Worm Breeder's Gazette 14(4): 46 (October 1, 1996)

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.

Mapping the C. elegans Src homologue, src-1.

Jennifer Dignan Hogan, John Collins

Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824

   The oncogene src encodes a tyrosine kinase implicated in signal
transduction pathways that control cell growth and development.
When activated by mutation, src can induce neoplastic
transformation.  Despite intensive study for many years in a variety
of organisms (almost exclusively vertebrates), a good understanding
of src function still eludes us.

   We are trying to knock out src-1, a C. elegans homologue of
vertebrate c-src, in order to characterize the role of src-1 in C.
elegans development.  Such knowledge will contribute to the
understanding of src function in all organisms.  We have isolated a
src-1::Tc5 mutant using the PCR / sib-selection screen of Rushforth
and Anderson (Zhang et al, C. elegans Meetings Abstracts - 1995).  In
this mutant, Tc5 resides within the fifth intron of src-1.  The
presence of Tc5 does not confer a visible phenotype because it is
spliced out with the intron leading to normal mRNA production.
Efforts to isolate a deletion caused by Tc5 excision are in progress.

   src-1 resides on a small "orphan" contig that has not been placed on
the physical map.  The mutant we isolated provides the opportunity
to map the gene via a PCR based approach using the src-1::Tc5 allele
as a molecular marker.  Using a primer within Tc5 and a primer in
exon 6, we are able to specifically amplify a 1.2 kb product only
from individuals heterozygous or homozygous for src-1::Tc5.

   We crossed strain TW399 [src-1 (cj290::Tc5)] by strains containing
markers near the center of each linkage group: LGI dpy-5, LGII dpy-10,
LGIII unc-36, LGIV unc-22, and LGV dpy-11.  Individual F2 animals
homozygous for the marker allele were picked and screened by PCR for
src-1::Tc5.  In each case, approximately 75% were positive indicating that
src-1 was not linked.  We established that src-1 is not X-linked by
following its inheritance through two generations of XO males.

   We continued our mapping efforts by crossing strain TW399 by strains
containing markers located on either arm of each linkage group: I L bli-3,
I R unc-54, II L sqt-2, II R unc-52, III L dpy-1, III R bli-5, IV L dpy-9,
IV R dpy-4, V L unc-34, and V R unc-51.  In each case but one,
approximately 75% of individual F2 animals homozygous for the marker
allele were positive for src-1::Tc5.  The one exception was F2 animals
homozygous for bli-3, a marker on the left arm of linkage group one.  Only
5% (3/60) were positive for src-1::Tc5.  Control reactions with the same
DNA confirmed that the negative reactions were in fact due to the absence
of src-1::Tc5.  Hence we conclude that src-1 is located near bli-3 on the
left arm of linkage group I.