Method for integrative transformation of yeast using dispersed repetitive elements
AssigneeChiron Corporation
InventorJeffrey R. Shuster
Life of this patent
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Abstract
Efficient integration of heterologous DNA into yeast genomic DNA is accomplished at high copy number by targeting integration vectors to dispersed repetitive elements such as DELTA sequences, Ty elements, or tRNA DNA sequences present in the host cell genome.
Claims20
A vector for introducing multiple copies of heterologous DNA into a yeast host cell genome, wherein said yeast host cell genome comprises a plurality of DELTA sequences, each of said DELTA sequences being homologous to the DELTA sequence occurring in plasmid pJS176 (ATCC Accession Number 68368), and wherein said vector comprises:
- a linear DNA molecule comprising first and second DNA sequences, wherein the first and second DNA sequences are homologous to the 5' end and 3' end, respectively, of each of the DELTA sequences in the host cell genome, whereby said vector is capable of homologous recombination with the host;
- and
- heterologous DNA flanked by said first and second DNA sequences.
A circular vector for introducing multiple copies of heterologous DNA into a yeast host cell genome, wherein said yeast host cell genome comprises a plurality of DELTA sequences, each of said DELTA sequences being homologous to the DELTA sequence occurring in plasmid pJS176 (ATCC Accession Number 68368), and wherein said vector comprises:
- a circular DNA molecule comprising first and second DNA sequences, wherein the first and second DNA sequences are homologous to the 5' end and 3' end, respectively, of each of the DELTA sequences in the host cell genome, whereby said vector is capable of homologous recombination with the host;
- and
- heterologous DNA flanked on the 5' side by said first DNA sequence, and on the 3' side by said second DNA sequence;
- wherein said circular DNA molecule has a unique restriction endonuclease recognition site positioned 5' of said first DNA sequence and 3' of said second DNA sequence.
A method for introducing multiple copies of heterologous DNA into a yeast host cell genome, wherein said yeast host cell genome comprises a plurality of DELTA sequences, each of said DELTA sequences being homologous to the DELTA sequence occurring in plasmid pJS176 (ATCC Accession Number 68368), wherein said method comprises:
- providing a linear DNA molecule comprising first and second DNA sequences, wherein the first and second DNA sequences are homologous to the 5' end and 3' end, respectively, of each of the DELTA sequences in the host cell genome, whereby said linear DNA molecule is capable of homologous recombination with the host, and heterologous DNA flanked by said first and second DNA sequences;
- and
- transforming a suitable yeast host cell with said linear DNA, wherein said linear DNA undergoes homologous recombination with a plurality of DELTA sequences in the host genome.
A method for introducing multiple copies of heterologous DNA into a yeast host cell genome, wherein said yeast host cell genome comprises a plurality of DELTA sequences, each of said DELTA sequences being homologous to the DELTA sequence occurring in plasmid pJS176 (ATCC Accession Number 68368), wherein said method comprises:
- providing a circular DNA molecule comprising first and second DNA sequences, wherein the first and second DNA sequences are homologous to the 5' end and 3' end, respectively, of each of the DELTA sequences in the host cell genome, whereby said circular DNA molecule is capable of homologous recombination with the host, and heterologous DNA flanked on the 5' side by said first DNA sequence, and on the 3' side by said second DNA sequence, wherein said circular DNA molecule has a unique restriction endonuclease recognition site positioned 5' of said first sequence and 3' of said second sequence;
- cleaving said circular DNA molecule at said unique restriction site;
- and
- transforming a suitable yeast host cell with said cleaved DNA molecule, wherein said cleaved DNA undergoes homologous recombination with a plurality of DELTA sequences in the host genome.
Dependencies are inferred from the claim text.