The Role of sV23 Sequences in Drosophila Eggshell Assembly

Phillip Romei
Marquette University
Milwaukee, WI
Mentor: Dr. Gail Waring

Our interests primarily lay in the means through which cells are able to construct extracellular structures. We are using the Drosophila eggshell to study the assembly of an extracellular structure in vivo. To understand how sV23 functions, we have altered specific sections of its coding region in order to observe differences in sV23’s ability to integrate into the eggshell. Specifically, we are studying the effect that the N-terminus has on sV23’s ability to integrate. 

A network of disulfide bonds has been suggested as a means for sV23 to integrate into the eggshell. An sV23 mutant protein with compromised disulfide bonding capability was able to integrate in early stages of eggshell development and then fell out of the protein network at later stages. It has been shown that sV23 is processed extracellularly; the N-terminus is cleaved after sV23 integrates into the eggshell. The timing of sV23’s loss of integration coincides with the cleavage of the N-terminus. This leads to our hypothesis that in the absence of normal disulfide bonding in late stage eggshells the release of sV23 is due to cleavage of the N-terminus. 

In order to test this hypothesis, we have created an sV23 gene in which the N-terminus is deleted and critical cysteine residues for disulfide bonding have been mutated. There will be no N-terminus to facilitate early integration, so we expect this sV23 double mutant will be unable to integrate at early and late stages of eggshell formation. 

To construct the mutant transgene, we used a subclone with an N-terminal deletion as a template for PCR mutagenesis. Through this process, two of the three critical cysteines were changed to serines. The PCR product with the N-terminal deletion and reduced disulfide bonding ability underwent a series of subclonings in order to include proper flanking sequences for expression in the flies as well as to insert the double mutant into the P-element transfer vector pCaSpeR 4, a vector able to insert the mutant into the fly genome. The next stage will be injection of the mutant into fly embryos, and through a series of genetic crosses, introduce the mutant into a sV23 null fly. 
 
 
 

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