deletion vs. disruption

David R. Nelson nelsondr at med.unc.edu
Mon Nov 15 10:11:29 EST 1993


	This is for the yeast group.  One problem with disruption of
a yeast gene that was not mentioned earlier is the problem of recombination
If a gene is disrupted and a mutant form is added on a plasmid, the 
mutation can be eliminated by gene conversion with the disrupted
chromosomal copy of the gene.  This will restore a wild type copy
of the gene either on the plasmid or the chromosome.  The result of 
this recombination event can be quite time wasting especially if you 
are trying to select for second site revertant mutations that restore 
function.  A lot of time can be wasted sequenceing wild type genes looking
for second site mutants.  Believe me, I have done it.  For more detail 
see Nelson et al. J. Molec. Biol. 230, 1159-1170 and 1171-1182 (1993).
A clean deletion of the gene of interest can eliminate this potential
problem, and it is well worth the time to do it.  

	A related problem involves gene families in yeast.  In the case
of the ADP/ATP carrier of mitochondria, there are three closely related
genes AAC1, AAC2, and AAC3.  Even after we completely deleted AAC1 and AAC2
recombination still occurs with AAC3 on the chromosom and AAC2 on a plasmid.
During selection for regain of function, hybrid genes have been produced
that have removed the mutation that destroys function.  This should serve 
as a warning to all site-directed mutagenesis experimenters in yeast. The
genome is very flexible, and it can often repair the damage we inflict by
our carefully placed mutations.  

David R. Nelson
nelsondr at med.unc.edu (until Jan. I am starting as Asst. prof. at the 
University of Tennessee at Memphis College of Medicine, Dept. of Biochemistry.


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