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(12) United States Patent (10) Patent No.: US 6,610,545 B2 Dujon Et Al USOO66 10545B2 (12) United States Patent (10) Patent No.: US 6,610,545 B2 Dujon et al. (45) Date of Patent: *Aug. 26, 2003 (54) NUCLEOTIDE SEQUENCE ENCODING THE Michel F, and Dujon B., Conservation of RNA Secondary ENZYME-SCE AND THE USES THEREOF Structure in Two Intron Families Including Mitochondrial-, Chloroplast- and Nuclear-Encloded Members, Embo Jour (75) Inventors: Bernard Dujon, Gif sur Yvette (FR); nal, vol. 2(1), pp. 33–38 (1983). Andre Choulika, Paris (FR); Laurence Dujon, B., and Jacquier, A., Mitochondria, 1983, Walter de Colleaux, Edinburgh (GB); Cecile Gruyter & Co., pp. 389-403. Fairhead, Malakoff (FR); Arnaud Jacquier, A. and Dujon, B., An Intron-Encoded Protein is Perrin, Paris (FR); Anne Plessis, Paris Active in a Gene Conversion Process that Spreads an Intron (FR); Agnes Thierry, Paris (FR) into a Mitochondrial Gene, Cell, vol. 41, pp. 383-394 (1985). (73) Assignees: Institut Pasteur, Paris (FR); University Dujon et al., “In Achievements and Perspective of Mito Paris VI, Paris (FR) chondrial Research', Biogenesis, vol. II, Elsevier Science Publishers, pp. 215-225 (1985). (*) Notice: Subject to any disclaimer, the term of this Colleaux, L. et al., Universal Code Equivalent of a Yeast patent is extended or adjusted under 35 Mitochondrial Intron Reading Frame is Expressed into E. U.S.C. 154(b) by 0 days. Coli as a Specific Double Strand Endonuclease, Cell, vol. 44:521-533 (1986). This patent is Subject to a terminal dis Michel, F. and Dujon, B., “Genetic Exchanges Between claimer. Bacteriophage T4 and Filamentous Fungi'?”, Cell, vol. 46, p. 323 (1986). (21) Appl. No.: 09/836,169 Colleaux, L., et al., Recognition and cleavage Site of the intron-encoded omega transposase, PrOC. Natl. Acad. Sci. (22) Filed: Apr. 18, 2001 USA, vol. 85:6022-6026 (1988). (65) Prior Publication Data Dujon B. et al., “Group I Introns as Mobile Genetic Ele ments: Facts and Mechanistic Speculations-A Review”, US 2003/0113887 A1 Jun. 19, 2003 Gene, vol. 82, pp. 91-114 (1989). Dujon et al., “Mobile Introns: Definition of Terms and Related U.S. Application Data Recommended Nomenclature,” Gene, vol. 82, pp. 115-118 (63) Continuation of application No. 09/196,131, filed on Nov. (1989). 20, 1998, now Pat. No. 6,238,924, which is a continuation Monteilhet C, et al., Purification and Characterization of the of application No. 08/417,226, filed on Apr. 5, 1995, now in vitro Activity of I-Sce I, a Novel and Highly Specific Pat. No. 5,962,327, which is a division of application No. Endonuclease Encoded by a Group 1 Intron, Nucl. Acids 07/971,160, filed on Nov. 5, 1992, now Pat. No. 5,474,896, which is a continuation-in-part of application No. 07/879, Res., vol. 18(6), pp. 1407–1413 (1990). 689, filed on May 5, 1992, now abandoned. Jacquier, A. and Dujon, B., The Intron of the Mitochrondrial 21s rRNA Gene: Distribution in Different Yeast Species and (51) Int. Cl. ................................................ C12N 15/55 Sequence Comparison Between Kluyveromyces thermotol erans and Saccharamyces cerevisiae, Mol. Gen. Genet., vol. (52) U.S. Cl. ........................................ 435/477; 435/478 192, pp. 487–499 (1983). (58) Field of Search .................................. 435/477,478 Colleaux, L. et al., The apocytochrome b gene of Chlamy domanas Smithli contains a mobile intron related to both (56) References Cited Saccharomyces and Neurospora introns, Mol. Gen. Genet., U.S. PATENT DOCUMENTS vol. 223:288–296 (1990). 5,474,896 A 12/1995 Dujon et al. ................... 435/6 Dujon, B. et al., in Extrachromosomal Elements in Lower 5,962,327 A 10/1999 Dujon et al. ...... ... 435/478 Eukaryotes (Plemum Publishing Corporation), pp. 5-27 6,238,924 B1 5/2001 Dujon et al. ...... ... 435/477 (1986). 6,395,959 B1 5/2002 Dujon et al. .................. 800/18 Thierry et al., Cleavage of Yeast and Bacteriophage T7 Genomes at a Single Site. Using the Rare Cutter Endonu OTHER PUBLICATIONS clease I-Sce I, Nucl. Acids Res., vol. 19(1), 1991, pp. Rouet et al., “Expression of a Site-specific endonuclease 189-190. Stimulates homologous recombination in mammalian cells,” (List continued on next page.) Proc. Natl. Acad. Sci, USA, vol. 91, Jun. 1994, pp. 6064-6068. Primary Examiner-Charles L. Patterson, Jr. Sharp, P.M., et al. (1988) Nucl. Acids Res. 16(17) (74) Attorney, Agent, or Firm-Finnegan, Henderson, 82O7-8211. Farabow, Garrett and Dunner, LLP Sharp, P.M., et al. (1987) Nucl. Acids Res. 15(3) 1281–1295. (57) ABSTRACT Gouy, M., et al., (1982) Nucl. Acids Res. 10, 7055–7074. Dujon et al., Sequence of the Intron and Flanking Exons of An isolated DNA encoding the enzyme I-Sce is provided. the Mitochondrial 21S rRNA Gene of Yeast Strains Having The DNA sequence can be incorporated in cloning and Different Alleles at the () and rib-1 Loci, Cell, vol. 20, pp. expression vectors, transformed cell lines and transgenic 185-187 (1980). animals. The vectors are useful in gene mapping and Site Michel F. et al., Comparison of fungal mitochondrial introns directed insertion of genes. reveals extensive homologies in RNA secondary Structure, Biochemie, vol. 64: 867–881 (1982). 27 Claims, 24 Drawing Sheets US 6,610,545 B2 Page 2 OTHER PUBLICATIONS Colleaux et al., “Rapid Physical Mapping of YAC Plessis et al., Site-Specific Recombination by I-Sce I: A inserts . .” Human Mol. Genet., 203):265-271 (1993). Mitochrondrial Group I Intron-Encoded Endonuclease Dujon et al., “Mobile Introns.” Abstract presented at EMBO Expressed in the Yeast Nucleus, Genetics, vol. 130(3), pp. Workshop, Roscoff, France, Jun. 24-28, 1990. 451–460 (1992). Tartof et al., “New cloning vectors and techniques . ss Dujon, B., “Des Introns Autonomes et Mobiles”, Annales de Gene 67.169-182 (1988). L'Institut Pasteur/Actualities, 1:181–194 (1990). U.S. Patent Aug. 26, 2003 Sheet 3 of 24 US 6,610,545 B2 T T GA severey offected A AAT GCT TT CCA A portially affected T GGC T G CT CA G c A AA " : t it.AA unoffected G U.S. Patent Aug. 26, 2003 Sheet 6 of 24 US 6,610,545 B2 -2 -l l 5 10 M H M K N K K N Q. W M N L G P N S 20 30 K L L K E Y K S Q L I E L N E Q F E A 40 50 G G L I L G D A Y I R S R D E 6 K T Y 60 70 C M Q F E W K N K A Y M D H W C L L Y C 80 90 Q W Y S P P H K K E R Y N H L G N L Y 100 110 I W G A Q T F K H Q A F N K . A N F 120 l30 V N N K K I P N N L V E N Y L T P M l40 50 G L A Y W P M D D G G K W D Y N K N S 160 70 N K S I W L N T Q S F T F E E W E Y L. W l80 190 K G L R N K F Q N C Y V K N K N K P 200 210 Y D S M S Y L. F Y N L I K P Y L. 220 230 P Q M M Y K L P N S S E T F L K POS it Ons that can be changed withOUt affecting enzyme SY (de?MOnStrated) positions - in -2 are not natural, The two amino acids are added due to ClOn ng Strategies positions to 10: can be deleted position 55: G is tolerated DOS ition 40; M. Or V are tolerated position 4:43: SA orStolerated N are tolerated position 46: W. Or N are tolerated position 9): A Stolerated positions 123 and 156: are tolerated position 223: A and S are tolerated Chan hat affect enzyme activity (demonstrated) position 9: to S position 38; I to S or N position 39: G to D or R position 40: to Q position 42: L to R DOSlt iOn 4: D to E G or H position 45: A to E or D position 46: Y to D position 47: I to R. Or N E33ApositionDOS 1. Of l l is5: D toO E F G. 5 position l l 6: G to E position l (17: G to S U.S. Patent Aug. 26, 2003 Sheet 7 of 24 US 6,610,545 B2 Group I intron Encoded Endonucleases and Related Endonucleases CLEAVAGE ENDONUCLEASE SE V INTRON SITE I-Sce I A TA GC (Saccharomyces mitochondrio) TAT T G T C C CAT TA T AT CG -Sce IV (Saccharomyces mitochondriaia) I-Sce II C (Saccharomyces mitochondrio) GA (ChlamydomonasI-Ceu chloroplast)I I-Ppo I (Physaru ?t UC leus (S?B)100dºy80)ATIW330{l({}d\O3000OM1 (SaccharomycesI-Sce mitochondrio) III (ChlamydomonasI-Cre chloroplast)I Endo. Sce I RF3 (Sacchgromyces inAlondi) ( Non intronic ) (SacchgromycesHO nucleus) (Non intronic) I-CSrn I (Ch lom domonas mitochondrio) (Pu gtive endonucleose ) -Pon I Podos Oro mitochondridig) ( (Puta E.e endonuclease) (Bacteriophage T4) FG, 6 U.S. Patent Aug. 26, 2003 Sheet 8 of 24 US 6,610,545 B2 EXPRESSION VECTORS EcoRI EcoRI 2 MICRON 2 MCRON pPEX 408 7.1 kb. Born EcoRI ORF-Sce FIG, 7 Hind 5457 Pyu 5224 EcoRI. 89 Nor I 5222 Hind II 4805 a Sce-I Pvu II. 4584 2 Pst I 4278 pRSW I Sce-I 5514 bp PolyASV40 Pst 1890 Born 5,595 Sco I.
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