Worm Breeder's Gazette 8(3): 23

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

ace-3, the Third Gene Affecting Acetylcholinesterase

C. Johnson, A. Journey

Using the unusual properties of class C acetylcholinesterase (AChE), 
the third AChE class described by Dennis Kolson at the last C.  
elegans meetings, -we devised an assay which is specific for this AChE 
class, as judged by the fact that the same AChE activity level is 
detected in wild type as in either single or double mutants affected 
in the previously described genes ace-1 and ace-2 (Johnson et al., 
Genetics 97, 261-279; Culotti et al., Genetics 97, 281-305).  This 
assay was then used to screen for mutants affected in class C AChE 
level, among the F2 progeny of cloned F1's from EMS-mutagenized N2 
parents.  Of 400 such progeny clones examined (2 animals each), 2 
independent isolates proved to have extremely low levels of AChE by 
this assay.  Enzymatic assay of male cross progeny from a cross 
between the two new strains indicates that they harbored allelic 
defects producing the enzymatic deficiency.  The new mutations were 
given the names dc2 and dc3, and the gene they identify was given the 
name ace-3.  Scoring always by enzymatic assay, ace-3 was mapped to 
the right end of chromosome II, near unc-52 and cad-1; 
unlinked to either ace-1 or ace-2.  The 
strain harboring dc2 was backcrossed twice to N2, then to a strain 
containing rol-1, to produce the rol-1 utant, 
and finally to N2, to reisolate ace-3 (final strain name PR1300).  The 
strain harboring dc3 was backcrossed twice to N2 (final strain name 
Extracts of the backcrossed ace-3 strain PR1300 were examined by 
velocity sedimentation and ion exchange chromatography, using the 
conditions and the selective assay which previously identified class C 
AChE in N2; no evidence for any class C AChE was found, and the lower 
detection limit was less than 5% of the wild type level.  Whether ace-
3 is the structural gene for class C AChE is uncertain; gene dosage 
experiments have not yet been done, and we have not yet detected any 
residual activity for qualitative characterization in a mutant strain. 
Neither of the two backcrossed ace-3 strains PR1300 and CD14 
exhibits any obvious abnormalities of either behavior or development; 
thus class C AChE, like class A and class B AChE, is dispensable in 
the presence of the other AChE classes.  ace-3; 
omozygotes are also behaviorally and 
developmentally normal, and ace-2; omozygotes 
are behaviorally grossly normal but have a slightly slower growth rate 
than wild type; thus in the absence of class C AChE, either class A or 
class B AChE alone is fully or almost fully adequate for normal growth 
and behavior.  
By contrast with these marginal deficiencies, apparent ace-2; 
ace-1 triple homozygotes are much more markedly 
affected.  These have been segregated from parents of two different 
genotypes, viz.  ace-2; ace-1/+ and ace-2; 
ace-1, and are recognized by the fact that they 
constitute approximately one-fourth of the progeny and are both 
developmentally and behaviorally arrested.  Such animals accumulate as 
eggs in which it is difficult to decide whether hatching has already 
occurred; the eggs are much less refractile than usual, but still have 
an apparent outer membrane, and give the impression of having been 
enzymatically digested from within without being punctured.  Attempts 
to move these eggs invariably rupture any outer membrane that was 
there originally, revealing inside a coiled offspring that only a 
worm's mother could love.  This poorest of attempts at an L1 is still 
bent into three parts, as required to fit into the egg shell, and 
remains similarly bent for several days thereafter; since it is 
essentially motionless when released (moving less than 1 worm diameter 
in 24 hours), it has also presumably been motionless in the egg, 
accounting for the failure of the shell to be mechanically ruptured.  
Surprisingly, these animals survive for several days, as judged by the 
production of a very occasional pharyngeal pump, but show absolutely 
no growth.  Internally, by electron microscopy, they exhibit the 
expected types of cells, at least broadly speaking, but structure is 
much distorted by the overall bent nature of the whole animal.  The 
difference in phenotype between these animals and ace-2; 
omozygotes indicates that in the absence of 
AChE classes A and B, class C AChE is essential for normal development,
and that the amount normally present is adequate for partial but not 
full coordination.  In support of this conclusion, animals of the 
genotype ace-2; ace-1 (one of the two parental 
types from which triple homozygotes have been segregated) are 
themselves small, considerably less fecund than normal, and even more 
uncoordinated than ace-2; omozygotes.  If, as 
we suspect, ace-3 is a structural gene for class C AChE, such animals 
should by gene dosage have only half the normal level of class C AChE, 
perhaps accounting for their more severe phenotype.  
Given the severity of the triple homozygote phenotype, we hope that 
with ace-3 we have identified the last of the structural genes for C.  
elegans AChE.