| Identification
of Drosophila Mutants with Altered Eggshell Protein Accumulation
Erin
Greene
Marquette
University
Summer
Mentor: Dr. Gail Waring
The
Drosophila
eggshell consists of two major layers, the vitelline membrane and the endochorion,
which are formed through the assembly of extracellular proteins that covalently
bond with one another to form a cross-linked eggshell. To better understand
this process, X-linked mutants with altered eggshell morphology were studied.
With these mutants, two questions were asked: (1) Is the cross-linking
of eggshell proteins altered? (2) Is eggshell protein accumulation altered?
Egg chambers, from early and late oogenesis, and laid eggs (when the protein-insoluble,
cross-linked eggshell has been formed) were collected and incubated in
2% SDS and ß-Mercaptoethanol and boiled. Proteins solubilized under
these conditions were separated by size on a SDS gel. Western Blot analysis
was used to look for alterations in the formation of non-reducible cross-links
in the laid eggs and eggshell protein accumulation in the egg chambers.
The antibodies used in the analysis detected the sv23 and dec-1 vitelline
membrane proteins and the s36 and s18 endochorion proteins.
One
mutant, DH28, demonstrated altered cross-linking in the laid egg through
the detection of the s36 eggshell protein. EK107, and its allele DJ192,
showed the absence of the s36 protein and a dramatic reduction in the accumulation
of the s18 protein during oogenesis. DC238, a fourth mutant, showed undetectable
accumulation of s36 and s18, and an apparent reduction in s60, a dec-1
derivative normally found in both the vitelline membrane and the endochorion
layers.
Complementation
analysis showed that EK107 and DJ192 are allelic to an existing mutant,
cor36; cor36 mutants have a mutation in the s36 structural gene. The reduction
in s18 in the EK107 and DJ192 mutants suggests a model in which these proteins
form a complex such that the absence of one may compromise the stability
of the other. The DC238 data suggests that the accumulation of several
endochorion proteins is altered in this mutant. Further studies will map
the DC238 mutation on the X chromosome and check for the presence of other
endochorion proteins. |
