Worm Breeder's Gazette 15(3): 26 (June 1, 1998)

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.

Cloning and Characterization of the Cytokinesis mutant stu-4.

Ananth Badrinath, John White

University of Wisconsin, 425 Henry Mall Rm. 5350, Madison, WI 53706

        We are characterizing the temperature-sensitive mutant stu-4
(e2406)  (originally isolated by David Livingstone) which maps to -4.42
on the left arm of LGI.  When gravid adults are shifted to the
non-permissive temperature 100% of the embryos die due to maternal
effect lethality.  We examined the cause of this lethality using 4-D
time-lapse (Nomarski) microscopy. The polar bodies fail to be extruded
resulting in several ooctye !pronuclei! migrating toward the posteriorly
located sperm pronucleus where they  generally meet at the center of the
embryo.  Spindle formation, and reformation of the nuclei following
chromosome segregation appears to be normal, but at no time point is
there any sign of furrowing at the cortex.  This results in a
multi-nucleated single celled embryo.  
        When L2 or L3 larval stage e2406 animals are shifted to the non
permissive temperature 100% of them become sterile and exhibit an
uncoordinated phenotype to a lesser extent.  These defects are
presumably due to failures in cell divisions in the post-embryonic
lineages giving rise to the gonad and ventral nerve cord respectively. 
At an intermediate temperature of 20 degrees only 7 per cent of the
homozygotes shifted as L2 or L3 larvae were fertile.  We were able to
rescue the post-embryonic defects with a small group of cosmids
containing K03E5, W05F2 and W07C11.  Up to 60% of the rolling
homozygotes shifted to 20 degrees as L2 or L3 larvae becoming fertile
        Of these three cosmids only K03E5 had been sequenced by the
genome consortium, and contains 5 predicted genes.  One of them
K03E5.cand3 codes for a protein with strong homology to calponins which
are actin-binding proteins that regulate acto-myosin contraction in a
calcium-dependent manner in smooth muscles.  When this gene!s function
was perturbed in N2 animals using double-stranded RNA mediated
interference we obtained early eggs that showed a strikingly similar
phenotype to e2406 embryos at the non-permissive temperature.  It is
possible that this gene may be a non-muscle isoform that plays an
important role in either the specification or regulation of the
acto-myosin contractile apparatus involved in cytokinesis.
        To confirm that this gene corresponds to stu-4 we would like to
sequence e2406, and further alleles which we are currently in the
process of recovering.  In a non-complementation screen singled F1 males
from EMS-mutagenized him-8 hermaphrodites were crossed to stu-4(e2406)
dyp-5(e61)/szT1 hermaphrodites and we looked for the presence of non dpy
sterile progeny.  From 740 haploid genomes screened we have obtained one
additional allele which we are currently in the process of backcrossing.