Worm Breeder's Gazette 10(2): 130

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.

Molecular Cloning of the unc-29 and unc-38 Levamisole Receptor Genes; RFLP's of These Genes Identified in Gamma Ray Mutants

J.A. Lewis

I have cloned fragments of the unc-29 and unc-38 genes by Tc1 
transposon tagging.  These genes are likely to encode structural 
peptides of the levamisole receptor, a nicotinic acetylcholine 
receptor present on nematode muscle.  Spontaneous, putative transposon-
induced mutants in these genes and other genes needed to make the 
receptor can be isolated by the drug resistance that mutationally-
induced receptor deficiency confers against the toxic muscle-
contracting effects of levamisole, a potent nicotinic acetylcholine 
Cloning the unc-38 receptor gene was very straightforward.  After 
backcrossing, 4 of 5 spontaneous unc-38 Bergerac mutants were found to 
possess the same novel 4.7 kb HindIII Tc1-containing restriction 
fragment.  Such a fragment was not present in 5 independent constructs 
containing unc-38+ from wild-type Bergerac (BO).  Comparison of 
flanking Tc1's present in mutant and control constructs indicated that 
the 4.7 kb HindIII fragment was in or close to unc-38 and unlikely to 
be a flanking Tc1.  When the genomic DNA flanking one of the mutant 
Tc1 inserts was used as a probe, the HindIII fragment under scrutiny 
was shown to be only 3.1 kb in size in Bergerac, in several control 
constructs, and in the Bristol N2 strain as compared to the 4.7 kb 
size fragment seen in the 4 mutants.  A fifth spontaneous unc-38 
Bergerac mutant contained a wild-type size 3.1 kb HindIII fragment.
As no full revertants of any spontaneous unc-38 mutants have yet 
been isolated, we examined homozygously viable gamma ray-induced unc-
38 mutants to confirm that the fragment which we cloned was part of 
the unc-38 gene.  Using a dose of 1500 rads and the same isolation 
technique used to obtain EMS-induced and spontaneous levamisole 
receptor mutants [Genetics 95: 905 (1980)], we isolated 34 mutants on 
40 selection plates: 7 unc-38, 5 unc-29, 8 unc-63, 8 unc-74, 2 unc-50, 
and 4 lev-1 mutants.  The EMS-induced forward mutation rate previously 
obtained indicates that these genes, with the possible exception of 
unc-50, are nonessential genes.  Possible deletions or rearrangements 
within a gene like unc-38 or unc-29 should thus be homozygously viable.
Restriction analysis of a homozygous mutant is simplified since 
there will be no background of completely normal restriction fragments 
contributed by a wild-type copy of the chromosome as there would be in 
the analysis of a balanced heterozygote.  Restriction fragment 
differences will show up as a clear absence of a fragment or a clear 
alteration in fragment mobility.  An even more important advantage in 
analyzing homozygotes, however, is that any gamma ray-induced 
deletions should extend no farther on the chromosome than the nearest 
left and right flanking essential genes bordering the nonessential 
gene targeted for analysis.  Any deletion going beyond the targeted 
gene into a nearby essential gene would be homozygously inviable.  
Thus, any restriction fragment differences observed in a homozygously 
viable gamma ray mutant can be presumed to arise in relatively close 
chromosomal proximity to the gene being mutated.  The disadvantage of 
this approach is the corresponding narrowing of all possible 
chromosomal abnormalities to only those that are homozygously viable.  
Of 6 gamma ray-induced unc-38 mutants so far examined, 3 mutants 
showed a difference from wild type in the size of the putative unc-38 
HindIII fragment initially identified by transposon tagging.  Fifteen 
lambda phage carrying this fragment have been isolated from an EMBL-4 
genomic library originating from Bill Wood's laboratory and the phage 
await further analysis.
Cloning unc-29 has been more difficult.  Jim Thomas (Horvitz lab) 
identified a 3.3 kb Tc1-containing EcoRI fragment that is closely 
linked to unc-29.  Counting Thomas' mapping results and my own, this 
fragment has not been separated from unc-29 in 22 recombination events 
with unc-13(e450) 1.1 map units to the left or in 18 recombination 
events with lin-11(n389) 1.6 map units to the right.  The presence of 
this fragment in an unc-29 control construct indicates that it is a 
flanking element.  I identified a novel 3.7 kb EcoRI Tc1-containing 
fragment not separable from unc-29 in 12 recombination events with unc-
13 or in 12 recombination events with lin-11.  The fragment was absent 
in an unc-29 control construct.  The genomic DNA flanking this Tc1 
insert used as a probe revealed that the parental size of the fragment 
was 2.0 kb in Bergerac and Bristol wild-type strains as opposed to 3.7 
kb in the x513 mutant strain.  In another spontaneous unc-29 mutant, 
the same EcoRI fragment was 2.5 kb bigger than in the wild type and 
the wild-type size of the fragment was restored in a revertant.  Nine 
phage containing the EcoRI fragment were pulled from the Wood lab EMBL-
4 genomic library.  From similarities and differences between the 
phage, the EcoRI fragments spanned by the phage were ordered.  Four 
additional spontaneous unc-29 mutants were probed with one of the 
phage.  Two of the additional mutants showed 1.6 kb 'inserts' in a 1.4 
kb EcoRI fragment immediately adjacent to the fragment initially 
cloned.  Two other mutants show 1.6 kb 'inserts' in the next 
contiguous EcoRI fragment, a 4.9 kb fragment, and the wild-type size 
of this fragment appears restored in revertants of these mutants.  A 
homozygously viable unc-29 gamma ray mutant is also altered in the 
size of the 4.9 kb fragment.  Thus, unc-29 gene expression is affected 
by alteration of any one of three contiguous EcoRI fragments.  I am 
attempting to detect an mRNA transcript from this region and am going 
to DNA sequence outward from the 'central' 1.4 kb fragment.
In summary, extremely useful ingredients in cloning the unc-29 and 
unc-38 genes were the generation of about a half dozen independent 
mutant isolates for each gene, the comparison of the Tc1 fragments 
found in mutants to the Tc1 fragments found in control constructs, and 
the examination of restriction fragment state in homozgyously viable 
gamma ray-induced mutants as well as in revertants of transposon-
induced mutants.