Automatic eukaryotic artificial chromosome vector
OwnerRonald A. Hitzeman, George E. Chisholm, IVinventors
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Abstract
The present invention is directed to novel recombinant nucleic acids for introducing yeast chromosomal elements into the genomes of bacteria. The invention provides methods to convert the modified bacterial genomes into artificial yeast chromosomes by fusing the bacteria with yeast that linearize the modified bacterial genomes, to produce artificial chromosomes.
Claims22
An automatic eukaryotic artificial chromosome vector comprising:
- (a) a prokaryotic selectable marker(s);
- (b) a DNA sequence selected from the group consisting of a DNA encoding a eukaryotic selectable marker(s), eukaryotic gene(s), eukaryotic cDNA(s), and combinations thereof;
- (c) a centromere;
- (d) a eukaryotic replication sequence(s);
- (e) at least two inverted telomeres;
- (f) restriction endonuclease site(s) at the functional chromosomal end of each telomere, wherein restriction at said site(s) converts said inverted telomeres into functional telomeres;
- and
- (g) a prokaryotic genome.
- 2.The automatic eukaryotic artificial chromosome vector according to claim 1 wherein said centromere, replication sequence(s), and telomeres function in a cell of an animal, plant, fungus, or a Protist.
A method of converting an automatic eukaryotic artificial chromosome vector into a eukaryotic artificial chromosome comprising:
- fusing a prokaryote with a eukaryotic cell, wherein said prokaryote comprises an automatic eukaryotic artificial chromosome vector comprising,
- (a) a prokaryotic selectable marker(s);
- (b) a DNA sequence selected from the group consisting of a DNA encoding a eukaryotic selectable marker(s), eukaryotic gene(s), eukaryotic cDNA(s), and combinations thereof;
- (c) a centromere;
- (d) a eukaryotic replication sequence(s);
- (e) at least two inverted telomeres;
- (f) restriction endonuclease site(s) at the functional chromosomal end of each telomere;
- and
- (g) optionally comprising at least a portion of a prokaryotic genome,
- wherein said eukaryotic cell expresses restriction endonuclease(s) that restricts said vector at said site(s), whereby said vector is converted into a eukaryotic artificial chromosome.
A method of altering a phenotype of a eukaryotic cell comprising:
- fusing a first prokaryote and a second prokaryote with a eukaryotic cell, wherein at least one of said prokaryotes comprises an automatic eukaryotic chromosome vector comprising,
- (a) a prokaryotic selectable marker(s);
- (b) a DNA sequence selected from the group consisting of a DNA encoding a eukaryotic selectable marker(s), eukaryotic gene(s), eukaryotic cDNA(s), and combinations thereof;
- (c) a centromere;
- (d) a eukaryotic replication sequence(s);
- (e) at least two inverted telomeres;
- (f) restriction endonuclease site(s) at the functional chromosomal end of each telomere;
- and
- (g) optionally comprising at least a portion of a prokaryotic genome,
- whereby said eukaryotic cell acquires an altered phenotype.
A method of converting an automatic eukaryotic artificial chromosome vector into a eukaryotic artificial chromosome comprising:
- fusing a prokaryote with a eukaryotic cell, wherein said prokaryote comprises an automatic eukaryotic artificial chromosome vector comprising,
- (a) a prokaryotic selectable marker(s);
- (b) a DNA sequence selected from the group consisting of a DNA encoding a eukaryotic selectable marker(s), eukaryotic gene(s), eukaryotic cDNA(s), and combinations thereof;
- (c) a centromere;
- (d) a eukaryotic replication sequence(s);
- (e) at least two inverted telomeres;
- (f) restriction endonuclease site(s) at the functional chromosomal end of each telomere;
- and
- (g) optionally comprising at least a portion of a prokaryotic genome,
- whereby said vector is converted into a eukaryotic artificial chromosome.
A method of converting an automatic eukaryotic artificial chromosome vector into an eukaryotic artificial chromosome comprising:
- restricting an automatic eukaryotic artificial chromosome vector, wherein said vector comprises,
- (a) a prokaryotic selectable marker(s);
- (b) a DNA sequence selected from the group consisting of a DNA encoding a eukaryotic selectable marker(s), eukaryotic gene(s), eukaryotic cDNA(s), and combinations thereof;
- (c) a centromere;
- (d) a eukaryotic replication sequence(s);
- (e) at least two inverted telomeres;
- (f) restriction endonuclease site(s) at the functional chromosomal end of each telomere;
- and
- (g) a prokaryotic genome,
- whereby said restricting at said sites said vector is converted into a eukaryotic artificial chromosome.
Dependencies are inferred from the claim text.