IIIHIII IIII US005474896A United States Patent (19) 11) Patent Number: 5,474,896 Dujon et al. (45. Date of Patent: Dec. 12, 1995 54 NUCLEOTIDE SEQUENCE ENCODING THE 185-187 (1980). ENZYME-SCE AND THE USES THEREOF Michel, F. et al., Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary 75) Inventors: Bernard Dujon, Gif sur Yvette; Andre structure, Biochimie, vol. 64: 867-881 (1982). Choulika, Paris, both of France; Michel, F. and Dujon, B., Conservation of RNA secondary Laurence Colleaux, Edinburgh, structures in two intron families including mitochondrial-, Scotland; Cecile Fairhead, Malakoff, chloroplast- and nuclear-encoded members, EMBO Jour France; Arnaud Perrin, Paris, France; nal, vol. 2(1): 33-38 (1983). Anne Plessis, Paris, France; Agnes Jacquier, A. and Dujon, B., The Intron of the Mitochondrial Thierry, Paris, France 21s rRNA Gene: Distribution in Different Yeast Species and Sequence Comparison Between Kluyveromyces thermotol 73 Assignees: Institut Pasteur; Université Paris-VI, erans and Saccharomyces cerevisiae, Mol. Gen. Genet., both of France 192: 487-499 (1983). Dujon, B. and Jacquier, A., Mitochondria 1983, Walter de (21) Appl. No.: 971,160 Gruyter & Co., pp. 389-403. Jacquier, A. and Dujon, B., An Intron-Encoded Protein is 22 Filed: Nov. 5, 1992 Active in a Gene Conversion Process That Spreads an Intron into a Mitochondrial Gene, CELL, vol. 41: 383-394 (1985). Related U.S. Application Data Dujon, B. et al., in Achievements and Perspectives of 63 Continuation-in-part of Ser. No. 879,689, May 5, 1992, Mitochondrial Research, vol. II: Biogenesis, Elsevier Sci abandoned. ence Publishers, pp. 215-225 (1985). Colleaux, L. et al., Universal Code Equivalent of a Yeast (51) Int. Cl. ............................. C12Q 1/68; C12N 15/70 52 U.S. Cl. ............... 435/6; 435/320.1; 935/78 Mitochondrial Intron Reading Frame is Expressed into E. 58) Field of Search ............................ 435/6, 255,320.1; coli as a Specific Double Strand Endonuclease, CELL, vol. 935/77, 78 44:521-533 (1986). Michel, F and Dujon, B., Genetic Exchanges between 56) References Cited Bacteriophase T4 and Filamentous Fungi?, CELL, vol. 46: PUBLICATIONS 323 (1986). Tartot et al., Gene 67:169-182 (1988) “New cloning vectors (List continued on next page.) and techniques . '. Primary Examiner-Margaret Parr Colleaux et al, Human Mol. Genet. 2(3):265-271 (1993) Assistant Examiner-Eggerton Campbell "Rapid Mapping of YAC inserts...'. Attorney, Agent, or Firm-Finnegan, Henderson, Farabow, Dujon, Bernard "Group I introns as mobile genetic ele Garrett & Dunner ments: facts and mechanistic speculations-a review,' GENE, vol. 82: 91-114 (1989). 57 ABSTRACT Dujon, Bernard “Mobile Introns', EMBO Workshop on An isolated DNA encoding the enzyme I-SceI is provided. Molecular Mechanisms of Transposition and Its Control, Roscoff, France, Jun. 24-28, 1990. The DNA sequence can be incorporated in cloning and Dujon, Bernard "Des Introns Autonomes et Mobiles', expression vectors, transformed cell lines and transgenic Annales de L'Institut Pasteur/Actualites, i:181-194 (1990). animals. The vectors are useful in gene mapping and site Dujon, B., Sequence of the Intron and Flanking Exons of the directed insertion of genes. Mitochondrial 21s rBNA Gene of Yeast Strains Having Different Alleles at the co and rib-1 Loci, CELL, vol. 20: 2 Claims, 22 Drawing Sheets AAAAATAAAATCAT AG AAA AAT ATT AAA AAA AA CAA GA ATC AAT CTC 66 CCAA TC M. K. N. I K K N G W M N G P N S AAA TTA TTA AAA GAA TAT AAA CA CAA TA AT GAA TA AAT ATT GAA CAA TT GAA GCA K K E Y K S Q E L E G F E A GGT AT GGTTTA AT TTA. GGA GAT GCTA A CGT AG CGT GAT GAA GGT AAA AC TAT G I. 6 L G D A Y R S R B E S K T Y GT AG CAA TTT CAC TCC AAA AA AAG GCA TAC ATG GAT CAT GA TGT TTA TTA, TA GAT C M F. E. W. K. N. K. A Y M D H W C Y D CAA TGG GTATTACA CCT CCT CAT AAA AAA GAA AGA is AA CA TTA. GGT AAT AGA Q W W L S P P H K K E R W N G. N. L. W. AT ACC TGG GGA GCT CAA ACT TT AAA CAT CAA GOT TAAT AAA TTA GCT. AAC A TT K A F M K L A AT GTA AAT AAT AAA AAA CT ACC AA AAT TA GT GAA AA TA TTA ACA CC ATG Y N N K K L P N N L W E N Y T P M AGT CG GCA TA, TGG TTT ATG GA GA GGA GGT AAA TGG GAAT AAAAA AAT C CT S A Y W F M D D G G K D Y N K. N S AA AAA AGT A GTA TTA AAT ACA CAA AG T ACT TT GAA GAA GTA GAA AA CT N K S W N T Q S F F E E W C Y W AAA GGT TA AGA AATAAA TTT CAA TTA AA TG TAT ST AAA ATT AATAAA AATAAA CCA K G R N K F Q C Y V K K N K P At A TATAT GAT TCT AG, AGT TAT. CTG AT TTT TAT AAT TA At AAA CCT TAT TTA Y I D S M S Y F Y N K P Y L. ATT CCT CAA AGATS TATAAA CTG CCAA AC AT TCA CC GAA ACTA AAA TAA I P G M M Y K L N S S E F L K 5,474,896 Page 2 OTHER PUBLICATIONS Saccharomyces and Neurospora introns, Mol. Gen. Genet., Colleaux, L. et al., Recognition and cleavage site of the vol. 223: 288-296 (1990). intron-encoded omega transposase, Proc. Natl. Acad. Sci. Dujon, B. et al., in Extrachromosomal Elements in Lower USA, vol. 85: 6022-6026 (1988). Eukaryotes, (Plenum Publishing Corporation 1986), pp. Dujon, B. et al., Mobile introns: definition of terms and 5-27. recommended nomenclature, GENE, vol. 82: 115-118 (1989). Thierry, A. et al., Cleavage of yeast and bacteriophage T7 Monteilhet, C. et al., Purification and characterization of the genomes at a single site using the rare cutter endonuclease in vitro activity of I-Scel, a novel and highly specific I-Sce, Nucleic Acids Research, vol. 19(1): 189-190 (1991). endonuclease encoded by a group 1 intron, Nucleic Acids Plessis, A. et al., Site-specific recombination by I-Scel; a Research, vol. 18(6), 1407-1413 (1990. mitochondrial group 1 intron-encoded endonuclease Colleaux, L. et al., The apocytochrome b gene of Chlamy expressed in the yeast nucleus, Genetics, vol. 130(3): domonas smithi contains a mobile intron related to both 451-460 (1992). U.S. Patent Dec. 12, 1995 Sheet 3 of 22 5,474,896 severely affected A AA i partially affected i unoffected 5' i:GT TA CGC III:ATAA 3' 3' CAAT GCGAT AT 5' U.S. Patent Dec. 12, 1995 Sheet 6 of 22 5,474,896 -2 -1 5 10 M. H M K N I K K N G V M N L G P N S 20 30 K K E Y K S Q L I E L N E Q F E A LO 50 G G L I L G D A Y I R S R D E G 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 L S P P H K K E R Y N H L G N L Y 100 10 I T W G A Q T F K H Q A F N K L A N L. F 120 130 I V N N K K I I P N N L V E N Y L T P M 140 150 G L A Y W P M D D G G K W D Y N K N S I 160 170 N K S I W L N T Q S F T F E E W E Y L. W 180 190 K G L R N K F Q. L N C Y V K N. K. N. K. P 200 20 I I Y D S M S Y L I F Y N I K P Y L. 220 230 P Q M M Y K L P N T S S E T F L K * POSitions that Can be Changed Without affecting enzyme activity (demonstrated) positions -l and -2 are not natural, The two amino acids are added due to ClOning Strategies pOSlt). OnS Ug 10; Can be deleted position 35: G is tolerated position 40: M or V are tolerated position 41: S or N are tolerated position 43: A is tolerated position 46: W or N are tolerated position 91A is tolerated positions 123 and 156; L are tolerated pOSition 225; A and S are tolerated Changes that affect enzyme activity (demon Strated) position 19; L to S position 38: t O S position 39: position 4Q: position 42: p0Sition 44: O H position 45: position 46: position pOSlton rr position | position F.G. 5 pOSition pOSiti On : U.S. Patent Dec. 12, 1995 Sheet 7 of 22 5,474,896 Group I intron Encoded Endonucleases and Redted Endonucleoses CLEAVAGE ENDONUCLEASE RECOGNITION SEQUENCE SITE V INTRON SITE I-Sce I-Sce IV (Saccharomyces mitochondrio) I Sce I (Saccharomyces mitochondria) (ChlamydomonasI-Ceu chloroplast)I I-Ppo I ( Phys OU C leus (SaccharomycesI-Sce mitochondrio) II (ChlamydomonasI-Cre chloroplast) Endo.