Submission Date

5-8-2022

Document Type

Paper- Restricted to Campus Access

Department

Biology

Adviser

Rebecca Lyczak

Committee Member

Dale Cameron

Committee Member

Michelle Vande Berg

Department Chair

Rebecca Lyczak

Project Description

Caenorhabditis elegans are microscopic worms that are a great model organism for examining genes and proteins involved in the reproductive system because of their fecundity, easy manipulation, and visible gonad. The Lyczak lab has studied two versions of a puromycin aminopeptidase known as PAM-1. We have identified a missense mutation pam-1(or347), a partial loss-of-function of pam-1 and a nonsense mutation, pam-1(or403), a complete loss of function. This PAM-1 protein is 36 – 37% similar to the humans Npepps protein. pam-1 mutants lay fewer viable embryos than wild-type worms because they have trouble completing meiosis. However, we have also found another gene that rescues pam-1 mutants' low hatch rate, known as wee-1.3. WEE-1.3 is involved in oocyte maturation. It inhibits the maturation-promoting factor (MPF) in oocytes to keep the oocytes immature prior to fertilization. A suppressing mutation in wee-1.3 raised the hatch rate of pam-1 embryos. Due to this, we believe PAM-1 has a direct or indirect effect on MPF. MPF is made up of cyclin B and cyclin-dependent kinase 1(CDK-1). To examine this interaction, I have conducted localization studies of CDK-1 in wild type, pam-1(or347), and pam-1(or403) worms using the confocal. I have also performed interaction studies of PAM-1 with MPF components in oocytes by using CDK-1 and WEE-1.3 RNAi with different variations of the strains. With these in hand, we have further characterized PAM-1 and MPF’s interaction. RNAi experiments have shown that pam-1(or347) has protection from the knockout of CDK-1. The localization studies have shown a significant difference between wild type’s and pam-1 mutants CDK-1 intensity, confirming that more CDK-1 is found at higher levels in the pam-1(or347) worms. To examine if CDK-1’s intensity changes based upon WEE–1.3 activation state, WEE-1.3 RNAi experiments were done upon worms with CDK-1 GFP. However, there was not much of significant difference found. Overall, pam-1(or347) mutants has shown that it has protection from WEE-1.3 knockout and protection from the loss of CDK-1 as well as a role in regulating CDK-1 levels. However, WEE-1.3 does not localize CDK-1. While the data supports a role for PAM-1 in regulating the MPF, future studies will be needed to determine how missense and nonsense mutations differentially affect the results.

Comments

This research was supported by an NIH grant.

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