusion with negatively charged cell membranes CFelgner and Ringold »Science 337: 387-388 (1989) 3. The use of lipofection to introduce exogenous genes into specific organs "in vivo" has certain practical advantages. The molecular determination of liposomes to specific cells "represents an area of benefit. It is clear that directing the transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity »such as the pancreas» the liver »the kidney and the brain. The lipids can be coupled chemically with other molecules "for the purpose of" making white "(see Mackey and coauthors, supra). Target peptides, eg, hormones or neurotransmitters, and proteins, such as antibodies, or molecules that are not peptides, could be chemically coupled to liposomes. It is also possible to introduce the vector in vivo as a naked DNA plasmid. Bare DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example »by transfection» electroporation »microinjection» transduction »cell fusion» DEAE dextran »calcium phosphate precipitation» use of a genetic cannon or the use of a transporter of the DNA vector Cvéase »for example, Wu and co-authors. J. Biol. Chem. 267: 963-967 (1992); Wu and Wu, J. Biol. Chem. 263: 14621-14624 (19BB); Har ut and co-inventors, Canadian Patent Application No. 2,012,311, filed March 15, 1990. In another embodiment, the present invention provides co-expression of a gene product that modulates the activity at the peptidyl 1-transferase site and a heterologous antisense or ribozyme gene, therapeutics »under the control of the specific sequence of DNA recognition» by providing a gene expression expression vector comprising both a gene encoding a peptidi 1-transferase center modulator (including »But without limitation» a gene for a mutant frame shift protein or mutant »mRNA degradation or an opposite sense RNA or a ribozyme specific for a mRNA that encodes said protein) with a gene for a nucleic acid or ribozyme of opposite sense, unrelated, under the control of coordinated expression. In one embodiment, these elements are provided in separate vectors; alternatively, those elements may be provided in a single expression vector. The present invention can be better understood by reference to the following non-limiting examples, »Which are provided as examples of the invention. 10 EXAMPLE mo 4-1 »an allele of the UPF1 / IFS2 gene» affects the change in mRNA and the efficiency of ribosomal displacement -1 The degradation of mRNA is a control point important in the regulation of the expression gene and has been shown to be connected to the translation process.

A clear example of this link is the observation that nonsense mutations accelerate the degradation of mRNAs.

This example shows that a subset of alleles mof (aintenance fíf Érame = frame maintenance) in yeast »that were isolated as chromosomal mutations that increased the efficiency of frame displacement at the site of frame shift of the L-A virus» and caused loss of satellite A virus from L-A. they also affect the trajectory of degradation of mRNA »mediated by nonsense. The mRNA levels containing nonsense were elevated in cells harboring the mof4-1 alleles. Additionally, mof4-1 is allelic with respect to UPF1, which has been shown to be involved in the degradation path of mRNA mediated by sinsent. Although the cells harboring the mof4-1 allele lose the M3- virus, the other alleles f (ie »upfl» up 2 and upf3) involved in the degradation of mRNA mediated by nonsense »maintain M. The ifsl and ifs2 »alleles identified precisely as mutations that increase the frame shift in the ribosomal frame shift signal -1, coming from mouse mammary tumor virus, proved to be allelic with respect to the UPF2 and UPF1 genes, respectively. and both ifs strains maintained MA. Strain mof4-1 is more sensitive to the aminoglycosome paronomycin than a strain upf1 / \ and increases the efficiency of frame shift in a strain mof4-l developed in the presence of paronomycin. These results indicate that Upflp has a double function both in the translation and in the change of mRNA.

MATERIALS AND METHODS Strain and media: The strains of Saccharomyces cerevisiae used are listed in Table 1. YPAD, YPG »SD» the complete medium of synthesis and the 4.7 MB plates to test the killer phenotype »were as previously reported (Dinman and co-authors »Genetics 136: 75-86 (1994) 3.

TABLE 1 CEPAS USED IN THIS STUDY Cells Genotype referenc a Y52- MATcr bl-l his4-5l9 ur 3-S UPFl :: hisG YGC106 MATa a_le2.3 ura3 leu2 hs7 canl sap3 UPFlülx? SG this study PLY36 MATo bi .4-38 sufW ura3-52 me.14 u? Fl-2 b PLY136 MATa bis4-38 sufl-1 ura3-52 ro «t4 trpl-1 upfl-2 b YGCU2 MATa -ÚS4-38 sufl-l ura3-52 toctH Upf 1-2 tipl-l leu2-3 UPF2 :: URA3 to PLY.39 MATa h_s4-38 su. l-l u? a3-52 UpG-1 b PLY133 MATo hi_4-38 sufl-1 ura3-52 mctH upf4-2 b 2907 MATa __is_W__2GQ leu2 trpl- < _901 _tt __-- 52 adc2-l0 K * c JD61 MATa t_i_4-644 _su2-l :: pJD85 K d ÍD75-1A MATa, eu2-l :: pJD85 t-is4-644 ura3 ade2 raofl-l K '4 JD65- 5C MATa leu2-l :: pJD85 his3.4-644 ura3 adc2 trpl-AMl mof3-l K 'd JD474-2C MATa lcu2-l: tp_T_ »85 ura 3 fipl-? 9Ql mof4-l K' d JD474-3D MATa leu2-l:; pT > 85 ura3 tüs4 mof4-l K 'c ID469-2C MATo leu2-l :: pJD85 or __? 3 trpl -_- 90l ade2 n. afd-1 K "d JD471-IA MATflr lcu2-l :: pJD8S u_a3 raof7-l K" d JD472-1 A MATo le? _2- i:: pJD85 ra3 mofS-l K "d JD472-9B MATa leu2-l :: pJD85 up? 3 bjs3.4 maG-l K "d" JD742-2D MATo leu2-i :: pJD85 ura 3 his3.4 L-AHN M, K * this study UID744-2C MATo teu2-l:; pJD8S ade2 his3,4 trpl ura3 n_o £ 3-l K. "d crass JD830 JD742-2D x JD474-3D this study 1074 MATa leu 1 L-AHN M, arl-i * d JD474-5A MATa leu2-l _: pJD85 ura3 his4 apl ade2 mof4 ~ l K- d 3164 MATa Jcarl-l argl L-AHN Mi K 'd 3165 MATa karl-1 argl tLU .x) L-AHN M, Kß d SX47 ATa MAT «hisl / + trpl / + ura3 / + KR- e L5a MATo cupl _.::up_3 uta3-52 h.s3 -__ 200 atle2 lys2 irpl leu2 - f ifsl MATe cupi_.::uu3 ura3-52 __. s3 -__ 2QQ ade2 lys2 irpl lcu2 ifel-2 K f ¡^ 2 MATo cupl /. :: u .__ 3 ura3-52 bis3- < _2QQ ade2 lys2 irpl leu2 i £ s2-l K f a) Cui and co-authors »Genes S Dev.» 9: 437-454 (1995) b) Leeds and co-authors »Genes &; Dev. »5: 2303-14 (1991) c) Dinman and coauthors» Proc. Nati Acad. Sci. USA. 88: 174-178 (1991); 5 d) Dimman and Wickner »Genetics» 136: 75-86 (1994) e) Dimman and Wickner, J. Virol. , 66: 3669-76 (1992) f) Lee and co-authors, Proc. Nati Acad. Sci. USA, 92: 6587 (1995). ^ T Genetic methods and analysis: Yeast transformation and E. coli were performed, as previously described CHagan and co-authors, Mol. Cel 1. Biol. , 15: B09-B23 (1995); Cui and co-authors, Genes S Dev., 7: 1737-1754 (1995); He and Jacobson, Genes & Dev., 9: 437-454 (1995) 3. He cured cells of the L-A virus by scratching individual colonies at 39 ° C and the loss of L-A was confirmed by agarose gel analysis. The cell generation k ~ rho °, the c onductions and the killer test were carried out as previously described CDimman and Wickner, J. Virol. , 66: 3669-3676 (1992) 3. Genetic crosses were made, sporulation and analysis of tetrada »analysis of β-galactosidase and the killer test, as described by CDinman and Wickner, Genetics, 136: 75-BS (1994) 3. DsRNA was prepared as described by CF ied and Fink »Proc. Nati Acad. Sei USES. 75: 4224-4228 (1978) 3 and analyzed by electro-ores s through agarose gels at 1.254. The test for paronomycin sensitivity of the various strains was carried out. as described by CCui and coauthors (1995), supra3.

The construction of plasmids The plasmids pJD107 and pJD108 were used for the analysis of β-galactosidase, from pF8 and pT125, respectively CDinman and co-authors, Proc. Nati Acad. Sci. USA, BB: 174-178 (1991) 3. In pJD107, the Hind fragment was ligated III of 4.9 kb of pFB »to pRS426 digested with Hind III ^ T CChristianson and coautoree »Gene, 110: 119-122 (1992) 3 and ÍO contains the promoter PGK1 »a translational start site» followed by a 218 base pair fragment of L-A cDNA containing the ribosomal frame shift signal -1. This is followed by the gene lacZ, which is in the -1 frame with respect to the initial site. pJDlOS contains the Hind fragment III of 4.7 kb of pT125, cloned into the HinglII site of pRS426 and the lacZ gene is in frame O without any intermediate fl sequence. PYCp33UPF1 and pYCp33UPF2 were constructed as previously described CCui and coauthors (1995) supra3. The plasmids pmof4AE »pmof4AB were constructed as follows and pmof4BE used to clone the mof4-1 allele: The Asp718-EcoRI and 1.47 kb fragment or the 2.6 kb Asp71B-BamHI fragment of pYCp33UPF1 containing the UPF1 gene was omitted. and it was replaced by the corresponding fragments of the mof4-l allele that were isolated by PCR (see below). HE cloned pmof4BE by inserting the 4.2 kb EcoRI-BamHI DNA fragment from roof4-len pyCplac33. Since pYCp33UPFl contains more than one BstXI site. was built pmof4XAE and pmof4XBE in two steps. A 978 base pair BstXI-Asp71B DNA fragment, from pPUC-UPF1 »was replaced by a BstXI-Aep718 DNA fragment» from pmo4AE and pmof4BE »respectively» forming pPUCmof4XAE and pPUCmof4XBt .. The BamHI-EcoRI fragments were cloned from 4.2 kb of estoe two plasmids, to pYCplac33.

Identification of the mof4-1 mutation: A PCR strategy was used to identify the mof4-1 allele. The sensitizers used for the PCR DNA fragments of the UPF1 gene were: sensitizer 1: 5 * -CCGGAATTCATGAACGGGAAA-3 '(SEQ ID NO: 8); senei bi 1 levator 2: 5'-GACCGGCCGTAACGGACGTTGTAATACAT-3r (SEQ ID NO: 9); sensibi 1 elevador 3: 5'-ATCCCCGCGGGAGTTGAAAGTTGCCATC-3r (SEQ ID NO: ÍO); 4: 5 sensitizer -GACGGATCCAAAGTATATTGGAC-3t (SEQ ID NO: 11) Genomic DNA (50-100 ng) CRose and co-authors, Methods Yeast Genetics, Cold Spring Harbor Press (1990) 3, were prepared from strain mof4 -1 and was used as a template in PCR The pairs of blinkers sens bl zador 1 and sens l zador 2 were used to synthesize the DNA fragments to build pmof4AE (figure 4), and sensib lizer 3 and sensib lizer 4 were used to synthesize the DNA fragment to construct pmof4AB (Figure 4), and sensibilizer 1 and seneib 1 elevator 4 were used to construct pmof4BE (Figure 4), respectively Two PCR products from two different reactions were used in the reaction of cloning to minimize CPR artifacts The CPR conditions used were as follows: 95 ° C - 5 minutes »94 ° C - 1 minute, 45 or 50 ° C - 1 minute, 72 ° C - 1.5 minutes, for 25 cycles, DNA fragments from PCR were purified from 154 gel of Garosa was used to exchange the corresponding DNA fragment of the UPFl gene of the silveetre type, which was in a YM plaplad33.

«RESULTS 10 Accumulation of CYH2 precursor and PGKI mRNA containing nonsense in a mof4-i strain: The eight previously identified mof mutants CDinman and Wickner (1994) eupra3 were analyzed for determine if they affected the activity of the degradation path of mRNA mediated by nonsense. It has been shown that the abundance of the inexactly spliced CYH2 RNA precursor "containing an intron near the 5T end" is a good monitor of the activity of this path of decomposing CHagan and coauthors (1995) supra »Cui and coauthors (1995) supra; He and coauthors, Proc. Nati Acad. Sci. USA, 90: 7034-7038 (19933. The state of the decomposition activity of the senseless mRNA media can be easily determined by comparing the ratio of the abundance of the CYH2 precursor to the mRNA of CYH2 in an RNA spot: Was the abundance of the CYH2 precursor and the mRNA observed? of CYH2 in cells harboring upfl-, UPF1- (wild type) and the various mof alleles by RNA staining analysis. As shown in Figure 2A, the abundance of the CYH2 precursor was low in UPF1 - * - MOF * e type 5 wild cells, but increased to at least five fold in the upfl ~ strain. An exploration of the abundance of CYH2 precursor in the mof mutants showed that the mof4-1 allele had an elevated level of CYH2 precursor, similar to the observed abundance ^ previously in the upf mutants (figure 2A). The abundance of ÍO precursor CYH2 was also elevated in the cells that housed alleles mof2-1, mof5-1 and mof8-1, although not to the same extent as in the upf or mof4-1 strains (figure 2A). The abundance of mRNA of CYH2 was not affected in those cells (Figure 2A). 15 The mof4-1 allele was further characterized because it caused the maximum accumulation of the CYH2 precursor. To determine if other mRNAs containing nonsense were affected by the mof4-1 allele, a mini-PGKl allele containing nonsense, whose abundance was found in the mof4-1 strain, was introduced. sensitive to UPF1 status in the CZhang cell and coauthors, Mol. Cell. Biol., 15: 2231-2244 (19953. The abundance of wild-type PGKl mRNA and containing nonsense, was determined by RNA staining and the results demonstrated that the PGKI transcript containing nonsense increased tenfold in an mof4-1 strain compared to the abundance in the svest-type cells »similarly to the level in upfl-2 (Figure 2B). The abundance of wild type PGK1 mRNA did not change in any of the cells (Figure 2B). mof4-1 is allelic to the UPFl gene 5 It was then determined whether mof4-1 is allelic to any of the UPF genes previously characterized. Strain mof4-1 was coupled with up l / \ or upf2 / \ and the abundance of CYH2 precursor in diploid cells was monitored. Abundance of _JP precursor CYH2 was low in the mof4-lxup 2 / \ cells (data not shown), but was high in mo-xupf / \ cells, its abundance being equivalent to that observed in an upfl / \ haploid strain (Figure 2C ). Additionally, a strain harboring the mof4-l allele was transformed with centromere-based plasmids harboring the UPF1 gene, the UPF2 gene or the vector alone, and the abundance of the CYH2 precursor was monitored. A mof4-l strain transformed with a single copy of the UPF1 gene reduces the concentration of the CYH2 precursor to the same niel observed in the wild type UPF1-1 cells (Figure 2C, lanes 1-2 and 7).

The plasmid harboring the UPF2 gene or the vector only, not 0 reduced the abundance of the CYH2 precursor in a mof -1 strain (Figure 2, lanes 3-4 and 5-6). Additionally, the efficiency of ribosomal frame shift -1, when determined by pJD107 and pJD108, was elevated in both the mof4-1 and upfl ~ cells, and the UPF1 gene was able to reduce the frame shift efficiency in a strain mof4-1 at wild type levels (data not shown). These results indicate that M0F4 is ale! co UPFl. Recently »Lee and his CLee colleagues and co-authors, Proc. Nati Acad. Sci. USA, 92: 65B7 (1995) 3 have identified eos mutants with increased frame shift (ifs, by "ncreased Frame Shift") in yeast. Using a construct containing the yeast CUP1 gene downstream of a ribosomal frame-shifting signal -1, from the gag-pol junction of mouse mammary tumor virus, the ifs mutants were identified by the loss of sensitivity to copper in the cells CUP! /. These two had efficiencies of ribosomal frame displacement -1 approximately twice as high as wild type cells, when measured by β-galactosidase activities. The IFS1 gene was cloned and its sequence was determined. The comparison of the sequences IFS1 and UPF2 shows that they are identical CCui and co-authors (1995) supra; He and Jacobson (1995) supra3. It has also been determined that ifs2 and mof4-1 remain in the same complementation group by means of β-galatosidase analysis and that the ifs-phenotype of fs2 med can be corrected before the UPF1 gene (data not shown). Both mutant strains ifsi and fs2 were able to propagate Ml (Table 2, see below) »indicating that these mutations affected the efficiency of ribosomal frame shift -1 by less than double.

* TABLE 2 CHARACTERIZATION OF THE MURDERER PHENOTYPE AND SENSITIVITIES TO THE DRUGS PHENOTYPE SENSITIVITY _5 NO. KILLER KILLED TO THE PAR0N0- HIS NICINA (Cl) ** 0 * L-A and M.,. in cells by cyto-tion. The killer phenotype was analyzed by killer plate analysis and the ability to maintain the 5 M dsRNA virus was monitored by RNA staining analysis. ** Strains No. i and No. 2 were developed in liquid medium-Leu and subsequently spread on plates on leucine that lacked minimal medium. A disk containing 1.0 mg of paronomic na was placed on the cell surface. The diameter of the zone of development inhibition was determined after the plates were incubated at 30 ° C for 4 days. Strains No. 3 »4, 5» 6 and 7 are mof4-1 or mof4-l (UPFi :: URA3 Commission of the mof4-1 allele of the chromosome) that host the indicated plasmids. These cells were tested in media lacking uracel and leucine. In contrast to the upf / \ or upflZ alleles, the mof4-l allele affects the maintenance of the virus M_,, It was then determined if the mutations that inactivate the path of decomposition of the mRNA "mediated by nonsense" were able to maintain the virus dsRNA of M .. L-A and Mp were introduced by cyto-duction in strains that host the mof -1 allele of the UPF1 gene »the alleles pfl / \, upf2-1. upf2 /. upf3-1 »upf4-1, ifsl-1 and ifs2-l, and these cells were spread on multiplycation plates in a field of cells that are sensitive to killer toxin. The cells that maintain P.x secrete the killer toxin and a ring of inhibition of development is observed CDinman and Wickner, (1992) supra3. The results of these experiments are summarized in Table 2 and show that only cells harboring the mof4-l allele were unable to maintain the killer phenotype. Consistent with the loss of the killer phenotype, the Ma. Ds RNA of 1.8 kb in the mof4-1 cells was not present, confirming the previous observation that the mutant cells mof4-l also have the Mak ~ phenotype (by MA ntenance of Killer = killer maintenance), that is, they can not propagate Ma- (frame 2 »figure 3). The cells that host the upfl alleles. upf2 »upf3» upf4 »ifsl and ifs2 and the upfl / \ and ifs2 alleles in keeping M. eugiere that the allele mof4-l ee a specific mutation in the UPF1 gene that alters both the degradation of mRNA and the efficiency in the displacement of ribosomal frame -1. Several results show that the Mak- phenotype is a consequence of the mof4 allele instead of a secondary mutation within the cell. A single copy of the UPF1 gene introduced into the mof4-1 cells in a centromere plasmid rescued the ability of the mof4-1 cells to maintain Mn. while cells transformed by vector had no effect (table 2, compare numbers 3 and 4). Additionally, omitting the UPF1 gene from the chromosome in the cells harboring the mole 4-1 allele restored the killer phenotype (Table 2 »No. 5). The tetrada analysis of the cells harboring the roof4-l allele crossed with a strain MOF * »LA *» jj .- * - »showed a 2: 2 segregation of the aseine - * - and killer - phenotype (figure 3) . Additionally »RNA analysis of total nucleic acids. from these spore clones, shows that the 1.8 kb band of dsRNA is present in the spore clones MOF * and is absent in the spore clones mof4-1 (figure 3). The cells that harbor the mof4-l allele are more senescent to paronomycin. Strains harboring mutations that lower translational fidelity have been shown to be hypersensitive to the antibiotic of amni nog and sewn paronomycin, a drug believed to increase the erroneous reading frequency in yeast (Palmer and coauthors »Nature» 277: 148 -150 (1979), Singh and coauthors, Nature »277: 146-148 (1979) .3 Sensitivity to paronomycin was determined for the strains containing the mof4-1 allele, the mof4-1 strain, the mof4 strain. -l which houses the UPFl gene in a plasmid and a mof4-1 strain in which the UPF1 gene (of4-1 (UPF:: URA3)) containing or lacking the mof4-1 allele in a plasmid focused »were developed and paronomycin sensitivity was monitored by comparing the zone of inhibition of development around a disc containing drug. strains harboring the mof4-l allele were more sensitive to paronomycin than the cells harboring the fl s lvestre-type gene pf1 or one allele up l / \ (table 2 »compare No. 2 with No. 1» No 4 with No. 3 or No. 5). Contrary to the strain mof4- 1 »the upfl / \ strain was not more sensitive to paronomycin than the strain UPFl "* - wild-type" which is consistent with previously reported results (Leeds and co-authors »Mol.Cell. Biol., 12: 2165-2177 (1992); Cui and coauthors (1995) supra3. The effect of these strains on paronomycin was a consequence of the mof4-1 allele, since the omission of UPFl of the chromosome of the strain mo 4-1 »makes it as resistant to paronomycin, as the strain mof4-l that hosts the UPFl gene of # type silveetre (table 2» No. 3 and No. 5). In addition »a paronomycin-resistant colony isolated from a parental mof4-l strain maintained Mx and had a wild-type ribosomal frame-shift efficiency (data not shown). The co-reversion of these three phenotypes indicates that they are linked to the mof4-l allele of the UPF1 gene. The effect of paronomycin on the expression of the LacZ gene in the wild type strains was also monitored. * -f mof4-1. It developed cells in liquid media in the presence of different concentrations of the drug and the β-galactosidase activities were determined normalized to the number of cells used in the analysis. The β-galactosidase activity was measured in a strain mof4-1 and the results showed that LacZ expression rose steadily with the increase in concentrations of parono icina (table 3). The activity of β-galactosidase in a wild type strain or in a strain -áBr mof4-1 that hosts the wild type UPFl gene. it was not affected by the addition of paronomycin (Table 3). Taken together. These results indicate that paronomycin exacerbates the defect in a strain mof4-1 and suggests that paronomycin can affect the efficiency of the ribosomal frame shift -1 in a strain mof4-1.

# TABLE 3 EFFECT OF PARONOMYCIN ON THE EXPRESSION OF THE GENE LacZ IN CEPAS mof4-l Paronom ci5 544 ddee --11 ri iboso ico Scrolling frame * na (μg / ml) mof4--1 + mof4-1 0 2.0 9.4 5 2.2 9.2 0 ÍOOOO 3 3.00 9.4 0 2 255 2 2..99 13.5 50 2.0 14.7 100 2.6 250 2.2 17.8 500 2.2 22.2 5 * The cells were JD474-5A containing pT125 (frame control O) or pFS (ribosomal frame-shift tester CDinman and co-authors (1991) supra3). Paronomycin was added to the cells inoculated at Ol D0as, E / ml and developed at 30 ° C for 4 hours. of 0 ß-galacosidase and the% ribosomal frame shift-l was calculated by (pF8 / pT125) x 10054. The average activities of β-galactosidase of the cells with pT125 and of the cells with pT125 + pUPFl was 50.1 ± 7.5 and 48.9 ± respectively 5 Identification of 1 mutation mof4-1 The UPF1 gene had been cloned and the sequence had been determined CLeeds and co-authors (1992) supra; Al tamura and co-authors »J. Mol. Biol., 224 : 575-587 (1992) 3. The amino acid sequence elicited from the UPF1 gene indicates that it encodes a 109 KD protein with motifs of zinc index near its amino terminus, and hosts the appropriate motifs that have to be classified as a member of group I of the ATP-binding RNA / DNA helicase superfamily CAltamura and co-authors (1992) supra; Koonin, TIBS, 17: 495-497 (1992) 3. Next, we wanted to identify the mutation or JT mutations that caused the mof4-1 phenotype. Using the appropriate sensitizers, the PCR products corresponding to the third 5r or two thirds of the UPF1 gene of the mof4-1 strain were isolated and the hybrid genes were prepared between the wild type UPF1 gene and the mof4- allele. 1 (figure 4A). In addition, the complete UPF1 gene of a strain mof4-1 5 was also synthesized by PCR. These plasmids were transformed to an epa upf / \ and the abundance of the CYH2 precursor was determined in these strains. The CYH2 precursor was abundant in cells containing a hybrid in which the 5T segment of the wild-type UPF1 gene was replaced with the 5 * fragment of the 0 mof4-1 allele (FIG. 4)., pmof4AEi_¡-,) »or in cells containing the plasmid pmof4BEA_SS» encoding the complete mof4-1 allele of the UPFl gene. The concentration of the CYH2 precursor was low in the cells harboring the plasmid pmof4ABi_s: »containing the hybrid gene UPFl in which the two thirds of gene 5 was replaced by the DNA fragment of the mof4- allele (figure 4 pmof4ABi__, ). The results indicate that the mutation in the mof4-l allele is located within the third 5T of the UPF1 gene. Consistent with these findings, "only the hybrids containing the portion of the 5 'third of the mof4-1 allele were seneible to paronomycin (Table 2» No. 6 and No. 7). 5 The DNA sequence of the 5 'region of the mof4 allele (1150 nt, a DNA fragment EcoRI-Asp71B) was determined from both plasmids pmof4AEA and pmof4BE (figure 4). The comparison of this section with the wild type UPFl J ^ T revealed that there was a single mutation from G to A in the nucleotide IO 586 in the cysteine rich region, which changes a codon cysteine to a tyrosine (figure 4). Both alleles mof4-l of plasmids pmof4AE3! and of pmof4BE5? they also contained the same mutation from G to A (data not shown). To confirm that the mutation of Cys to Tyr resulted in the mof4 phenotype, a BstXI-Asp71B DNA fragment of 9O0 base pairs, of the wild-type UPF1 gene, with an analogous DNA fragment, of the plasmid pmof4AE.j. or of the plasmid pmof4BE, which hosts the muof4-1 mutation (figure 4 »pMF4XAE and pMF4XBE). The cells that host the hybrid UPFl gene had the same characteristics that strain mof4-1 »had high abundance of CYH2 precursor and were more sensitive to paronomycin (Table 2» Figure 4).

DISCUSSION 25 The results presented here imply that the alleles mo 2-1. mo 4-1. mo 5-1 and mof8-1. which were identified as mutations that increased ribosomal frame displacement efficiencies -1 programmed in the L-A frame displacement site CDinman and Wickner. (1994) supra3 »also increase the abundance of the CYH2 precursor that contains the synenate and the mini-PKK1 mRNA, which suggests that these mutations partially or completely abrogate the ^ _ activity of the degradation path of mRNA mediated by ¥ the nonsense (figure 2A). The strains that contain the allele mof4-1 had the maximum effect on the mRNA degradation activity mediated by nonsense »and it was shown to be an allele of the UPF1 gene (figure 2). The UPF1 gene sequence has been determined and hosts the zinc index motifs »NTP hydrolysis and CAlta ura helicase and co-authors (1992) »supra3. An alteration in the UPF1 gene results in the stabilization of the mRNAs containing the synenate and leads to an eupresssion phenotype of nonsense CLeeds and co-authors »Genes S Dev .. 5: 2303-2314 (1991); Cui and coauthors »(1995). supra Although strains containing the mof4-l and? Pfl / \ alleles both increase the abundance of mRNAs containing nonsense, strains harboring these alleles have signifi- cantly different phenotypes. For example, the strain mo 4-1 is more sensitive to the am nogl sewn paronomycin than a strain up l / \ table 2). Additionally, unlike strain m or 4-1, the up / l strains can support the killer dsRNA * M virus (table 2). The viral particle encoded L-A of 39 nm »has icosahedral symmetry and is composed of 59 dimers Gag and 1 dimer of Gag-Pol CCheng and co-authors, J. Mo1. Biol., 224: 255-258 (1994) 3. Frame shift efficiency ribosomal -1 determines the stoichiometry of the Gag protein a Gag-Pol. The change in efficiency of ribosomal frame shift -1 affects the ability of cells to _ propagar * - CDinman and Wickner, (1992) supra. The loss of Ma ^ 0 in strains mof4-1 can not be explained by stabilizing the mRNA of L- A that contains the frame offset. Excess of the L-A mRNA from a cDNA clone confers a superaseein phenotype (Ski-) in the CWickner yeast cells and co-authors »J. Virol. , 65: 151-161 (1991); Madison and co-authors, Mol. Cell. Biol. , 15: 2763-2771 (1995) 3, the opposite of Mak ~ phenotype. These results suggest that the mof4-1 allele of the UPF1 gene specifically affects the programmed ribosomal 1 frame shift efficiency, changing the ratio of the Gag to Gag-ol products synthesized. It is interesting that the strains containing the upfl / \ alleles, upfl-2, upf2 / \, upf3-1, all of which inactivate the mRNA decomposition pathway mediated by nonsense, equivalent to allele mof4-1, do not promote the loss of A (table 2). This is consistent with the notion that simply stabilizing the L-A mRNA does not promote loss of M RNA virus. Taken together, these results suggest that mof4-1 is a unique allele of the UPF1 gene that raises the abundance of mRNAs containing nonsense. increases the efficiency of the directed ribosomal-1 frame shift and sensitizes those cells to paronomycin. A single change from Cys to Tyr at codon 62. in the UPFl gene. account for the mof4-1 allele of the UPFl gene (figure 4). This mutation is in the region rich in cysteine. of the amino terminal of the UPFl gene (figure 4). A genetic analysis of the UPF1 gene that will investigate the role of upflp in the change of mRNA and the suppression of nonsense. It would show that an omission at the amino terminal that eliminated the cysteine-rich region of the UPF1 gene separated its decomposing activity from its function at the translation end. Cells harboring an upflen allele in which the zinc index region was omitted were able to readily degrade nonsense-containing transcripts but activated their translational termination activity, which allowed for the suppression of alleles in their sense. Taken together, these observations further demonstrate that hpflp has activities involved in mRNA decomposition as well as in the modulation of various aspects of the translation procedure. The mof4-l allele is unique because this lesion inactivates mRNA degradation activity mediated by nonsense and alters the programmed translational frame displacement. The results described here demonstrate that using only frameshift informant debugging is insufficient to identify mutants of ribosomal frame shift β. The mutants upfl / \ »upfl- 2. up 2-1. upf2 / \, upf3-1 »ifsl» ifs2 and mof8 are positive in the informant purifications »but they do not affect the maintenance of the dsRNA virus of M (table 2 and data no 5 shown). Thus, the MAK phenotype allows to distinguish between mutations that affect the efficiencies of ribosomal frame displacement of those mutations that only affect the change of mRNA. ^ mF Viral packaging requires stoichiometry The appropriate amount of Gag to Gag-ol proteins is synthesized. This is often achieved by displacing the ribosomal framework or suppressing nonsense mutations. It is important that the efficiency of the displacement of the ribosomal framework -1 in the mof4-1 cells is high in response to The increasing doses of paronomycin, since this demonstrates that a drug can modulate efficiencies of displacement and framework, supports the notion that displacement of the ribosomal framework can serve as a target or potential target for anti-viral compounds. is. HE anticipates that the identification and characterization of the gene products involved in these procedures and of the drugs that modulate this procedure will lead to therapeutic products to combat viral diseases.

EXAMPLE 2 ANALYSIS OF mof2-l AND SUIL-1 AT THE GENETIC AND MOLECULAR LEVELS mof2-1 is of great interest because: 1) it confers the maximum increase in ribosomal frame shift efficiency -1 of all mutants mof »2) can not propagate satellite virus M» 3) is senescent at temperature »having a classical cell cycle that depends on the high phenotype; and 4) also has a degradation phenotype of mRNA mediated by nonsense mutant (Upf). A clone of the SUI1 gene was able to complement the temperature sensitivity »frame shift and the Upf phenotypes of mo mutants 2-1. Based on these results »this example is proposed: A) analyze mof2-1 and suil-la the genetic and molecular levels; B) determine if the human homologue of the SUI1 yeast gene. HISOSUIl can complement the various mutant phenotypes of mo cells 2-1 »and C) test the hypothesis that the Mo 2 protein (Mof2p) is a general regulator of translational fidelity» that increases the restart regimes of Translation and / or as a stimulator factor on the hydrolysis of nucleotide triphosphate (NTP).

Genetic and molecular analysis of mof2-1 Figure 5A illustrates a map of the original clone (pIB) which was able to complement the mof2-1 phenotype. The omission analysis revealed that a subclone of plS »that hosts the gene * SUI1» was able to complement 1) the growth defect ts ~ »2) the mRNA defect mediated by nonsense; and 3) the Mof phenotype of the mof2-1 cells. The first series of experiments was designed to determine if M0F2 is allelic to 5 SUI1. All the spore clones generated from a genetic cross of mof2-l with mullions suil-1 »were sensitive to the temperature to develop at 37 ° C» evidence that f .m _-__- or ___ f ____-_ 2 _______-- --1 ____ - _ and s _______ u__il-1 belong to the same complementation group. In a second experiment, an integrating vector of IO yeast based on URA3, using a yeast DNA fragment that extends downstream of the SUI1 gene (the H3 fragment to Salí from pl8.) This was linearized with Sphl and integrated into the DNA of a diploid strain mof2- 1 / wild type After the analysis of southern to determine that the fragment had integrated into the correct location of one of the chromosomes »the diploid was sporulated and the resulting spore clones analyzed. The Ura3 - * - phenotype was always cosegregó with the _ * phenotype Mof2-l »which indicates that the sequence of pIB (which is physically linked to SUI11) was also linked physically to the mof2-1 gene. Taken together. these data prove that mof2-1 is an allele of SUI1. It could also be demonstrated that a plasmid harboring the SUI1 gene allowed the mof2-1 mutants to propagate the satellite virus M ,,. The allele mof2-l of SUI1 was cloned using techniques from pol erase chain reaction (PCR) and the sequence of the whole gene could be determined. Sequence analysis revealed that 100 the mof2-1 allele was the result of a base G-> mutation. A »which changes the amino acid residue 115 of a highly conserved glycine to arginine (Figure 5B). This represents a single sui allele: all other suil alleles are grouped into 5 highly conserved amino acid residues D81 and Q82 within the context of LQGDQR (SEQ ID NO: 12) CYoon and Donahue »Mol. Cell. Biol. »12: 248-260 (1992) 3. This figure also illustrates a comparative alignment of the SUI1 homologs of humans (SEQ ID _ T NO: i), of Aedes sp (mosquitoes) (SEQ ID NO. 2) »of rice (SEQ ID NO: 3)» of S cerevis ae (SEQ ID NO: 4) and Methanococcus sp (SEQ ID NO: 5). The human homolog of the yeast SUI1 gene can complement the mutant phenotypes of Mof mediated by Mof and by nonsense of the mof2-1 cells. It has been shown that several mammalian onset factors are able to complement the counterparty defects in the yeast cells and the human SUI1 homologue (hSUIHSOl) has been cloned and its sequence determined. Suip and hSUIHSOlp share an identity of 6054 and 0 a similarity of 8054 (31). hSUIHSOl is able to complement the sensitivity to temperature »the increased efficiency of ribosomal frame shift -1» mRNA degradation mediated by nonsense and the maintenance phenotypes of M of the mof2-1 mutants. These experiments demonstrate the conservation of function between human and yeast homologous genes, which serve as yet another example of the basic biological similarity of these two organisms. These observations are of great help with respect to the investigations herein between the connection between translation initiation, translational lengthening and degradation of mRNA mediated by nonsense in mammalian systems; and it also helps in the process of identifying proteins that can serve as potential targets for the rational design of antiviral agents that affect ribosomal frame displacement - or genetic investigations using the different forms of Suil. Having shown that mof2-1 is a unique allele of SUI1 and that the human homolog can complement all the phenotypes of mof2-1, a series of experiments was carried out. to examine the genetic links between the ribosomal frame shift. viral maintenance and erroneous translation reading that results in the suppression of mutations at the start of translation. To manage this well, we built solid strains of yeast. She turned a haploid cell harboring the wild type SUI1 gene in a low copy URA3 vector, with an integrator vector of its yeast, which interrupts the chromosomal copy of the SUI1 gene. After the selection and confirmation of the alteration by analysis of southern »the vectors of low copy TRP1"that host the wild-type SUI1 gene, the mof2-l allele, the l-1 allele, or hSUIHSOl. in this string.

* Subsequently, the URA3 base vector of the cell was cured using 5-fluorootic acid (5-FOA), leaving only the TRP1 vectors in the cells. Other genetic analyzes examined comparatively the Mof and Sui phenotypes of mof2-1, suil-1 and hISOSUIl (Tables 4 and 5).

TABLE 4 CHARACTERIZATION OF MARKING DISPLACEMENT EFFICIENCIES IN CELLS M0F2 / SUI1 ISOGENAS ÍO Strain ß-alactosidase activity * »54 frame displacement efficiency (5) 15 +1 -1 +1 Y218 (WT) 43.48 0.91 1.39 2.1 3.2 Y219 (mof2-1) 45.46 4.86 1.64 10.7 3.6 Y220 (SU 1-1) 44.70 2.41 1.3B 5.4 3.1 __¿ 20 Y221 (hISOSOUI) 32.38 0.65 0.97 2.0 3.0 TABLE 4 CONTINUED 25 Strain Maintenance Displacement (+) frame (type of that loss (-) tre folded) of M-. dsARN 30 Z [_ Y21B (WT) 1.0 1.0 Y219 (mof2-1) 5.1 1.2 Y220 (su 1-1) 2.6 1.0 35 Y221 (hISOSOUI) 1.0 1.0 * These experiments were carried out in at least three different colonies of each strain. The ß-galactosidase activity unit was presented as activity / DO ^ oo / hour and -5 ranged more than 1554. Maintenance or loss of MA dsRNA virus was determined by both M2 killer analysis and agarose gel RNA electrophoresis.

TABLE 5 10 SUPPRESSION OF His4u "« IN CEPAS SUI1 / M0F2 Strains (alel o) 4 Activity Activity Proportion Suppression of ß-gal of ß-gal from UUG / AUG of UUG in HIS4AU < a in s4uu «(X) (type 15 l acZ lacZ sil bending folded Y218 (WT) 8.6 0.22 2.5 l.O Y219 (mof2-1) 7.1 1.14 16.0 6.4 Y220 (suil-1) 4.6 0.68 15.6 6.2 Y221 (hIS0SUIl) 6.1 0.19 3.0 1.2 * These experiments were carried out in SUI1 / M0F2 isogenic strains. The numbers were the average of the measurements in three ndependent colonies. The activity of ß- was presented galactosidase as activity / DO ^ oo / hour with variations of no more than 1554. Table 4 shows the characterization of the displacement efficiencies of frame of S0F2 / SUI1 solid cells. The efficiency of frame travel Ribosomal-1 of mof2-l cells is approximately five times larger than their wild-type counterparts. The sui1-1 mutants also increase the ribosomal frame shift efficiency -l about 2.5 times above the wild-type levels. However, this is not a sufficient increase to promote the loss of the satellite A virus, that is, only the mof2-1 mutation confers the Mof phenotype on the cells. The human homologue seems to be fully capable of replacing the yeast gene »further supporting the notion of evolutionary conservation of the Suilp function. It is interesting that none of the forms of His tests had no effect on the displacement of ribosomal frame +1 »promoted by Tyl. Table 5 reports the Sui phenotypes of the constructions herein. This analysis shows that mo cells 2-1 also have a complete Sui phenotype »promoting the efficient suppression of the his4UiJ < to.

Again the human sonogen is able to complement the yeast gene.

Characterization of the GCN4 depression phenotype of sui and mof2-1 mutants The GCN4 gene is repressed by a translation control mechanism in which the short open reading frames »upstream of the gene (uORFs) are transited in the presence of overflows abundant from tRNA to noac sides. This causes the ribosomes to be transected to dissociate from the mRNA before being able to restart at the beginning of the GCN4 message. Under conditions of amino acid depletion, GCN4 is repressed by means of the increased restart levels at the start codon of GCN4. The sui2 (elF-2) and 5 SUI3 (eIF-2ß) mutants abolish the repression of GCN4"which leads to constitutively depressed GCN4 expression" and the transcription activation of HIS4 independent of amino acid availability. * F It was tested if the forms mof2-1, suil-1 and hSUIHSOl IO were able to de-repressor GCN4 by measuring the β-gal activity expressed from the GCN4-lacZ fusion constructs. The activity of the enzyme GCN4-1acZ is de-repressed approximately 10-fold in response to histidine depletion in wild-type cells (38). If the mutations suil-1 and mo 2-1 would have produced increased expression of GCN4-lacZ, it would be suggested that those mutations "like mutations sui2 and SUI3, al were the mARM translation control of GCN4. Figure 7 shows that neither mof2-1 nor suil-1 de-represses the expression of GCN4. This suggests that Apparent increases in ribosomal frame shift -1 are not due to increased levels of ribosome clearance or internal start in the lacZ reporter mRNA outside of frame, further supporting the conclusion that mof2- mutants 1 y. to a lesser degree suil- 25 1 »actually exert their effects at the level of the ribosomal frame shift -1.

Experiments directed to the biochemical characterization of Suilp. SUI »mof2-1 had been prepared. suil-1 and hSUIlISOl, which contain the FLAG epitope marked at its N-terminal ends to evaluate biochemically SUIlp. These clones are able to support the growth of yeast cells in the absence of other SUI1 forms, which indicates that the addition of the epitope tags does not interfere ÍO with the Suil-p function. These recombinant proteins had also been expressed using high copy vectors in E. coli and are capable of isolating large amounts of pure protein using immunoaffinity columns. An analysis of displacement of ribosomal frame -1 in vitro using extracts of yeast cells. Preliminary experiments using the isogenic strains described above have shown that the general pattern observed in vivo remains applicable for the in vitro system, ie that the efficiency of the ribosomal frame shift -1 is approximately 5 times greater in extracts of mof2-l cells and 2 times higher in extracts of sui1-l cells (data not shown).

Investigations about the possible ribosomal association of 25 Suilp, Mof2-lP, Suil-lp and hISQSUIlP. The fact that mof2-l has effects on both the ribosomal frame-1 shift and the nonsense-mediated mRNA decomposition suggests that Suup may be involved in some translational processes beyond the start. Western blots of 5-fold fractions can be probed for Suilp to determine if this protein is present in elongating ribosomes. For example, an anti-FLAG antibody can be used to visualize the bands in cells harboring Su? L proteins tagged with FLAG. ^ f Suilp. Suilp in the fractions corresponding to the IO lengthening ribosomes can be compared to those that shine from extracts of cells harboring mof2- 1 and suil-l mutations marked with FLAG. It is possible to carry out the binoculars profiles under different conditions »for example» growth rate »temperature» treatment with drugs (cycloheximide »puromycin) and salt concentrations. An alternative approach is to subject cells growing logarithmically to moderate lysis, and to cluster them using gel filtration techniques. An anti-TCM1 antibody (ribosomal L3 protein) can be used to identify the various fractions of ribosome. The polysome fractions, which must be correlated with the ribosomes in the elongation phase, "will be larger than the monosomic fractions and" in such a way "they must be eluted faster from the column. These fractions can be probed for different Suilp isoforms marked with FLAG »using the anti-FLAG antibody. Quantitative Western spotting techniques can be used »in an effort by ÍOB discern the qualitative differences in the union of the different Suilp isoforms.

Examine the effects of Mof2- P «suil-IP and hSUIHSQp type 5 wild, purified. on the hydrolysis of GTP with purified G proteins »is known to be involved in the lengthening phase of the translation Normal analyzes were used to determine the Jjf basic line speeds of hydrolysis with GTP of EF-la and EF-2B CKinzy and Coolfors, Genetics, in press; Merrick » Enzymol. 60: 108-123 (1979); Perentesis and co-authors »J. Biol.

Chem .. 267: 1190-1197 (1992) 3. The different purified isoforms of Suilp are added to these reactions »to determine their effects on the rates of hydrolysis in GTP. Hydrolysis in GTP increased with Suilp »by one or both proteins, would support the conclusion that Suilp also > • 'acts during the lengthening of translation. »The examination of the effects of Mof2-lP» Suil-lp and hSUIHSOp of wild type »purified» on the hydrolysis of ATP »using Upflp. it is known that it is involved in the lengthening phase of the translation The observation of the stimulatory or inhibitory effects substance the turnover of Suilp in the passage of peptidyl transfer during the translation (suppression of sense occurs at the end »what happens in the peptidyl transfer step). In case the effects of Suilp on ATP hydrolysis mediated by Upflp are observed, the effects of the Suilp isoforms can also be examined. If Suilp affects the ATP hydrolysis mediated by Upflp »any change in the stimulatory / inhibitory effects of Suilp on the Upflp mutants will be of particular interest.

Gene dosage experiments 10 These experiments are designed to determine whether excessive expression of EF-the »EF-2T or Upflp can suppress Tas mof2-1 or suil-1 mutations. The mof2-1 and sui1-1 cells can be transformed with high copy plasmids harboring the TEF2 »EFT1 or UPF1 genes. The deficiencies of displacement of ribosomal framework -1, the ability to maintain M ^. "The decomposition phenotypes mediated by 0 'nonsense and the ability to suppress the allele his4ULJa, can be evacuated.

EXAMPLE 3 IDENTIFICATION OF DRUGS THAT ALTER RIBOSOMAL DISPLACEMENT AND INHIBIT VIRAL PROPAGATION A critical step in viral propagation is the assembly of the viral particle »that partially depends on the availability of the correct relative amounts of viral proteins. Thus, although the maintenance of the translation reading frame is considered to be fundamental for the integrity of the translation process and "finally" for the development and viability of the cells, many cases have been described in which the ribosomes they are aimed at displacing the reading frame by means of visual signals »in order to guarantee the appropriate proportions of structural proteins (Gag) to enzymatic (Gag-pol)» available for the assembly of viral particles. Said ribosomal frame shift signals »for the most part» are seen in RNA viruses »for example» retroviruses and in the retrotransportables elements »coronaviruses» astroviruses, totivirus and some non-segmented (+) ssRNA viruses of plants . The ribosomal frame release also has been described in bacteriophage T7 and a number of bacterial transposons »as well as in a few bacterial cell genes and in mammalian chromosomal genes. Viral frame shift events produce fusion proteins in which the domains of the C terminal and the N terminals are coded by two open structure frames - different overlaps. The ribosomal frame shift in the -1 direction requires a heptamera sequence, X XXY YYZ (the O frame is indicated by the spaces) called the "slip site" CJacks and Varmus »Science 230: 1237 (1985) 3. The simultaneous sliding of site A and site P tRNAs linked by ribosome "by a base in the direction 5", it still leaves its non-tilted bases correctly paired in the new reading frame. The ribosome-bound tRNAs must first disappear from the 0-frame and then re-pair with their non-tilted bases "to the frame-1. A second frame-displacement promoter element" usually a pseudonudo RNA, is immediately located 3T with respect to the site. Sliding. The pseudoknot of mRNA causes the ribosomal pause on the displacement site while the ribosomal sites A and P are occupied. respectively »by the aminoacyl-tRNA species (aa-tRNA) and 1-tRNA peptidi. It is believed that the RNA pseudonym promotes the ribosomal pause on the site of displacement, which increases the probability of ribosomal movement 5"CSomogyi and coauthors" Mo. Cel 1. Biol. 13: 6931 (1993); You and co-authors »Proc. Nati Acad. Sci. USA B9: B636 (1992) 3. The effects of two inhibitors of pep id l-transferase »anisomycin and sparsomycin» on ribosomal frame shift efficiencies »and on the spread of yeast dsRNA viruses» were examined. These drugs specifically alter the efficiency of the ribosomal frame shift -1 but not +1 »both in vivo and in vitro» and promote the loss of two viruses (LA and L-BC) »that use a ribosomal frame shift -1 in yeast cells grown at sublethal doses of these drugs. Both drugs also change the efficiency of ribosomal frame shift -1 in a rabbit reticulocyte system, suggesting that they may have applications for RNA viruses with higher eukaryotic content, which also use this translational regulatory mechanism. The results of the present offer a new series of compounds to the arsenal of antiviral agents, which have a potentially wide scale of applications in the clinical, veterinary and agricultural fields. To examine the effects of these drugs on efficiencies in ribosomal frame shift and vivo, the selective medium containing the indicated concentrations of anisomycin or sparsomycin was inoculated with "equal amounts of logarithmic growth yeast cells" that harbored the control of frame O (pT125). the ribosomal frame shift test plasmids -1 »derived from L-A (pFB) or the ribosomal frame shift test plasmids derived from Tyx (pJD104) CBalasumdaram and co-authors» Proc. Nati Acad. Sci. USA »91: 172 (1994) 3 and incubated at 30 ° C for specific times» after which the activities of β-galactosidase (β-gal) were determined. Two important series of information were obtained from these experiments (figure B). First, as indicators of the effects of these drugs on translation in general, the activities of ß-gal were monitored from cells containing pT125 developed in different concentrations of drug. This series of data shows that these drug concentrations do not decrease the overall translation to less than 8054 (of aniso icine) (figure BA) or less than 5054 (sparsomycin.) (Figure BB) of the controls without drug. of data »the measurement of ribosomal frame shift efficiencies -1 and +1» was determined by calculating the proportions of ß-gal produced from cells harboring pFB or PJD104 »divided by the β-gal activities of the cells harboring pT125 »developed at equivalent concentrations of drugs Here the data show that» in general »increasing the concentrations of sparsomine tends to increase the efficiency of the ribosomal frame shift -1 (figure BC), while anisomycin has the opposite effect, that is to say »decreases the deficiencies in the displacement of the ribosomal frame -l (figure BD) As predicted» no drug had any effect on the displacement of The osomic kidney frame +1 To determine if the changes in the values of the crude ß-gal activity were a consequence or not of the half-lives of ß-gal diminished (with anisomycin) or increased (with esparsom c na) was measured the effects of these drugs after numerous different periods of incubation. The results remained consistent, ie the longer incubation times did not result in major changes in the efficiencies of the ribosomal frame shift -1 (data not shown). To follow consistent »all subsequent in vivo measurements of ribosomal frame displacement were analyzed after 6-hour incubations with these drugs. Apparent changes in the efficiencies of the ribosomal frame shift could also be due to reduced (with anisomycin) or increased (with sparsomycin) abundances of the frame shift reporter mRNAs. To face this, RNA was extracted from cells at half logarithmic growth phase at different concentrations of anisomycin or sparsomycin »equal amounts of RNA were separated by means of a denaturing agarose gel» was transferred to nitrocellulose and probed with the radiolabelled lacZ probe. The abundances of lacZ mRNA were equivalent to all tested drug concentrations "suggesting that drug-induced changes in the abundance of reporter mRNA was not responsible for apparent changes in ribosomal frame shift deficiencies -1 (data not shown). The model also predicts that host cells harboring mutations in the gene products involved in peptide l-transferase center formation must: (1) have ribosomal frame shift efficiencies different from those of wild-type cells; (2) have viral propagation defects and (3) neither anisomycin nor sparsomycin should have any additional effects on the efficiencies in the projection of the ribosomal framework -1. The TCM1 gene (MAK8) encodes the ribosomal protein L3 CFried and Warner »Proc. Nati Acad. Sci. USA 78: 238 (19B1); Fried and Warner Nucí. Acids Res. »10: 3133 (1982); Wickner and co-authors »Proc. Nati Acad. Sci. USA, 79: 4706 (19B2) 3 and the makB mutants provide an appropriate test for these criteria. The tcml / makB mutants can not maintain Ma._ "and are resistant to anisomycin and a variety of other peptidyl transferase inhibitors Jiménez and co-authors» Biochim. Biophys. Acta 383: 427 (1985) 3. The efficiency of the ribosomal frame shift -1 in the makB-2 cells »in the absence of drugs» was determined at approximately 5254 (figures 9A »B without drug). This value is 2.9 times higher than the cells of wild type (1,854) »which is consistent with the notion that conditions affecting the peptidyl trans erasa center should change the efficiency of the ribosomal frame shift -1. As predicted »none of these drugs affects the efficiency of frame travel ribosomal -1 (figures 9C »D). Although increasing doses of anisomycin or fl ^ * sparsomycin tend to inhibit translation in wild-type cells in vivo, they have opposite effects on the efficiency of ribosomal frame shift -1. For To determine if these drugs are acting at the same step in the translation "a mixing experiment was analyzed (figure 10). These results show that »with respect to the effects on the ribosomal frame shift -1» the anisomycin and the sparsomycin cancel each other out. This is consistent with The fact that both act in the passage of peptidi 1-transferase in the translation "but do not distinguish between them compete for the same sites" by overlapping sites or by separate binding sites. By changing the efficiency in the march of ribosomal -1 »these drugs should change the ratio of Gag to Gag-pol proteins» obtainable for the assembly of viral particles »interfering consequently with viral propagation. To cope with this, wild-type cells were cultured in a rich medium containing different concentrations of anisomycin or of sparsomycin. After 24, 48 »72» 96 or 120 hours »aliquots of cells were scratched for individual colonies» in a rich medium »which were then reproduced in plaques on indicator to mark their killer phenotypes. Figures HA »B» show that the loss of the killer phenotype was correlated with both increased drug dosage and multiple changes in ribosomal frame shift efficiencies -1. Although the killer phenotype is maintained stably in JDBB cells. in the absence of drugs »the development in anisomycin 3.8 μmol resulted in approximately 5354 loss of the" total "colony forming units of the killer tract" after only 48 to 72 hours (figure HA). More dramatically, delearning in the presence of 2.6 μmol of esparsomine resulted in a loss of 70 to 7554 in the killer phenotype (Figure 11B). To determine if the loss of satellite virus Mn. was responsible for the loss of the killer phenotype »a non-killer colony (K ~) of each drug concentration was collected in the 72-hour data series» randomly »and extracted total nucleic acids (TNA) and CPark co-authors» Virology 216: 451 (1996) 3. Approximately equal amounts of TNA were separated through a non-denaturing TAE-agarose gel at 154 ° and stained with ethidium bromide (Figure 12A). As expected "is not present to M band. dsRNA of l.B. kb in the K ~ samples (treated with drug). Additionally »it seems that L-A has also been cured with these drugs. For confirm this "the RNA was denatured in the gel" was transferred to nitrocellulose and hybridized with LA probes labeled with C3a! P CTP and filament RNA (+), from Ma .. Figure 12B confirms that these samples do not hybridize with any of the probes »which confirms that the loss of killer phenotype was a consequence of the loss of L-A »what < supports the notion that drug-induced changes in the efficiency of ribosomal frame shift-1 interfered with the assembly and propagation of viral particles of L-A. 20 Recently »the L-BC cDNA sequence (also called La), a ubiquitous minor dsRNA virus from yeasts, has been determined by CPark and co-authors supra. L-BC also uses the classic -1 ribosomal frame shift mechanism in the production of its Gag-pol function.

However, L-BC has shown that it is much more difficult to cure cells than L-A CHopper and co-authors, J. Biol. Chem. # 252: 9010 (1977) 3. It is interesting that after 72 hours at concentrations of 3.8 μmoles of anisomycin »the L-BC band dsRNA also seems to be absent (Figure 12A). However, "no specific probe for L-BC is available" and this could not be confirmed by Northern blotting. However, these data suggest that in addition to promoting the loss of L-A and M, the reductions induced by anisomycin in the ribosomal frame shift -1 also promotes the ^ 0 ^ loss of L-BC. The specific actions of antibiotic on the translational apparatus were classically analyzed using in vitro systems. To test the effects of anisomycin and sparsomycin in vitro, an in vitro yeast-based translation system was used to monitor the efficiency of ribosomal frame shift -1 »using a luciferase-based reporter system. The system consisted of yeast extracts competent for the translation »from an L-A-strain or» L-BC-or wild-type strain, to which control was added for March O or a mRNA Luciferase reporter, ribosomal frame displacement tester -1. Figure 13 shows that the trends observed in vivo were reproducible using the in vi ro system, that is, by increasing the concentrations of anisomycin, the efficiency of the displacement of ribosomal framework -1 »while increasing the concentrations of sparsomycin increased ribosomal frame shift efficiencies -1. Some interesting differences were observed between in vivo and in vitro systems. First, "the effective concentrations of these drugs were three orders of magnitude lower in vitro" very likely a reflection of drug absorption and metabolism in intact cells. Secondly "at those lower doses of drug" both drugs stimulated ___ initially the production of luciferase from the mRNA ^ ¥ ^ LUCO reporter. However, on the stimulatory scale »the trends in the ribosomal frame shift remained the same. Third, the basic line efficiency of ribosomal frame firing -1 is significantly greater in vitro than in vivo. This could indicate differences in the stability of the reporting mRNA in the two systems. Since the ribosomal frame shift reporter mRNA has two frame termination codons within the first 200 bases of its 3.3 kb mRNA, it constitutes a sinsent do-mRNA. This makes it a substrate for attack by the trajectory of displacement of mRNA mediated by nonsense. In fact, cells harboring mutations in the genes involved in this pathway establish this reporter mRNA and produce increased apparent efficiencies of ribosomal frame shifting -1 in vivo. The differences observed in lae Baseline efficiencies of ribosomal frame shift -1 may be an indication that the mRNA degradation pathway mediated by nonsense may be partially or totally inactivated in an in vitro translation system. The in vitro system also differs in that it contains "RNase inhibitor" RNAs which can help stabilize the LUC-1 reporter mRNA against general nucleotide attack. These factors are most likely responsible for the higher baseline efficiencies of the ribosomal frame shift -1 observed in this ^^ * "report and in other reports using systems in vitro translation, and underlines the importance of using efficiencies in ribosomal frame displacement »determined in vivo» as the most reliable indicators of Gas-to-Gag-pol ratios determined virally. Anisomycin and sparsomycin affect the efficiency of ribosomal frame shift -1 and viral propagation in yeast; But these results translate into higher eukaryotes ?. As a first step, LUCO mRNA and LUC-1 were used to measure the effects of these two drugs on translation and frame displacement. ribosomal -1, and in the reticulocyte translation system of rabbit, in vitro. Figure 14 shows that the same general trends that were seen in yeast-based systems are recapitulated in the reticulocyte system, ie »anisomycin inhibits and sparsomycin stimulates the ribosomal frame offset -1. There were some notable differences; Firstly, reticulocyte extracts were approximately 10 times more sensitive to anisomycin and 100 times more sensitive to sparsomycin than yeast extracts. At that concentration scale, both drugs stimulated the total production of luciferase to from the LUCO mRNA »which is not consistent with the lower-scale drug concentration data of yeast extract. With respect to the frame shift _ ribosomal -1 »the reticulocyte system was much more ^ F ^ sensitive to both drugs. The greater the concentration of anisomycin (15.2 nanomoles) plus reduced the efficiency in ribosomal frame shift ~ 1 »to only 354 of the control without drug» which closed it. The maximum concentration of sparsomycin stimulated the displacement of the ribosomal framework -1 14 times »or from approximately 654 in the control without drug until almost B45I »that is to say» almost as many ribosomes were induced to move the reading frame »as they were in the frame. As with the in-troduction system of the yeast, the initial efficiencies of the ribosomal frame shift -1 were greater than the observed in vivo and again most likely due to the stabilization of LUCO mRNA in this system. The antiviral activities of these antibiotics against HIV were also tested. Figure 15 shows that these drugs decreased HIV viral titers Approximately 3054 of the controls without drug at concentrations of anisomycin that were 1500 times lower than the minimum inhibitory concentration of anisomycin (the MIC of anisomycin is approximately 1.5 μg / ml) and 50 times lower than the MIC of sparsomycin (the MIC of sparsomycin = 50 nanograms / l). Thus, these two peptide inhibitors have antiviral properties predicted by the model presented in Figure 1 to concentrations that are well below toxic levels for human host cells.

EXAMPLE 4 DEMONSTRATION OF NON-REDUNDANT PHENOTYPES OF PROTEINS UPf The TCM1 gene encodes the L3 protein of the subunit of ribosome 60S, which has been implicated as a component involved in the activity of peptid 1-transferase (Shulz and Nierhaus, 19B2). The TCM1 is equal to MAK8 »a gene necessary for the maintenance and propagation of the satellite virus of double-strand RNA Ma .. A genomic fragment of TCM1 / MAK8 (L3 / \) encoding the N-terminal 100 amino acids of L3 »was isolated as an upf suppressant -» a mutant allele that stabilizes mRNAs containing nonsense. This fragment can also act as an antisupresor of upf1-2, but not of upf3-1, two other alleles that are involved in the degradation path of mRNA, mediated by nonsense. A full-length clone of TCM1 / MAK8 did not have this anti-suppressive activity. The expression of L3 / \ has no effect on the stability of the nonsense mRNA, neither in the wild type nor in the upf mutant cells. Expression of the L3 / \ fragment conferred an Mof phenotype on the cells of wild type »which increased the efficiency of the ribosomal frame shift -1» but not +1 »and the efficiency of the ribosomal frame shift -1 also rises in the ^ strain makB. The polysomic profiles of strains that host the ^^ fragment L3 / \ showed reduced levels of 60S subunits » compared to wild-type cells »characteristic with most mak mutants and killer assays demonstrated that episomal expression of the L3 / \ fragment confer a" dominant "MaK-negative phenotype on the wild type" and the Ski- strains, the upfl and upf2 are normally capable of A and the expression of L3 / \ increases the killer loss rates in that cell. The upf3 mutants have a Mak- »phenotype that becomes complete in the presence of L3 /. These data imply the involvement of the peptidyl transferase center in the maintenance of the framework in the independent translation of the degradation path of mRNA mediated by nonsense.

THE MATERIALS AND THE METHODS Restriction enzymes from Boehringer were obtained Mannheim »New England Biolabs and The radioactive JI nucleotides of NEN or Amersham were obtained. The oligonucleotides used in these studies were acquired from the DNA synthesis center UMDNJ-RWJ.

Isolation and characterization of clone L3 / \ Ycp50 was used in these studies (Ausubel and coauthors » 1992) and YCplac33 (Gietz and Sugino »1988). Clone L3 / \ was isolated from a yeast genomic bank YCp50 (purchased from ATCC) »that ^^ was prepared from a partial Sau3A digest »as it was? previously described (Cui and co-authors »1995). Briefly »strain PLY136 of upf2-1 his4-3B SUFl-l was transformed with this bank and a total of 5000 Ura - * - transformants were purified by means of plate reproduction» on minimum medium lacking uracil and histidine »and developed at 30 ° C or 37 ° C for 3 days. The colonies that grew at 30 ° C but not at 37 ° C were collected and tested again. We isolated 9 strains that harbor the plasmids (YPF2-1 to YPF2-9). To confirm that the loss of suppression was due to the plasmid, the transformant strains were cured with 5-fluoro-orotic acid (5-FOA) (Rose and co-authors »1990) and was tested for its ability to suppress 37 ° C development» in selective media. Then the plasmids of those strains were isolated and propagated in E. coli. It was found that YPF2-4 and YPF2-9 were identical (they were named YcpA9) »each of which contained the gene fragment.

L3 /.

Sublongation of gene fragment L3 / \ YcpA9 was analyzed by restriction endonuclease mapping. Fragments of the DNA insert in YcpA9 were subcloned into yeast centromere plasmid YCplac33: YCpESp (2.7 kb EcoRI-Sphl DNA fragment) »YCpESn (4.2 kb EcoRI-SnaBI DNA fragment), YCpBS (BglII-DNA fragment) Exit of 4.3 kb) »YCpNS (3.5 kb Ncol-Sall DNA fragment)» YCpPS (PvuII-SalI DNA fragment of O.9 kb). Plasmid YCpdP is a derivative of YCpA9 in which the PvuII DNA fragment was omitted.

Marking of the L3 / \ fragment with the FLAG epitope »creation of a 13 / \ -B-gal fusion protein and sublining The Oligo 5'-ATAGGATCCTTAACCGGCCGGACAGTAATA-3 * (SEQ ID) was used for the polymerase chain reaction (PCR) NO: 13) corresponding to the 5 'of the gene TCM1"and the oligonucleotide 5" -ATAGGATCCTGTCATCGTCGTCCTTGTAGTCTCTCAAACCTCTTGGGGTT-3 * (SEQ ID NO: 14) "containing the FLAG epitope sequence and the complementary sequence at the 3' end of the region encoder L3 / \ »using genomic DNA from wild-type cells» as a template, the PCR products were digested with Bgl II and BaroHI »and cloned into vector YCplac33.The complete TCMl / MAKB gene was also cloned by chain reaction Polymerase was subcloned into YCplac33 and YEplacl95.PJD134 was constructed by cloning a BglII / HindlII blunt end fragment containing the transcription terminator PGK1"to a blunt-ended NorI site of pRS3l5 (Si orski and Hieter» 19B9). subcloned the frag ment L3 / \ BamHl »marked with FLAG, in pJD134 to create pJD138. a CEN6 LEU2 vector containing the transcription termination sequence PGK1. downstream of the L3 / \ fragment tagged with FLAG »from terminal C. PJD139 contains the transcription terminator L3V-FLAT-PHK1» subcloned into the URA3 vector of 2 micras pRS426 (Christianson and co-authors »1992).

Cloning of upf3 The strategy used to clone the UPF3 gene was the same as that used to clone UPF2 (REF) and the Ltt / fragment. The strain PLY139 of upf3-1 was transformed with a bank Ycp50 (Rose and co-authors) and a total of 2 x 10"URA * transformants was purified by plaque reproduction on minimal medium lacking uracil and histidine and developed at 30 ° C or at 37 ° C for 3 days. that only grew at 30 ° C were selected for further analysis and a YCpBl plasmid that harbored the UPF3 gene was confirmed by common and current genetic methods "including the construction of alleles of gene folding." Subsequent subelonation revealed that an Asp7l8 fragment -B lII of 2.1 kb was sufficient to complement the upf3 mutations and the sequence analysis of this clone showed that it was identical to the previously reported upf3 sequence (REF).

Preparation of polysome profiles Poly isome extracts were prepared according to Baim and co-authors (1985), but modified as follows. Cycloheximide was added to 200 ml of cultures (DOβoo = 0.7) of cells harboring pJD139 or pRS426"at a final concentration of 100 μg / ml" and mixed with 100 ml of ice-cold medium containing 100 μg / ml of cyclohexane. ? imida. The cells were immediately harvested by centrifugation and washed twice with extract buffer (10 mmoles Tris »pH 7.4» 100 mmoles NaCl, 5 mmoles MgCl-s »100 μg / ml cycloheximide, 200 μg / ml heparin). The cell pellets were resuspended in an equal amount (weight / volume) of the extract regulator. About 0.25 ml of acid was washed, glass beads were added (Thomas Scientific Co.) and the cells were lysed by vortexing the suspensions eight times for 15 seconds "with a 30 second cooling period on ice afterwards. of each agitation. 1 ml of the lysate extract regulator was added and the lysate centrifuged once at 5,000 g for 5 minutes and then once at 10,000 g for 10 minutes. 30 units of D02 (SO of the supernatant were applied to 12 ml of sucrose linear gradient of 754 to 4754 »containing 50 mmoles of Tris-acetate (pH 7.4) 50 mmoles of NH ^ Cl» 12 mmoles »of MgCl ^ and 2 mmoles of 2-mercaptoethanol The sucrose gradient was centrifuged at 39,000 rpm for 150 minutes in a SW41 (Backman) rotor and the polysome profiles were collected using a density gradient fractionator »model 640 (ISCO) and recorded by a VA-6 UV / VIS detector (ISCO).

Assay analysis, frame displacement analysis and total nucleic acid extraction and analysis. Yeast strains harboring the L-A and M viruses were transformed. with the L3 / \ fragment in high copy or low copy plasmids or with the vector only. The killer analysis was carried out as previously described (see example 1), by means of colonies that reproduce in plates on plates of 4.7 MB freshly seeded with a seeding of 5 x 47 killer indicator cells (0.5 ml of a suspension at 1 optical density unit at 550 nm per ml per plate). After 2 days at 20 ° C the killer activity was observed as a clear area around the killer colonies. To quantify the loss of killer activity, the colonies that had been identified as killer * were re-scored for individual colonies and the percentage of killer colonies was determined. The efficiencies of ribosomal frame displacement -1 and +1 were determined, as previously described »using the O frame control, the ribosomal frame shift test plasmids -1 and +1. Analysis of β-galactosidase, using the fusion reporter L3 / \ - lacZ, also used this method. Total nucleic acids (TNA) were extracted from the # cells as previously described (Dinman and Wickner »1992» 1994). Equal amounts of TNA were separated by means of 1.054 agarose gels and visualized with ethidium bromide. TNA was denatured in the gels at 45 ° C for 30 minutes in 5054 formamide »9.2554 formaldehyde» lxTAE »the gels were washed with water and the nucleic acids were transferred to nitrocellulose.

I-? Preparation of radioactive probes 10 DNA probes with high specific activity were labeled with Ca ZP3dCTP, by extension of random hexonucleotide sensitizer. A 0.6 kb EcoRI-HindlII fragment of the CYH2 gene was used as a probe to monitor the abundance of the CYH2 precursor and the CYH2 mRNA. A fragment Bgl-SalI of 1.2 kb »of the HIS4 gene was used as a probe to monitor the mRNA abundance of his4-38. A Smal / HindIII fragment of 3.1 kb »of pT125» was used as a probe to monitor the abundance of lacZ mRNA. The RNA probes of strain L-A and Ma (+) were formed » as previously described (Dinman and Wickner »1994). using T3 RNA polymerase shifts transcripts. marked with Ca3StP3CTP. of the pLMl digest of EcoRV (to hybridize the filament L-A (-)) and the strain p596 (M? (-)) was digested with PstI. The membranes were prehybridized for 5 hours at 55 ° C in formamide to 5054 »5X SSC. 50 mmoles of NaHPO ^, pH 6.8 »0.154 SDS» 1 mmole of EDTA »0.0554 each of BSA» Ficoll and polyvinylpyrrolidone »and hybridized at 10" 7 cpm of each probe in the same buffer at 55 ° C »overnight The membranes were washed in five changes of O.IX SSC »0.154 SDS at 65 ° C for 20 minutes, and exposed for autoradiography.

THE RESULTS Previous results have shown that the mutation or omission of UPF1 genes. UPF2 »UPF3» result in the sensitization of mRNAs that contain nonsense. Combinations of mutations or omissions of UPF genes do not result in additional establishment of mRNAs containing nonsense, which indicates that these proteins can function as a complex. Consistent with this notion » recent results have shown that Upf2p interacts with Upflp and Upf3p. Two series of results that fw * monitor the programmed ribosomal frame shift and the frame shift suppression, which indicate the mutation or omission of the UPF3 gene, will be presented. that show unique phenotypes that do not are observed in strains that harbor mutations or omissions of the UPF2 or UPF3 genes.

Identification of an antisupressor of his4-38 in upf2-1 and upfl- 2 »but not in upf3-1 mutant cells. 25 The omission or mutation of the UPF2 gene results in the stabilization of the mRNAs containing the synendido »the suppression of nonsense and» in combination with the phenotypes 2 »the wild type UPF2 gene was isolated by purifying a strain that housed upf2- l his4-3B SUFl-l »with a genomic bank» and identifying cells that no longer grew at 37 ° C in a medium lacking histidine. At that time », another clone (YCpA9) was isolated which also prevented the growth of the upf2-1 mutant strain at 37 ° C» in a histidine lacking medium. Since this did not code UPF2, the ability of this clone was tested to act as an ant suppressor of upf1-2 and upf3-l 10 in strains harboring the his4-3B SUFl-1 alleles. YCpA9 was able to act as an antisupresor of upfl-2 and upf2-1 »but not upf3-1.

The N-terminal one hundred amino acids of the gene product TCMl / MAKB are responsible for the antisupreßor phenotype upfl-1 and upf2-l YCpA9 contains a yeast genomic DNA insert of 6.4 kb. Several low-copy plasmid subclones based on Ycplac33 were constructed and transformed into strain PLY36 of upf2 »in order to locate the functional fragment. A 600 nt fragment at one end was sufficient to abrogate the alosuppression phenotype of upf2-1 and upfl-2. Sequence analysis showed that this fragment contains the complete non-transiated 5 * region and the first 300 nt (100 amino acids) that is to say »the fourth part of the N terminal of the TCMl / MAKB gene that encodes the L3 protein of the 60S ribosomal subunit» and which is thought to be involved in the formation of the 1-trans erasa peptidi center. This fragment was called L3 /. The effects of episomal expression of the full TCM1 / MAKB gene were also tested "and it was found that it conferred the same types of development as the strains transformed with the vector only" that is "no antisuppression of upfl-2 or upf2- 1. Thus »the expression of the L3 / \ fragment is responsible for the antisuppression of upf1-2 and upf2-1.

IO The episomal expression of the L3 /? Fragment affects the rates of cell growth. It was noted that the cells transformed with the plasmids bearing L3 / \ grew slower than the control cells. To quantify the effect of this clone on the The growth of the cells was determined by the times in which the cells containing low copy or high copy plasmids that housed the fragment or vector L3 / \ were only bent. Bending times of wild-type cells expressing L3 /? In low copy or high copy plasmids were approximately twice that of the cells harboring the vector alone (8 hours versus 4 hours at 30 ° C). The growth rates of the cells harboring the full-length TCMl / MAKB gene "originated in the plasmid" had no effect on cell growth rates (data not shown).

The antisuppression of upf1-2 and upf2-1 by L3 / \ is independent of the state of the decomposition path of the mRNA. mediated by nonsense The different effects of the L3 / \ fragment in the strains upl- »upf2 ~ and upf3 ~ could be due to changes in the degradation rates of the nonsense mRNA of s4-3B or effects at the level of translation. In order to distinguish between these two possibilities, the sustained abundance state of his4-38 mRNA in these strains and in wild-type strains was determined by Northern blot analysis. Total RNA was isolated »separated through agarose gel and formaldehyde» was transferred to a nylon membrane and the mRNA was probed using the HIS4 fragment. As controls, the mRNAs of the CYH2 precursor and CYH2 were also probed using a radiolabelled CYH2 fragment. In all upf strains transformed with the L3 /? Fragment or with a control vector, the mRNA abundance of hie4-38 remained high. The mRNA level of hi84-38 in the wild-type strains (upf strains transformed with the UPF *. Plasmid) were low. Thus, the growth defect of the strain harboring an episomally expressed copy of the L3 /? Strains upfl- and upf2 ~ 'but not in the upf3 ~ »strain was not due to a decrease in the level of mRNA degradation. of h? s4-3B.

The L3 fragment encoded by L3 / \ decreases the free 60S ribosomal subunits Since L3 / \ has the complete 5T leader sequence and approximately 300 nucleotides of the N-terminal coding region »this genomic fragment must be transcribed and transladated. In an attempt to determine if this small fragment was expressed and if it was assembled on the ribosomes, a FLAG epitope was inserted at the 3"end of the L3 / fragment.The L3 fragment labeled with the epitope had the same phenotype as the L3 fragment as it was judged by the killer data (see below) and the antisuppression of h S4-3B in the upf2-l mutant strain (data not shown). The polyoma profiles of a wild-type strain transformed with L3 / labeled on a high copy plasmid (pJD139) or a vector control (pRS426) Examination of the polysome profiles showed that episomal expression of the L3 /? fragment resulted in a decrease in the peak height of the ribosomal subunits. 60S The fractions were collected along the gradient and the equal amounts of protein were separated from each fraction by SDS-PAGE with gradient from 1054 to 2054. Although an attempt was made to monitor the amount of labeled L3 fragment by analogy Western blot analysis "could not be detected in any of the polysomatic fractions" or in the total cell lysates. It is not known whether this is a consequence of the poor translation of the fragment, of the inaccessibility of the FLAG epitope to the antibody or of the post-translational degradation to the FLAG epitope. We also tried to create a fusion protein of L3 / \ / ß-galactosidase, with the purpose of analyzing ß-galactosidase by means of poly isome gradients. Although the sequence analysis confirmed that the correct clones had been constructed, it was found that these constructs were maintained in an unstable manner in yeast cells (data not shown). Thus, it can not be definitively stated that the L3 /? Protein is actually expressed, even though the multiple phenotypes that are observed in the cells transformed with these plasmids are strong evidence of this. Additionally, the data do not allow to determine if the L3 /? Fragment is associated with ribosomes.

Expression of the fragment L3 / \ causes loss of killer activity and dsARN virus M, the satellite virus M? of L-A requires the products of the MAK genes of at least 30 chromosomes for maintenance and reproduction, including TCM1 (MAKB) Previous studies have shown that in most mak ~ mutants the level of 60S subunits decreased in comparison With the cells of the wild type, the L3 / e expression also reduced the levels of 60S ribosomal subunits in the wild-type cells to determine if the episomal expression of the L3 / fragment can recapitulate the Mak8 phenotype., high copy and low copy plasmids that harbored the L3 /? gene fragment were transformed into killer cells * of the wild type and the killer phenotype loss was determined by plaque reproduction of the transformants on killer indicator plates. Although the cells transformed with vector lost only a small fraction of the killer activity, the cells transformed with the original L3 / o gene fragment or marked with FLAG had significant loss rates of the killer phenotype. An analysis of killer loss shows IO that the cells that had lost most of the murderous activity had also lost ^ dsRNA. It is notable that the expression of L3 / \ promoted the loss of killer (and M &) even when supported by the CB3 isotype of L-A, which is able to derive the cell requirement for most of the MAK genes. including MAKB. The effects of the expression L3 / \ with respect to killer loss in the upf and ski mutants were also examined and it was found that this also exacerbated killer loss in these contexts. It is interesting that mutants by omission of upf3 (upf3 / \) have intrinsically high killer loss speeds »that are reduced to nothing in the presence of L3 /. The ski mutants normally have the opposite phenotype of the mak mutants, ie they increase the copy number of the L-A and M viruses and the ski mutants are generally dominant to the mak mutants in that the double mutants mak / ski are K ~ (Toh-E and Wickner »1980). This is particularly true with respect to the double mutants s / raakS. However, when it was transformed with a high copy vector that housed the L3 / \ fragment (pJD139), even the s i mutants exhibited high rates of killer loss due to the loss of M,. Thus, the expression of the L3 /? Fragment is episodic with respect to the ski mutants.

The expression of L3 / \ confers an Mof phenotype. The proportion of Gag / Gag-pol fusion proteins encoded by LA, available, as determined by the efficiency of ribosomal frame shift -1, is critical for the maintenance of MA "and the mutations that affect the efficiency of shift shift of ribosomal framework change the Gag / Gag-pol protein ratio, which promotes the loss of Ma. To determine whether the expression of L3 / \ affects this procedure, the efficiency of the ribosomal frame shift in various wild-type strains harboring the L3 /? Fragment, derived from LA or ribosomal frame shift reporters -1 or + was measured. 1 »derivative of Tyl. The episomal expression of the L3 /? Fragment increases the efficiency of the ribosomal frame shift -1"but not programmed +1, in wild-type cells. The 2.3-fold increase in a ribosomal frame-shift efficiency of -1 is at the upper limit of the tolerated frame displacement efficiency for the minimum maintenance of M. The intrinsic efficiency of the ^ W ribosomal frame shift -1 is 5,354 in the ma 8-2 mutants, ie »2.9 times larger than in the wild type cells. In such a way »the maKB-2 mutation is also an mof mutation. We also investigated the effects of L3 / \ expression on upf mutants. He found numerous interesting pieces of information. First, in the strains u fl ^ and upf2 ^, the intrinsic efficiency of the ribosomal frame shift -1 appears to be approximately twice as great as in the cells of the wild type. This is due to the fact that the frame shift reporter -1 is a nonsense mRNA »which is stabilized in these cells» which allows a larger overall production of the β-gal reporter protein. as opposed to the increased efficiencies in the ribosomal frame offset -1. This is consistent with the data presented previously (example 1). Additionally, the expression of L3 / \ increases the efficiency of the ribosomal march shift in these cells to the same extent as in wild-type cells »ie» approximately triple. The apparent efficiency of ribosomal frame shift -1 in up3f cells _.._. is intrinsically elevated (754) and the additional expression of Ltt / \ only increases this to 9,454. This explains why upf3x cells show high intrinsic velocities of killer loss: they are also mof mutants since they increase the efficiency of ribosomal frame shift-1 independently of their ability to stabilize nonsense mRNA.

DISCUSSION 5 The L3 /? Fragment acts as a suppressor of the his4-38 frame shift allele of the upf1-2 and upf2-1 ^ áFr mutant cells. The L3 /? Fragment was identified as an ant suppressor. upf2-l by loss of the capacity of upf2-l his4-3B SUFl-l cells, transformed with a plasmid containing this gene fragment "to develop at 37 ° C in medium lacking histidine. The fact that a full length TCMl / MAKB clone does not have this activity »suggests that the The expression of the N-terminal 100 amino acids of this protein is responsible for the phenotypes observed. The antisuppression of upf1-2 and upf2-1 by L3 / \ may be the result of the decrease in the overall amount of the functional His4 protein »transiated from the mRNA of HIS4-38 to 37 ° C. This could be due to: (1) the abrogation of the capacity of the glycine tRNA encoded with SUFl-l to suppress the frame shift at 37 ° C »(2) the reactivation of the path of decomposition of the mRNA» mediated by nonsense » at the temperature not permissible; or (3) a defect in This translation is the result of the inability of the upfl-2 and upf2-1 mutants to act as alosuppressors with the SUF1-l gl cina tRNA at 37 ° C. The fact that the expression of the fragment L3 / \, in combination with the mutation upf3-1 does not abrogate the deletion of SUFl-l at 37 ° C »argues against a defect in the functioning of glycine tRNA» coded by SJLJF1- 1 »at that temperature. Additionally, since the abundances of His4-38 mRNA and mRNA of the Cyh2 precursor were equal in the cells with or without a L3 /? Gene fragment, it argues directly against the reactivation of the mRNA-mediated degradation path. for the nonsense. Thus »it is likely that the L3 / \ fragment exerts its anti-suppressive activity at the level of translation» decreasing the translation of the functional His4 product »from the mRNA of H1S4-38. TCMl / MAKB encodes the kidney protein L3 »which is involved in the formation of the peptidyl transferase center. The transcription of TCM1 is under the control of a single upstream activation sequence recognized by a multifunctional transcription factor (Dorsman and coauthors »19B9» Hamil and co-authors »1988) and the L3 /? Fragment contains all the upstream sequence required for his transcript. Moreland and coauthors (1985) examined the nuclear localization signal at the TCM1 dine. performing various omissions in the 3"coding region, fused in frame with the lacZ gene, and detected the location of the β-galactosidase by immunofluorescence microscopy.The results suggest that the expression of the first 21 amino acids of L3 was sufficient to direct the fusion protein to the nucleus, so it is quite possible that the L3 fragment produced by L3 / \ is transported to the nucleus and subsequently to the nucleolus by the assembly on the ribosome.5 like other ribosomal proteins »the expression of the protein L3 is regulated and the level of the L3 protein remains the same even when the cells have extra copies of the TCM1 gene (Pearson and coauthors »19B2» Beus and co-authors »1994) i ^ r Reducing the growth rates of the cells IO expressing the L3 / \ fragment and the decrease in peak heights of the 60S ribosomal subunits "support the notion that the expression of this gene fragment affects translation. Two mutually exclusive models can be proposed to take into account the observations of the present: (1) that the decreased levels of the 60S subunit could be due to the incorporation of the L3 protein fragment into the ribosomes "which directly affects the process of peptide transfer; or »alternatively» (2) that the effects observed could be due to indirect effects of the expression of the peptide fragment on the biogenesis of the ribosome. If the L3 / \ »fragment is translocated but the ribosomes are not assembled» the peptide fragment can decrease the regularization of the expression of the wild-type TCM1 gene. This could be taken into account for the reduction of the peak heights of the 60S subunit in the polysome profiles »from cells harboring the L3 / fragment.

Unfortunately »these data do not allow to distinguish between these possibilities.

The L3 /? Fragment recapitulates a makB mutation 5 That the cells expressing the L3 / V fragment lose M dsARN »indicates that the loss of killer activity was not due to translational defects» the processing or export of the killer toxin . Additionally, since '^ ¥ 1a reduction in 60S ribosomal subunit levels is a IO phenomenon characteristic of most mak mutants (Ohtake and Wickner »1995)» the results presented here clearly indicate that the extrachromosomal expression of the L3 / \ fragment confers a "dominant" Mak-negative phenotype on the wild-type cells. The observation that The expression of the fragment L3 / \ results in a 2.3-fold increase in the efficiency of the ribosomal frame shift -1 is interesting »since it is just at the limit where the loss of M ^ is observed and shows that the expression of this fragment confers a Mof-dominant phenotype as well.

Two other pieces of data support this notion. First »the expression of this clone leads to the exclusion of M ^ from the cells harboring the" Derivation "isotype CB3 of L-A (strains JDB8 and JD111 are L-AHNB Mx). While most of the mak »agents include makB. they can be derived by CB3 (ie »L-AHNB can support Ma. And murderer in these mutants m? I <)» those mof mutants that can not support the killer »lose Ma.» Independently of the isotype L-A. That the efficiency of ribosomal frame shift -1 is significantly elevated in the ma 8-2 mutants, suggests that mutations in the ribosomal protein L3 and "consequently" in the center of peptidi 1-transferase »may affect maintenance of translation of the reading frame also. An important finding is the expression of the L3 /? Fragment which resulted in an increase in the efficiency of ribosomal frame shift -1 »driven by L-A» but did not affect the ribosomal frame shift -1 promoted by Tyl.

EXAMPLE 5 MUTANTS AFFECT THE ACTIVITY OF THE CENTER OF PEPTIDIL- TRANSFERASA The isosis experiments have shown that certain mof mutants affect the activity center of peptidi 1-transferase. Two antibiotics »trichodermin and anisomycin» both peptidyl transferase inhibitors »have been shown to be ineffective in the tcml mutants (which were first identified by their resistance to trichodermin). None of these drugs has any effect on the displacement of the ribosomal framework -1 in the mutants maKB-2 »and their efficiencies in the displacement of frame 1AA ribosomal -1 are also high »which makes these cells are also mutants mof. Other mof mutants show some rather interesting tendencies. Like mof8-2, none of these drugs affect the efficiency of ribosomal frame shift -1 in the mutants mof1-1 and mof? - »suggesting that defects in the peptidyl transferase transfer reaction respond to» or they contribute factors for »the Mof phenotypes of these cells. Interestingly, anisomycin does affect frame-1 shift efficiencies in mof6-1 cells, but sparsomycin does not have this effect (Figures 16A-B). Conversely, »sparsomycin changes efficiency in the displacement of ribosomal frame -1 in cells mof -1» while there is no effect with anisomycin in these cells (figures 16A »B). These data suggest that mutations mof6-l and mof9-1 can be used as probes in the dissection of two different sites of action of these drugs on the peptidyl transferase reaction.

EXAMPLE 6 ANISOMYCIN AND SPARSOMYCIN SUPPRESS MUTATIONS WITHOUT SENSE The UPF1 + and UPF1- strains were treated with 5 μg / ml of sparsoramycin »anisomycin and paromomycin» and tested for the suppression of nonsense. As shown in figure 17, these drugs were able to suppress nonsense mutations. The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art., from the preceding description and the attached figures.

It is intended that such modifications be within the scope of the .e.as.-e.v.nC.cac.one. -nexeß. Various publications are cited herein, and their descriptions are incorporated by reference in their entirety.

Claims (27)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a drug that affects the center of eukaryotic pep idil-trans erasa, in the manufacture of a drug to modulate the function of a eukaryotic peptidyl transferase center.

2. The use according to claim 1"" further characterized in that the drug is an antibiotic

3. The use according to claim 1 or 2"further characterized in that the drug is an inhibitor of the peptidyl transferase center.

4. The use according to the re-indication 1 »2 or 3» further characterized in that the drug is selected from the group consisting of sparsomycin and anisomycin.

5. The use according to any of claims 1 to 4 »to treat a viral infection.

6. The use according to any of claims 1 to 4. to treat an HIV infection.

7. The use according to any of claims 1 to 4 »to treat a disease associated with a nonsense mutation in a gene.

8. A mutant gene that encodes a protein involved in the ribosomal frame shift, characterized in that the mutation results in modulation in the efficiency of the ribosomal frame shift.

9. - The mutant gene according to claim B »further characterized in that it increases the efficiency of ribosomal frame displacement.

10. The mutant gene according to claim B or 9 »further characterized in that it modulates the degradation of nonsense mRNA.

11. The mutant gene according to claim B »9 or 10» further characterized in that it is selected from the group consisting of mof4-1 »mof2-1 and mof5-1.

12. An expression vector characterized in that it comprises a mutant gene according to any of claims 8 to 11 »operatively associated with an expression control sequence.

13. The use of an expression vector according to claim 12, for modulating the ribosomal frame shift or the degradation of mRNA.

14. The use according to claim 13, to treat a viral infection.

15. The use according to claim 13, to treat a disease associated with a nonsense mutation in a gene.

16. A hybridizable nucleic acid in vivo with a mRNA that encodes a protein involved in the programmed ribosomal frame-1 displacement characterized in that a mutation of a gene encoding the protein results in the modulation of displacement efficiency. of frame # ribosomico.

17. The nucleic acid according to claim 16 »further characterized in that it is selected from the group consisting of a nucleic acid of opposite sense and a ribozyme.

18. The nucleic acid according to claim 16 or 17 »further characterized in that the gene is selected from the group consisting of mof4-1» mof2-1 and mof5-1. IO 19.- An expression vector »characterized in that it comprises a nucleic acid according to the rei indication 16, 17 or B» operatively associated with an expression control sequence. 20. The use of an expression vector according to claim 19 for the manufacture of a medicament for modulating ribosomal frame displacement or degradation of mRNA. 21. The use according to claim 20 »for treating a viral infection. 22. Use according to claim 20 to treat a disease associated with a nonsense mutation in a gene. 23.- A method to purify an active drug in the center of eukaryotic peptidi-1-transferase »characterized 25 because it comprises: (a) contacting cells with a candidate drug "and (b) determining the modulation of the peptidyl transferases; wherein the drug that modulates the peptidi 1-transferases is active in the eukaryotic peptidyl transferase center. 24. The method according to claim 23 »further characterized in that the modulation of the peptidyl transferases is determined by a method selected from the group consisting of: (i) identifying a phenotype associated with a mutation selected from the group consisting of mof1-1, mof4-1, mof2-1, mof5-1, mof6-1 and his4; (ii) detecting the increased stability of nonsense mRNA or short mRNA transcripts; (iii) cultivate a line of mutant cells »deficient in tyrosine and leucine. by biosthesis in a tyrosine and leucine-deficient culture medium »(iv) detecting an altered ratio of Gag proteins to Gag-pol in a cell infected with a virus; and (v) join a protein that modulates a frame shift event. 25.- A polypeptide corresponding to the N-terminal 100 amino acids of the ribosomal binding protein L3. 26.- A pharmaceutical composition to increase the efficiency of ribosomal frame shift -1 but not +1"characterized in that it comprises the polypeptide of claim 25 and a pharmaceutically acceptable carrier. 27. The use of a polypeptide according to claim 25 for the manufacture of a medicament for treating a viral infection. 2B.- A nucleic acid encoding the polypeptide according to claim 25. 29.- An expression vector, characterized in that it comprises a nucleic acid of claim 28, operatively associated with an expression control sequence. 30. The use of an expression vector according to claim 29, for modulating the ribosomal frame shift. 31. The use of claim 30, to treat a viral infection. 32. A method for treating a disease associated with a nonsense mutation in a gene "characterized in that it comprises modulating the degradation of mRNA in accordance with the method of claim 20. 33.- A method for purifying an active drug in a center of eukaryotic 1-transferase peptidi, characterized in that it comprises: (a) contacting cells with a candidate drug; and (b) determining the modulation of the peptidyl transferases; wherein a drug that modulates the peptidyl transferases is active in the center of eukaryotic peptidi 1-transferase. 34.- The method according to claim 23 »further characterized in that the modulation of the peptidyl-traneferaeae is determined by a method selected from the group consisting of: (i) identifying a phenotype associated with a mutation selected from the group consisting of mofl-l »mof4-1» mof2-1 »mof5-1» mof6-1 and his4 »(ii) detect increased stability of nonsense mRNA or short mRNA transcripts» (iii) culture a line of mutant »defective cells in tyrosine and leucine "by biosynthesis in a culture medium deficient in tyrosine and leucine; (iv) detect an altered ratio of Gag proteins to Gag-pol in a cell infected with viruses »and (v) bind a protein that modulates a frame shift event. 35.- A polypeptide corresponding to the N-terminal 100 amino acids of the ribosomal binding protein L3. 36.- A method to increase the efficiency of displacement of ribosomal frame -1 »but not of +1» characterized in that it comprises introducing the polypeptide of the re vindication into a cell. 37.- A method to treat a viral infection »characterized in that it comprises modulating the ribosomal frame displacement according to the method of claim 26. 38.- A nucleic acid» characterized in that it encodes the polypeptide of claim 25. 39.- An "expression vector" characterized in that it comprises a nucleic acid of claim 28, operatively associated with an expression control sequence. 40.- A method for modulating ribosomal frame displacement, characterized in that it comprises introducing an expression vector of claim 29 into cells. 41. A method for treating a viral infection, "further characterized in that it comprises modulating the ribosomal frame shift according to the method of claim 30. SUMMARY OF THE INVENTION The degradation of mRNA is an important control point of gene expression and has been shown to bind the translation procedure; a clear example of this union is the observation that nonsense mutations accelerate the degradation of mRNA; This report demonstrates that a subset of mof alleles (frame maintenance) in yeast, which is isolated as chromosomal mutations that increase the efficiency of frame change at the site of the LA virus frame change and caused the loss of the LA satellite of the M virus, also affects the degradation pathway of mRNA mediated by mutations in eent? do; mRNA levels containing nonsense mutations were elevated in the cells that reach the roof4-1 alleles; in addition, mof41 is allelic to UPF1, which has been shown to be involved in the degradation pathway of mRNA mediated by nonsense mutations; although the cells that arrive at the roof4-a allele lose the virus A the other alleles f (for example? fp, upf2, upf3) involved in the maintenance of mRNA degradation mediated by nonsense mutations maintains x; The ifsl and if82 alleles previously identified as mutations that improve the frame change in the ribosomal frame change signal from the mutations that originate in the mouse mammary tumor virus were shown to be all cos to the UPF2 genes and UPFl, respectively, and both ifs strains maintained MA strain roof4-l is more senescent to amynoglotype paromomycin than an upflg strain (D) and increases the eff ect of frame change in a mof4-a strain that grows in the presence from paromornícina; These results indicate that the? pfl-p has a dual function in translation and spin of mRNA. fifteen twenty 25 CR / cgt P98 / 375F