homologous recombination in fungi

ROBERT PROCTOR rproctor at ASRR.ARSUSDA.GOV
Thu Apr 7 16:00:27 EST 1994


Hello:

Does anyone out there know anything about the frequency 
of homologous recombination during fungal 
transformation varying with the size of the piece of 
the target gene in the disruption vector?  I seem to 
remember hearing a talk where someone disrupted a 
fungal gene with several different sized fragments of 
the same target gene, the larger the fragment of the 
target gene in the transformation vector the greater 
the frequency of disruptants among the transformants.  
If anyone has seen any published work on this, could 
you provide me with a reference or two?  If you've had 
any personal, non-published experiences like this I'd 
be glad to hear about them too.  Thanks in advance for 
your help.

The reason I'm asking is that I disrupted a toxin 
biosynthetic gene, Tri6, in Fusarium sporotrichioides 
using the procedure in which the isolated coding region 
of the gene was truncated at both ends, ligated into a 
transformation vector, and then transformed into 
Fusarium.  The frequency of disruptants (homologous 
recombination) among the transformants was quite low, 
about 1%.  When we have disrupted other genes in F. 
sporotrichioides using this same approach, homologous 
recombination frequencies are higher, sometimes as high 
as 40%.  One difference between these other genes and 
Tri6 is size.  Tri6 is quite small and the truncated 
version used in the transformation vector was only 468 
bp.  The truncated versions of the other genes were 
about 1 kb, sometimes greater.  So, my thought was that 
maybe the homologous recombination frequency for Tri6 
was low because of the small size of the truncated copy 
of Tri6 in the transformation vector.  Does this sound 
plausible?

Thanks again.
Bob




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