Method of selecting recombinant DNA-containing streptomyces
AssigneeEli Lilly and Company
InventorsR. Nagaraja Rao, Richard K. Stanzak
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Abstract
A novel method of selecting Streptomyces recombinant DNA-containing host cells and vectors useful in exemplifying the method are described. The vectors confer apramycin resistance to sensitive Streptomyces host cells and thus provide a convenient method of selecting Streptomyces transformants. The apramycin resistance-conferring gene used in the method is an acetyltransferase aac(3)IV gene and can be isolated from E. coli K12 BE1041/pKC309 (NRRL B-15827) on an .about.1.5 kb PstI-EcoRI restriction fragment.
Claims38
A method for selecting a recombinant DNA-containing Streptomyces host cell, said method comprising:
- (a) transforming an apramycin-sensitive, restrictionless Streptomyces host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Streptomyces host cell, said vector comprising the apramycin resistance-conferring gene of plasmid pKC309, and
- (b) culturing said transformed cell under conditions suitable for selection for apramycin resistance,
- subject to the limitation that expression of apramycin resistance-conferring activity is controlled by the promoter of said apramycin resistance-conferring gene of plasmid pKC309 and that said host cell in susceptible to transformation, cell division and culture.
A recombinant DNA vector capable of transforming an apramycin-sensitive restrictionless Streptomyces host cell and conferring apramycin resistance thereto, said vector comprising the apramycin resistance-conferring gene of plasmid pKC309, subject to the limitation that expression of apramycin resistance-conferring activity is controlled by the promoter of said apramycin resistance-conferring gene and subject to the further limitation that said vector does not comprise DNA having (1) the same order and relative position of restriction sites as depicted in the map of FIG. 5, or (2) the same linear order and relative position of restriction sites as would occur upon interrupting the map depicted in FIG. 5 at any single point.
A method for selecting a recombinant DNA-containing Nocardia host cell, said method comprising:
- (a) transforming an apramycin-sensitive, restrictionless Nocardia host cell with a recombinant DNA cloning vector capable of autonomous replication or integration in said Nocardia host cell, said vector comprising the apramycin resistance-conferring gene of plasmid pKC309, and
- (b) culturing said transformed cell under conditions suitable for selection for apramycin resistance,
- subject to the limitation that expression of apramycin resistance-conferring activity is controlled by the promoter of said apramycin resistance-conferring gene of plasmid pKC309 and that said host cell is susceptible to transformation, cell division and culture.
Dependencies are inferred from the claim text.