| 摘要 |
A DNA cassette (I) for the transformation of yeast not including any exogenous DNA other than the gene of interest is new and comprises at least: (a) a dominant negative marker; (b) two non-exogenous and non-recombinant, directly repeated sequences (DRS) with the genome of the host strain; (c) eventually at least a gene of interest, with the necessary elements for expression in the host cell. A DNA cassette (I) for the transformation of yeast not including any exogenous DNA other than the gene of interest is new and comprises at least: (a) a dominant negative marker; (b) two non-exogenous and non-recombinant, directly repeated sequences (DRS) with the genome of the host strain, the two sequences are positioned in either part of the fragment and contain the dominant positive marker; (c) eventually at least a gene of interest, with the necessary elements for expression in the host cell. Independent claims are also included for the following: (1) a process (II) for the integration of a target gene or the inactivation of a gene in the yeast comprising: (a) transforming the yeast with the help of a DNA construct comprising (I); (b) selecting the yeast comprising the integrated cassette using the positive marker; (c) then, after culture, the yeast is selected comprising the cassettes that has been excised using DRS in selecting yeast deprived of the negative marker; (2) a process for the transformation of an auxotrophic yeast with a plasmid shuttle to obtain expression of an elevated number of copies of at least a gene of interest comprising: (i) transforming the yeast with a plasmid shuttle; (ii) selecting on minimal medium allowing for the induction of the negative marker, the cells not comprising the plasmids in which the part corresponding to Escherichia coli fragments and to the negative marker has been excised; and (3) yeast transformed using (I) obtained by (II) and only contain the DNA of the yeast with the eventual exception of some of the gene(s) coding for a protein of interest and non-coding DRS sequences. |
