Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient. |
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Authors: | Hugues Blanc Claude Gerbaud P Piotr Slonimski and Michel Guérineau |
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Institution: | (1) Laboratoire de Biochimie Enzymologie, Institut Gustave Roussy, F-94800 Villejuif, France;(2) Centre de Génétique Moléculaire du C.N.R.S., F-91190 Gif sur Yvette, France;(3) Laboratoire de Génétique, Institut National Agronomique, F-75005 Paris, France |
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Abstract: | Summary By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 m yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 m DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 m-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura+ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5 monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type.These features should offer new possibilities for cloning with yeast. |
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