ts mutant by N-degron method

[Tien-Hsien Chang]

     We have tried a new technology to generate temperature-sensitive (ts) 
mutants [see Dohmen, R. J., Wu, P. and Varshavsky, A. (1994) Heat-inducible 
degron, a method for constructing temperature-sensitive mutants. Science, 263, 
1273-1276].  Basically one simply constructs a fusion consisting of a portable 
ts protein-degradation cassette fused to the 5' end of the gene of interest.  
This construct is then integrated to the chromosomal site of the gene of 
interest, creating a truncated version and the recombinant 
temperature-sensitive "degradable" (td) version flanking the plasmid DNA on the
chromosome.

     The gene of interest in our case is DBP5 (DEAD-Box protein; an essential 
putative RNA helicase gene of unknown function; cellular localization unknown).
 At the first glance, this technology works extremely well, in that the dbp5-td
strain failed to grow on either YPD or SD-Ura solid media at 37oC (analyzed by 
streak-out or spotting method).  Although after 3-5 days' incubation at this 
temperature, there are a small number (1 to 6 in each streak out) of revertants
(?) popping out on the plate.  Introduction of the ubr1-deletion allows the td 
strain to regain growth at 37oC, demostrating the temperature-sensitivity is 
due to N-degron protein degradation pathway.

     But the good news ends here, because we could not reproduce the plate 
results in the liquid culture.  We have tried several times for the temperature
shift growth curve studies.  In all cases, the td strain grows equally well as 
the wild type strain (in selective medium) for the first 9-10 hours after being
shifted to the nonpermissive temperature.  We have tried shifting at various 
OD600, such as 1 (mid-log phase), 0.8, or 0.3 (early log phase), and obtained 
practically the same results.  The td strain showed approximately 50% reduction
of growth (1.0 versus 1.6 of wild-type strain) for a period of at least 7-8 
hours subsequent to the first 10 hr of shifting to 37oC.  However, the td 
strain still eventually reach near saturation (with comparable OD as the wt) 30
hr after the temperature shift.  This result is completely at odds with the 
tight growth arrest phenotype on the plate.

     We have measured the spontaneous reversion frequency of the td strains in 
the liquid culture.  The numbers we got were 5.4x10e-7, 7.2x10e-7, and 3x10e-6 
from 3 independent experiments using two td isolates.  Thus, we believe that 
the problem is unlikely due to exceptionally high spontaneous reversion 
frequency (derived from the the N-degron pathway).

     We have inspected the cell morphology 10 to 30 hours after the shift in 
the liquid culture and found no obvious signs of strange shapes (such as 
super-enlarged shapes) which may explain the high OD after shift.

     At this moment, we are still scratching our heads.  Would any one out 
there like to venture some explanations of this seemingly strange observation?
The central question is how do the cell growth (physiology) differ from each 
other on plate and in the liquid medium?  Could it be due to the difference of 
osmolarity, nutrient diffusion rate, or something else?

          Tien-Hsien Chang
          Department of Molecular Genetics
          The Ohio State University
          484 West 12th Ave.
          Columbus, OH 43210

          (614)-292-0631
          (614)-292-4466 [fax]
          chang.108 at osu.edu