Molecular Biology

The myo-3p::FLAG::pab-1 contruct (pPRSK9) was assembled by ligating the myo3 promoter from a 2498 bp HindIII/Asp718 fragment from pPD95.86 (a gift from A. Fire1), a FLAG epitope encoded by oligonucleotides, and a PCR fragment containing genomic pab-1 (accession number U24123.1) into pBluescript II SK (Stratagene) cut with SacI/HindIII (see supplemental web site for more details).  Previous work has shown that amino-terminal epitopes do not to interfere with the function of PABP2,3, and that the PABP-bound RNA best represents the mRNA made in a cell of several candidate mRNA-binding proteins 4.

A transgenic strain expressing myo-3p::FLAG::pab-1 was made by microinjecting5 wild-type animals with myo-3p::flag::pab-1 (25 ng/ul) and sur-5::gfp (pTG96; 100 ng/ul; a gift from Min Han)6, generating an extrachromosomal array, and then randomly integrating the extrachromosomal array into the genome using g-irradiation5.  Transgenic worms containing the integrated array (gaIs146) were back-crossed to wild-type twice, resulting in strain SD1075.

 

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2.         de Melo Neto, O. P., Standart, N. & Martins de Sa, C. Autoregulation of poly(A)-binding protein synthesis in vitro. Nucleic Acids Res 23, 2198-205 (1995).

3.         Craig, A. W., Haghighat, A., Yu, A. T. & Sonenberg, N. Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392, 520-3 (1998).

4.         Tenenbaum, S. A., Carson, C. C., Lager, P. J. & Keene, J. D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci U S A 97, 14085-90 (2000).

5.         Mello, C. & Fire, A. DNA transformation. Methods Cell Biol 48, 451-82 (1995).

6.         Yochem, J., Gu, T. & Han, M. A new marker for mosaic analysis in Caenorhabditis elegans indicates a fusion between hyp6 and hyp7, two major components of the hypodermis. Genetics 149, 1323-34 (1998).