From owner-celegans@net.bio.net Sun Dec 15 22:00:00 1996 Path: biosci!BIOSCIENCE.BIOLOGY.UTAH.EDU!Jorgensen From: Jorgensen@BIOSCIENCE.BIOLOGY.UTAH.EDU ("Jorgensen") Newsgroups: bionet.celegans Subject: summary of ts mutations Date: 16 Dec 1996 06:10:14 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 146 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net 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 Anderson. 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 promoter. 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: none 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 nonsense 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 splicing lag-2(q420ts) mutation at splice acceptor lin-39(n1490ts); lin-39(n1872ts): both G to A transition at splice donor let-23(n1045cs) .