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Resources » Paper

Yingsong Hao et al. (2001) International C. elegans Meeting "USING YEAST-TWO HYBRID AND RNA-MEDIATED INTERFERENCE TO IDENTIFY PROTEINS ESSENTIAL FOR PIE-1 ACTIVITY AND LOCALIZATION IN EARLY EMBRYOS"

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  • Comments on Yingsong Hao et al. (2001) International C. elegans Meeting "USING YEAST-TWO HYBRID AND RNA-MEDIATED INTERFERENCE TO IDENTIFY PROTEINS ESSENTIAL FOR PIE-1 ACTIVITY AND LOCALIZATION IN EARLY EMBRYOS" (0)

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    Status:
    Publication type:
    Meeting_abstract
    WormBase ID:
    WBPaper00018611

    Yingsong Hao, & Geraldine Seydoux (2001). USING YEAST-TWO HYBRID AND RNA-MEDIATED INTERFERENCE TO IDENTIFY PROTEINS ESSENTIAL FOR PIE-1 ACTIVITY AND LOCALIZATION IN EARLY EMBRYOS presented in International C. elegans Meeting. Unpublished information; cite only with author permission.

    The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage in early C. elegans embryos. PIE-1 has at least two functions in the embryonic germ lineage: 1) inhibition of mRNA transcription, and 2) promotion of efficient NOS-2 expression, a maternal protein required for primordial germ cell development. Structure-function studies have indicated that these two functions require sequences in the C-terminal region of PIE-1 (Tenenhaus et al., 2001). The C-terminus of PIE-1 is also required for asymmetric segregation of PIE-1 in dividing germline blastomeres (Reese et al., 2000). We have used the C-terminus of PIE-1 as a bait in a yeast two hybrid screen in the hope of identifying proteins required for PIE-1 activity and/or localization. Screening of 900,000 transformants yielded 365 candidate interactors (thanks to Zheng Zhou and Bob Horvitz for their excellent library). To quickly identify functionally relevant candidates, we designed a simple protocol to test the in vivo function of each candidate by RNAi. We first isolate the "prey" plasmids from the positive yeast transformants by selection on an auxotrophic E. coli strain. The prey inserts are then PCR amplified and transferred by recombinational cloning into Lisa Timmons and Andy Fire’s feeding vector and transformed into E. coli strain HT115. Resulting transformants are used to "feed" three tester worm strains: 1) a MED-1:GFP line (thanks to Morris Maduro and Joel Rothman) to assay for defects in transcriptional repression, 2) a GFP: nos-2 3’UTR line to assay for defects in NOS-2 expression, and 3) a PIE-1:GFP line to assay for defects in PIE-1 localization. The screen is ongoing and progress will be reported at the meeting.


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