Worm Breeder's Gazette 13(4): 28 (October 1, 1994)

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.

Mutagenesis of C. elegans Using N-ethyl-N-nitrosourea.

Elizabeth De Stasio, Dinesh Stanislaus, Catherine Lephoto

Department of Biology, Lawrence University, Appleton, Wl 54911

  Published sequences of EMS-induced mutations in C. elegans are 95%GC->AT
transitions with the remaining 5% evenly split between AT->TA and GC->TA
transversions. Due to frequent production of A-T base pairs, EMS is quite useful for
the production of stop codons. For studies of protein interactions however, it is
desirable to produce non-null mutations with higher frequency. A literature search
revealed that N-ethyl-N-nitrosourea (ENU) may be a useful mutagen. In Drosophila,
21% of ENU-induced mutations are AT->GC transitions; 4% are GC>CG
transversions(1). Neither category of mutation has been found to be induced by EMS in
C. elegans.
  Our studies indicate that ENU may be an appropriate mutagen for C. elegans.
We determined ENU-induced lethality and sterility using N2 animals. Toxicity
increases dramatically with increasing ENU concentration. At ENU concentrations
below 30 mM however, viability remains greater than 80%. We also tested the ability of
mutagenized worms to produce viable eggs after exposure to increasing ENU
concentrations. A sharp increase in sterility is seen between 40 and 50 mM ENU.
Below 30 mM ENU however, sterility is less than 20%.
  To determine the frequency of mutagenesis induced by low concentrations of
ENU, we chose reversion of the rubberband allele, unc-93 ( e1500 ).Suppression of the
paralyzed, rubberband phenotype can be accomplished by null mutations in one of
several known genes, including sup-10 and unc-932 .Sequence data are available for
both genes(3,4) making the molecular determination of ENU-induced mutations
possible. In addition, this reversion strategy has been used to test the frequency of
reversion induced by other mutagens(2).
  We found the ENU-induced reversion frequency at concentrations below 30 mM is
no different from that of 50 mM EMS, 3 X 10+E-4 per haploid genome. Our values are
3-fold lower than the published e1500 reversion frequency using EMS(2), most likely
due to our demand that revertants exhibit fully wild type motility. A total of 155 ENU-
and 20 EMS-induced revertants of unc-93 ( e1500 )were isolated and saved.
Complementation tests of 95 strains show 48% to be putative null alleles of unc-93 ,41%
putative null alleles of sup-9 ,while 4 strains contain sup-10 mutations and 7% are
dominant suppressors, likely sup-11 alleles.
  We are finishing the complementation tests and are sequencing the putative
unc-93 null alleles to determine which base changes have been induced by ENU. We
are using PCR amplification of genomic unc-93 followed by cycle sequencing. A full
report will be available at the spring worm meeting.
 (1) Pastink, et al. (1989) Genetics 123:123-129.
 (2) Greenwald & Horvitz (1980) Genetics 96:147-164.
 (3) Cummins, Levin, Horvitz, Albertson & Anderson, unpublished.
 (4) Levin & Horvitz (1992) J. Cell Biol. 117:143-155.