Worm Breeder's Gazette 13(4): 88 (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.

Analysis of the Worm Laminin B Gene.

Gautam Kao1, Edward Hedgecock2, Bill Wadsworth1

1 Dept. of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
2 Dept. of Biology, Johns Hopkins University, Baltimore, MD 21218.

  The basement membrane is a sheath of extracellular material that provides a
connection between cells and the external environment. The heterotrimeric
glycoprotein, laminin forms part of the scaffold structure of the basement membrane
and interacts with cells via receptors such as integrins. The analysis of the function of
laminin will provide some insights into the crucial roles the extracellular matrix has
in controlling neuronal pathfinding, organogenesis, muscle positioning, tissue
separation and the distribution of developmental signals.
  The three subunits of laminin: alpha, beta, gamma (previously known as A, B1
and B2 respectively) have been cloned from a number of organisms. There is a high
degree of sequence conservation for each subunit across the phylogenetic spectrum.
The 337 subunit consists of two globular domains ( the N-terminal domain VI and
domain IV) two stretches of EGF like repeats (domain III and domain IV) and two
a-helical regions (domains II and I).
  We have previously described the isolation of a DNA fragment that shows
homology with the domain VI region of vertebrate and insect laminin 337 genes. This
DNA was physically mapped to cosmid C24D10 on chromosome IV near dpy-13 (1).
  A homozygous viable mutation in the gene lam-1 ( rh219 )was isolated in a screen
for new mutants linked to dpy-13 . rh219 mutants share phenotypic characteristics
with mutants of epi-1 the laminin a chain gene (2). Furthermore, genetic interactions
have been observed between epi-1 and lam-1 mutants. rh219 is a
reduction-of-function mutation since rh219 /Dfanimals die as larvae. These
molecular and genetic observations suggest that there is a laminin, 337 chain gene on
C24D l0 and that it corresponds to lam-1 .
  We tested the ability C24D10 to rescue rh219 defects by crossing a cosmid bearing
array into mutant animals. Array containing rh219 animals are fully rescued for egg
laying ability and locomotion. Sequencing of the domain VI region of the gene shows
greater than 50% sequence identity with the fruitfly and mouse genes. This sequence
includes a well conserved transition from domain VI to domain V.
  In order to better understand the phenotypic consequences of the rh219 mutation,
we are examining the mutant by electron microscopy. Preliminary results revealed
the presence of several defects that are reminiscent of the phenotype of epi-1 mutants
and are consistent with the notion that the basement membrane is defective. These
defects include the following: a) Whorls of matrix material are seen delaminated from
the basement membrane. b) Muscle cells are seen in abnormal positions. c) The
excretory canals are abnormal, either having a completely collapsed lumen, or failing
to grow out properly. d) There appears to be abnormally high levels of yolk in the
intestine perhaps as a result of decreased transport of yolk to the gonad. e) The sheath
cells are abnormal in structure. In addition the proximal region of the gonad that
normally contains meiotic germ cells, instead contains immature mitotic cells.
Thanks to David Hall for his help in interpreting the EM pictures.
 (l) L. Gong, W. Wadsworth, and E. Hedgecock ( l991 ).8th International C. elegans. Meeting, pg
 (2) K. Joh, and E. Hedgecock (1993). 9th International C. elegans. Meeting, pg 222