ISSN:
0749-503X
Keywords:
Transformation
;
oligonucleotides
;
site-directed mutagenesis
;
co-transformation
;
cytochrome c
;
RAD genes
;
CYC1 mutants
;
Life and Medical Sciences
;
Genetics
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially fuctional iso-1-cytochrome c. Aproximately 3 × 104 transformanrs, constituting 0·1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogenous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double-strand plasmid, was efficient in a rad52- strain, ssuggesting that the pathway for transformation with oligonucleotides is different from that with linearized double-strand plasmid. We describe a procedure of co-transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amono acid alteration elsewhere in the iso-1-cytochrome c molecule; approximately 20% of the transformants obtained by co-transformation contained these desired second alterations.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/yea.320081104
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