Worm Breeder's Gazette 13(4): 94 (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.

Correlating the Physical and Genetic Maps on Chromosome III (Left) - A First Step.

Diana Collins, Helen I. Stewart, David L. Baillie

Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC

Figure 1

As reported in the June 1994 issue of the WBG, the sequencing
consortium is rapidly completing the sequence of chromosome
III. Concurrently, our lab has been generating a large number of new
mutations in the region covered by the free duplication sDp3
(approximately 75% of chromosome III, left) (1). These mutations
include EMS induced point mutations in essential genes and small,
UV/Gamma induced deletions (see H. Stewart et al, this issue). In
collaboration with the C. elegans genome sequencing consortium,
specifically the St. Louis lab, our lab has begun the task of
correlating the genetic and physical maps on the left arm of
chromosome III. As a first step toward this goal, I have been micro
injecting cosmid DNA received from St. Louis (my thanks to
P. Latreille) into N2 hermaphrodites to create stable transgenic
strains carrying specific sequenced cosmids. It is our intention to
use these transgenic strains to cross these cosmids into various
mutant strains and observe if the DNA is able to rescue the mutant
phenotype. In this manner we will be able to place a large number of
genetic markers on the physical map. Germline transformation is
performed as per Mello et al. (1991), with cosmid DNA normally being
injected at an approximate concentration of 20 ng/265l. Prior to
injection, cosmid DNA is mixed with the plasmid pCes 1943 to form an
injection mixture having a total DNA concentration of approximately
100 ng/265l. pCes 1943 is derived from the plasmid pRF4 (2). It
contains the dominant rol-6 ( su1006 )allele (3), and has been
modified to contain a kanR cassette. Transformant strains which
stabily give rol-6 progeny are PCR tested to confirm the presence of
cosmid DNA. It has been my observation that approximately one stable
rol-6 transformant in four does not contain cosmid DNA. So far I have
produced transgenic strains from 22 cosmids: C06E8 , C06G4 , C14B9
C18H2 , C29E4 , C30C5 , D2007 , F08F8 , F11H8 , F31E3 , F37C12 , F44B9
, K06H7 , K07D8 , R151 , R13A5 , T04A6 , T20B12 , T20H4 , T21D11 ,
ZK652 ,and ZK688 (see figure). Although there are a few cosmids left
of mec-14 which I have not been able to successfully inject (due to
problems with DNA availability or possible dosage effects), these
transgenic strains represent a reasonably continuous stretch of DNA
approximately 654 000 bp long. Preliminary crosses between lethal
bearing strains and cosmid-containing transformants have yet to reveal
a cosmid rescue. However, as our newly generated mutations are further
mapped with respect to previously generated physical markers, the task
of choosing cosmids which are likely to rescue particular mutants will
become easier. [see figure] In the meantime we are making these
transformant strains available to the C. elegans community. If you
would like us to send you any of these strains please contact me at:
dcollins@darwin.mbb.sfu.ca This research is being funded by a grant
from the Canadian Genome and Associated Technologies (CGAT)

 (1) Collins, D., H. Stewart and D. L. Baillie (1994). WBG Vol. 13 No. 2: 66

Figure 1