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summary of ts mutations

Mon Dec 16 09:10:14 EST 1996

Thanks all for the robust response to my enquiry into temperature sensitive
mutations.  Here is my summary.

For a while now I have been irritated by the difficulties we face when
analyzing functional defects in mutants that may have developmental or
compensatory changes because of those mutations.  Temperature sensitive
mutations avoid these pitfalls but we have to depend on good fortune and the
vagaries of mutagenesis for the rare temperature sensitive mutant.  I have
been wondering lately whether one could engineer temperature sensitive
mutations.  Scott Beeser, a graduate student here at the University of Utah,
provided me with the following paper:

Rennell, D., Bouvier, S.S, Hardy, L.W. & Poteete, A.R. (1991).  Systematic
Mutation of Bacteriophage T4 Lysozyme. J. Mol. Biol. 222, p 67-87.

These authors engineered stop codons at each position in the open reading
frame of this gene.  They then provided the strain with suppressor tRNAs that
could replace this stop codon with any amino acid.  They thereby exhaustively
substituted all amino acids into each residue and tested whether each
substitution could provide a functional protein.  In the table they remarked
whether these substitutions created a heat sensitive protein at 37#161#
compared to 30#161#.  In summary, a valine, leucine or isoleucine in a
hydrophobic region changed to a charged amino acid created a heat sensitive
protein; that is, nonpolar to charged substitutions created heat sensitive
proteins.  This temperature sensitivity is presumably caused by the protein
unfolding due to a buried charge.  It is a bit surprising that these
substitutions are functional even at lower temperatures.

The question remained whether such changes would be heat sensitive in C.
elegans, particularly since the range of temperatures at which we like to
work is 20#161# to 25#161#.  I surveyed the community and cataloged 43
temperature sensitive mutations (see below).  Although there were 7 changes
from nonpolar residues to charged residues, only one of these mutations was a
change from a V, L or I to a D, E, K, or R.  So why aren't these expected
substitutions found in ts mutations in C. elegans?  Basically because you
can't get there from here.  The genetic code is arranged so that transitions
produced by errors in replication (or by EMS) make conservative
substitutions.  To convert a codon for any of those hydrophobic residues to
one of the charged residues would require at least one transversion.  So I
think that the method may still work but we just don't have the empirical
data to support or contradict these observations in C. elegans.

A number people came up with their own suggestions for how to design
temperature sensitive mutations and I have listed these here.  

1) designer ts mutations in yeast can be made by attaching an amino terminal
ts fragment that targets the protein for degradation at elevated
temperatures.  Roy Parker (Univ Ariz) has used this method for some of his
proteins in yeast but he said that it is not always dependable.
Ref: Dohmen Wu Varshavsky (1994) Heat-inducible N-degron activates ubiquitin
degradation. Science 263, 1273-1276.  

2) several people suggested the alanine scanning technique originally
published by the Botstein group.  As I remember alanine scanning replaces
charged residues with alanines, with the idea that these changes would
destroy salt bridges with interacting proteins.  I would not expect such
mutations to have a high likliehood of being temperature sensitive but I
can't find my file with those papers so I couldn't check this out.

3) ts Smg system.  Leon Avery wrote: Richard Zwaal is working on a general
method based on a ts smg mutation.  The idea is to append the 3' unc-54
sequence after the termination codon of your gene.  The unc-54 sequence is
the segment that follows an unc-54 nonsense mutation that produces ~5% of
normal message levels in a wild-type background, but high levels in a smg
background.  This should make your constructed message unstable in a smg+
background, but stable in smg-.  There is a smg-7(ts), so you expect in that
background to get high expression at restrictive T, but only about 5% at
permissive.  I got this idea from a talk someone in Victor Ambros's Lab gave
at the last International meeting, and Richard talked it over with Phil

4) ts factor X site.  I was thinking that one could engineer a factorX site
into the middle of a protein and then induce factorX under a heatshock

5) Finally, there is the hydrophobic to charged amino acid model.  Here are
the mutations which I collected:

C. elegans temperature sensitive mutations obtained by mutagenesis:
amino acid groupings follow Alberts et al.  Molecular Biology of the Cell

nonpolar to nonpolar:
unc-4(e2322ts) 	L to F
glp-1(e2141)   	L to F
glp-1(e2144)   	L to F
unc-25(n2569)  	L to F 
lin-14(n530ts) 	L to F
fer-1(hc24)    	L to F
let-60(?)      	L to F
fer-1(hc13)    	A to V
deg-1(u38)     	A to V
unc-25(sa94)   	M to I  
fem-3(ts)      	M to I
cha-1(cx52)    	P to L
mpk-1(ga111)   	V to G 
pha-1(ts)      	C to Y

nonpolar to polar:
cha-1(cx48)   	P to S
fem-3(ts)     	P to S
glp-1(bn18)   	A to T
ced-9(n1653ts)	Y to N

nonpolar to charged:
glp-1(q224)   	G to E
glp-1(q231)   	G to E
unc-25(n2379) 	G to E
cha-1(cx49) 	  G to R
egl-19(n2368cs)	G to R
cha-1(md39)	   A to D
lin-14(n679ts) V to D

polar to nonpolar:
cha-1(y226)	   T to I
tax-2(ts)     	T to I

polar to polar:
fer-1(b232)   	S to N

polar to charged:

charged to nonpolar
glp-1(sy56)   	R to W
lin-14(n179ts)	R to G

charged to polar
cha-1(cn101)  	D to N

charged to charged
cha-1(cx50)   	R to K
fem-3(ts)     	E to K

lin-2(n105): stop about 25 aa from the C terminus.
mpk-1(ga110), mpk-1(oz140): stop 26 aa from the C terminus. 
glp-1(q35) stop 122aa from C terminus.
egl-15(n1477ts) stop
egl-10(n692) stop

lag-2(q420ts) mutation at splice acceptor
lin-39(n1490ts); lin-39(n1872ts): both G to A transition at splice donor

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