The construction of mobilizable YACs and their direct conjugative transfer from E. coli to yeasts

Abstract

YAC, yeast artificial chromosome, consists of Saccharomyces cerevisiae's ARS, CEN and nutrient requirement genes and TEL from Tetrahymena with ori and ampicillin resistant genes of pBR322. we have constructed two mobilizable pAY-YACs plasmids by inserting oriT at different positions. pAY-YAC-B (12.2 kbp) containes oriT in between two telomeres, whereas in pAY-YAC-E (14 kbp) oriT has been inserted at the cloning site of pYAC4. Both plasmids were mediated from E.coli to S.cerevisiae and S.kluyveri with the aid of helper plasmid pRH220 which resides mob and tra genes. The results of conjugal transmission, restriction enzyme digestion and Southern blotting analyses indicated that both plasmids were able to maintained their size and structure, thus reflected the faithfull accomplishment of nicking and resealing of plasmids during conjugation. The direct transformation of plasmid DNA into yeast showed that pAY-YACs plasmids, may be due to their small size, have tendency of circularization.