Construction of a large plasmid lacking linearizing single restriction sites by simultaneous in vivo recombination and plasmid shuffling in yeast.
Creation of large ( approximately 15 kb) recombinant plasmids can be done in a single step by co-transformation of yeast cells with a partial restriction digest of a plasmid vector and a linear insert whose ends overlap one of the vector restriction sites. This method is used to generate a plasmid expressing the Saccharomyces cerevisiae rRNA genes containing the Ca.LSU group I intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme.
Miletti
K E
KE
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635, USA.
Leibowitz
M J
MJ
D002176:Candida albicans / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D016337:RNA, Catalytic; D012331:RNA, Fungal; D012335:RNA, Ribosomal; C065039:RNA, ribosomal, 25S