Fa
ctors influen
cing the dire
ct transformation of the yeast Sa
ccharomy
ces
cerevisiae with syntheti
c oligonu
cleotides were investigated by sele
cting for
cy
c1 transformants that
contained at least partially fun
ctional iso-1-
cyto
chrome
c. Approximately 3 x 10(4) transformants,
constituting 0.1% of the
cells, were obtained by using 1 mg of oligonu
cleotide in the rea
ction mixture. Carrier, su
ch as heterogeneous oligonu
cleotides, enhan
ced transformation frequen
cies. Transformation frequen
cies were dramati
cally redu
ced if the oligonu
cleotides had a large number of mismat
ches or had terminally lo
cated mismat
ches. Transformation with oligonu
cleotides, but not with linearized double-strand plasmid, was effi
cient in a rad52- strain, suggesting that the pathway for transformation with oligonu
cleotides is different from that with linearized double-strand plasmid. We des
cribe a pro
cedure of
co-transformation with two oligonu
cleotides, one
corre
cting the
cy
c1 defe
ct of the target allele in the host strain, and the other produ
cing a desired amino a
cid alteration elsewhere in the iso-1-
cyto
chrome
c mole
cule; approximately 20% of the transformants obtained by
co-transformation
contained these desired se
cond alterations.
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