Ts allele both heat- and cold-sensitive?

Model Organism: Worms Scientific Discussion
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Hi! I recently found out that a certain recessive ts mutant is doubly ts: it shows the Loss of Function phenotype at too-cool temp as well as too-warm. This mutant is actually a sort of instant double mutant: EMS gave it two missense lesions, in different parts of the gene. Maybe one substitution loosens things (making the protein heat-sensitive), and the other one tightens things (making it cold-sensitive)? I’m sure there’s lots of, millions or billions of examples of cold-sensitive mutations. WT Taq polymerase (and every other ORF in Thermus aquaticus) has a bunch of them, obviously (assuming that T. aquaticus evolved from a not-so-thermophilic ancestor).

I do know that neither missense is in the background strain.

I don’t know if the cold-sensitivity is recessive or not.

I’m unable to CRISPR-in or CRISPR-out the individual missenses to find out if one is heat-sensitive and the other is cold-sensitive. Alternatively, could a single missense somehow cause both of these sensitivities? I’m a protein structure dilettante, I mean ignoramus, but that intuitively seems far-fetched. I wonder if anyone has ever heard of such a thing?? That is my question.

(It’s possible that this is a case of evolution in the lab. This mutant was isolated in a screen that happened years before I got my hands on the 6x backcrossedxWT homozygous strain, and it might have eked along at partially-restrictive temperature for a total of hundreds of rounds of DNA replication, so a spontaneous mutation that counteracts the original heat-sensitive one might well have become fixed in the little population. I don’t think it was ever customary to freeze newly isolated mutants in this lab, so I guess we’ll never know.)

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Look into the n1045 allele in let-23, analyzed in a 1991 Genetics paper by Raffi Aroian in Paul Sternberg’s lab. At 15C n1045 animals are vulvaless, at 25C n1045 animals multivulva (Simplified). Gene dosage analysis with null alleles suggests that there is more let-23 function at 25C than at 15C, but that function is compromised at both.

So is n1045 both cs and ts? Or does LET-23 have two activities revealed at different levels of function? Aroian and Sternberg argue that LET-23 both stimulates vulval induction, revealed by stronger loss of function, and inhibits vulval induction, revealed by weaker loss of function. So functionality is a continuum, not both cs and ts. A background of reduced function but also cs. So reduced function at higher temperature and more reduced function at lower temperature, but the Vulvaless at lower temperature masks the Multivulva of higher temperature. Is that clear? I have not read the paper in many years.

The molecular nature is also known (Aroian Kramer Sternberg 1993). n1045 alters a splice acceptor site. (They have a similar allele in dpy-10, but the phenotpes are not as interesting from your perspective.) Gene product is reduced but some still present via cryptic splicing events. Presumably you get more functional LET-23 at higher temps than lower temps, matching the phenotypes.