Worm Breeder's Gazette 11(5): 76

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New Alleles of lin-4

Rhonda L. Feinbaum and Victor Ambros

lin-4 is a heterochronic gene that controls the timing of a number 
of developmental events in C.  elegans.  The lin-4 allele (e912) 
causes a retarded phenotype that is virtually identical to that caused 
by lin-14(sd) mutations.  Genetic epistasis experiments have suggested 
that lin-4 acts upstream of lin-14 in the heterochronic regulatory 
pathway.  Gary Ruvkun's laboratory has shown that the temporal 
gradient of lin-14 protein, high to low from early to later in 
development, is disrupted in lin-4(e912) animals.  In lin-4(e912) 
animals, the level of lin-14 protein remains high throughout 
development causing the reiteration of early cell fates.  These data 
suggest that lin-4 is, at least in part, responsible for generating 
the temporal gradient of lin-14 activity.
In order to further understand the role of lin-4 in the temporal 
control of development we have begun to isolate new alleles of this 
gene.  The original lin-4(e912) allele was generated by [32P] decay 
over 10 years ago.  Because no new alleles of lin-4 have been 
identified in numerous general screens for egl or vul animals (where 
they would have been expected to turn up) we wanted to establish the 
null phenotype of lin-4 prior to screening for point mutations in this 
gene.  Unfortunately, no deficiencies existed that covered the lin-4 
region of chromosome II.  Therefore our first non-complementation 
screen was aimed at isolating a deficiency of the lin-4 locus.  Wild 
type N2 males were mutagenized with gamma rays, mated by lin-4(e912)
dpy-10/mnc1; 79ts) hermaphrodites and the F1 
hermaphrodite progeny were screened at 20 C for non-dpy lin-4 like 
animals.  Out of 7,000 F1 hermaphrodites screened, one lin-4 deletion 
mutant (maDf4) was identified.  maDf4 is homozygous inviable and fails 
to complement lin-4, bli-2,  
analysis using a dissecting microscope, the phenotype of lin-4(e912) 
Df4 animals is indistinguishable from that of lin-4(
e912)/lin-4(e912) animals (with the exception of an enhanced sterility 
of lin-4(e912) Df4 animals).  These data suggest 
that the original lin-4(e912) allele is not a gain-of-function allele 
and indicates that lin-4(e912)/null is fertile and viable.  Since lin-
4(e912)/maDf4 is not significantly different from lin-4(e912)/lin-4(
e912), lin-4(e912) is probably a null allele.  However, maDf4 does not 
delete spe-3, a gene that maps only 0.1 mu to the right of lin-4 and 
thus we are not yet certain that maDf4 completely deletes lin-4.  Now 
that we have a lin-4 clone it will be possible to test this directly (
see Lee, Feinbaum and Ambros, this WBG).
Encouraged that we knew the expected phenotype of lin-4(e912)/lin-4 
null we have initiated a new series of non-complementation screens to 
identify EMS induced lin-4 alleles.  A non-complementation screen 
identical to the one outlined above, except that the males were 
treated with EMS instead of gamma rays, was undertaken.  Out of 8,000 
F1 hermaphrodites screened we have identified 1 new lin-4 allele (
ma161).  ma161 looks phenotypically similar to e912 but Southern blot 
analysis with our lin-4 clone has confirmed that it is in fact a new 
allele of lin-4.  We intend to continue isolating new lin-4 alleles 
utilizing this non-complementation strategy.