Worm Breeder's Gazette 9(1): 47

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Relationship between Phenotypes and ChAT Activity of the cha-1 ts Allele cn101

R. Hosono, T. Sassa, and Sigeru Kuno

Figure 1

The cn101 mutation that was originally isolated as a temperature 
dependent paralytic mutant, was found to be an allele of the cha-1 
gene, the structural gene for choline acetyltransferase (ChAT).  The 
strain is normal in its phenotypes (development, behavior and drug 
sensitivity) at 16 C but abnormal at restrictive temperature : slow 
growth and small adult size at 25 C, resistance to cholinesterase 
inhibitors and paralysis by coiling its body at 30 C.  J. B.  Rand 
mapped many alleles of genes cha-1 and unc-17.  The relative position 
of the cn101 mutation was determined based on complementation for 
behavioral deficiency with available markers as follows (distances 
between markers are arbitrarily positioned).
[See Figure 1]
ChAT activity of crude extracts from cn101 grown at permissive 
temperature (16 C) was only 10% of wild-type ChAT activity.  ChAT from 
the mutant was very temperature sensitive 
that is, 
the activity was maximal at 10 C but not detectable at 25 C, while 
wild-type enzyme activity was maximal at 25 C.
In order to know the relationship between paralytic behavior and 
enzyme level caused by this mutation, we purified the mutant ChAT 
about 80-fold using (NH4)2SO4 fractionation, Sephacryl and DEAE 
chromatography.  The partially purified enzyme was still temperature 
sensitive as in the crude enzyme.  The differences between both 
enzymes in the affinity for choline and acetyl-CoA were not remarkable.
However, the affinities for the substrates were greatly reduced by 
high concentration of NaCl in the mutant enzyme as summerized below.
These results are consistent with that cn101 is the mutation of the 
structural gene for ChAT whose activity is greatly affected by 
temperature and salt concentration.

Figure 1