Nucleotide sequence encoding the enzyme I-SceI and the uses thereof
AssigneeInstitut Pasteur Universite Paris-VI
InventorsBernard Dujon, Andre Choulika, Laurence Colleaux, Cecile Fairhead, Arnaud Perrin, Anne Plessis, Agnes Thierry
Life of this patent
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Abstract
An-isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.
Claims26
An isolated DNA encoding the enzyme I-SceI, wherein the DNA has the nucleotide squence:
- 9 ATC CAT ATG AAA AAC ATC AAA AAA AAC CAG GTA ATG 2670 M H M K N I K K N Q V M 12 2671 AAC CTC GGT CCG AAC TCT AAA CTG CTG AAA GAA TAC AAA TCC CAG CTG ATC GAA CTG AAC 2730 13 N L G P N S K L L K E Y K S Q L I E L N 32 2731 ATC GAA CAG TTC GAA GCA GGT ATC GGT CTG ATC CTG GGT GAT GCT TAC ATC CGT TCT CGT 2790 33 I E Q F E A G I G L I L G D A Y I R S R 52 2791 GAT GAA GGT AAA ACC TAC TGT ATG CAG TTC GAG TGG AAA AAC AAA GCA TAC ATG GAC CAC 2850 53 D E G K T Y C M Q F E W K N K A Y M D S 72 2851 GTA TGT CTG CTG TAC GAT CAG TGG GTA CTG TCC CCG CCG CAC AAA AAA GAA CGT GTT AAC 2910 73 V C L L Y D Q W V L S P P H K K E R V N 92 2911 CAC CTG GGT AAC CTG GTA ATC ACC TGG GGC GCC CAG ACT TTC AAA CAC CAA GCT TTC AAC 2970 93 H L G N L V I T W G A Q T F K H Q A F H 112 2971 AAA CTG GCT AAC CTG TTC ATC GTT AAC AAC AAA AAA ACC ATC CCG AAC AAC CTG GTT GAA 3030 113 K L A N L F I V N N K K T I P N N L V E 132 3031 AAC TAC CTG ACC CCG ATG TCT CTG GCA TAC TGG TTC ATG GAT GAT GGT GGT AAA TGG GAT 3092 133 N Y L T P M S L A T W F M D D G G K W D 152 3091 TAC AAC AAA AAC TCT ACC AAC AAA TCG ATC GTA CTG AAC ACC CAG TCT TTC ACT TTC GAA 3150 153 Y N K N S T N K S I V L N T Q S F T F E 172 3151 GAA GTA GAA TAC CTG GTT AAG GGT CTG CGT AAC AAA TTC CAA CTG AAC TGT TAC GTA AAA 3210 173 E V E Y L V K G L R H K F Q L N C Y V K 192 3211 ATC AAC AAA AAC AAA CCG ATC ATC TAC ATC GAT TCT ATG TCT TAC CTG ATC TTC TAC AAC 3270 193 I N K N K P I I Y I D S M S Y L I F Y N 212 3271 CTG ATC AAA CCG TAC CTG ATC CCG CAG ATG ATG TAC AAA CTG CCG AAC ACT ATC TCC TCC 3330 213 L I K P Y L I P Q M M Y K L P N T I S S 232 3331 GAA ACT TTC CTG AAA TAA 233 E T F L K *
A method of genetically mapping a eukaryotic genome that does not contain a natural restriction site for I-SceI, comprising the steps of:
- (a) artificially inserting one or more I-SceI sites at various positions in the genome;
- (b) completely cleaving said genome at the inserted I-SceI sites, with the restriction enzyme I-SceI, to produce nested chromosomal fragments;
- (c) purifying said fragments of step (b) by pulsed field gel electrophoresis (PFG);
- (d) transferring the fragments to a solid membrane;
- (e) hybridizing the fragments bound to said membrane to a labelled probe containing DNA complementary to said fragments;
- (f) detecting the hybridization banding patterns;
- and
- (g) mapping said eukaryotic genome based on the hybridization banding patterns observed in step (f).
A method for in vivo site directed genetic recombination in an organism using enzyme I-SceI, comprising the steps of:
- (a) introducing a synthetic gene encoding the I-SceI endonuclease into an expression vector;
- (b) inserting a I-SceI restriction site next to or within a gene of interest carried on a plasmid;
- (c) co-transforming the cells of said organism with said expression vector of step (a) and said plasmid of step (b), whereby said gene of interest, carried by said plasmid of step (b), is inserted into a chromosome of said organism at a specific site.
A method of genetically mapping a genome that does not contain a natural restriction site for I-SceI, comprising the steps of:
- (a) artificially inserting one or more I-SceI sites at various positions in the genome;
- (b) completely cleaving said genome at the inserted I-SceI sites, with the restriction enzyme I-SceI, to produce nested chromosomal fragments;
- (c) purifying said fragments of step (b);
- and
- (d) mapping said eukaryotic genome by detecting said fragments.
Dependencies are inferred from the claim text.