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Some bcy1 mutants in yeast could be lethal??

Marta marta at dada.jci.tju.edu
Mon Mar 15 17:34:52 EST 1999


Does anybody know if it is possible obtain lethal phenotype in some
Saccharomyces cerevisiae genetic background by knocking-out that gene?.
The inactivation of BCY1 encoding the regulatory subunit of cAPK is
supposed to be viable as has been extensively demonstrated in the
literature so this question can seem aparently stupid but I'm having
some problems at this respect at the time I'm tracting to delete this
gene. My strategy consists in generate by PCR a fragment that contains
an auxotrofic marker flanked in each extreme by sequences near or inside
the ORF of that gene, in order to replace the complete sequence with
that by genetic transformation. I'm using diploids for that experiment.
After select the transformants in the appropiate selective medium I can
confirm the presence of the deletion in at least one of the chromosomes
but when I tract to dissect the transformed diploids the tetrads are 2
viable: 2 non viable. Precisely the non viable are which are supposed to
heritate the deletion. Any ideas about what is happening or about an
alternative way to get this kind of mutation?
Every sugestion will be wellcome.Thank's a lot !

Marta Rubio-Texeira
Thomas Jefferson University
Kimmel Cancer Center
233 S 10th St. rm. 826
Philadelphia PA 19107
Tel: (215) 503-4594
Facsimile: (215) 923-2117





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