Worm Breeder's Gazette 15(2): 42 (February 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.

A mutation in alpha-spectrin affects elongation of the embryo and myofilament organization.

Ken Norman, Don Moerman

Department of Zoology, University of British Columbia, Vancouver, Canada

     An L1 lethal mutant ra409 was isolated from a screen designed to
isolate embryonic lethals with defects in muscle development. ra409
animals have a unique phenotype in that mutant animals do not complete
morphogenesis and do not elongate entirely.  Although all tissues types
examined have differentiated in ra409 homozygotes, many do not appear
wild type.  In particular, the body wall muscle and the overlying
basement membrane are 2-fold wider than in wild type animals.  In
addition, muscle filaments in ra409 animals are oriented at a steeper
angle to the longitudinal axis of the embryo than what is seen in wild
type.  This may be a consequence of either the lack of complete
elongation or the expansion of muscle cells or basement membrane.  We
have also observed that the pharynx is much shorter in these mutant
animals and labors during contraction.  The actin filament organization
and the antigen recognized by MH4 (intermediate filament like antigen)
in the hypodermis both appear normal, with the exception that the MH4
staining pattern is two fold wider corresponding to the wider muscle
quadrants in this mutant.
     Recent characterization and cloning of sma-1 (McKeown, C., Praitis,
V. And Austin, J. 1997 Worm Meeting) provided a clue to aid in the
identification of the ra409 gene.  sma-1 is a viable mutant that has
defects during morphogenesis similar to ra409 mutants.  At hatching
sma-1 mutants are half the length of wild type, a phenotype that is
identical to ra409 homozygotes.  Transformation rescue of sma-1 animals
revealed that sma-1 encodes a beta-spectrin (McKeown, C., Praitis, V.
And Austin, J. 1997 Worm Meeting).  We have genetically mapped ra409 to
a region on the X chromosome containing an alpha-spectrin (C. elegans
genome sequencing consortium). Spectrins are a major components of the
membrane cytoskeleton and are important for maintaining cell shape and
polarity. Since beta-spectrin forms heterodimers with alpha-spectrin, we
felt this alpha-spectrin might be a likely candidate gene for ra409.
     To determine if the ra409 lesion is in the alpha-spectrin gene we
initially took an antisense RNA approach using a 2.8 kb partial cDNA
obtained from Y. Kohara.  Antisense as well as RNA injection into the
gonad of wild type worms has been shown to phenocopy the mutant
phenotype of several genes in the progeny of injected worms (Driver,
S.E., Ali, M., Mello, C.C. 1997 Worm Meeting).  The 2.8 kb cDNA to the
3! end of the alpha spectrin gene was used to generate RNA and this RNA
was injected into wild type animals and the progeny were screened for
the presence of the ra409 phenotype.  Several L1 larvae from the
injected wild type hermaphrodite resembled ra409 homozygotes, but unlike
ra409 animals these were viable.  Next, in an attempt to rescue ra409
animals we transformed animals with a purified cosmid clone, M01F12,
that contains the entire alpha-spectrin gene (supplied from the Sanger
Center).  We obtained transformation rescue of the ra409 mutants thus
demonstrating that ra409 is an allele of the alpha-spectrin gene.
     This alpha-spectrin, asp-1 (alpha spectrin), is 58% identical to
the vertebrate non-erythroid alpha-spectrin and 63% identical to the
Drosophila alpha-spectrin. The non-erythroid alpha-spectrins and ASP-1
contain an SH3 domain, a calmodulin binding domain and a calcium binding
region. Now that the gene is identified the goal will be to determine
how it functions during development.  One area we will investigate is
what role spectrin plays in the patterning of the muscle