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The 6 kbp flp-1 upstream region was amplified from wild-type genomic DNA and inserted into a construct with the flp-1 coding region to yield LN/B3-SB. flp-1(yn2); lin-15(n765ts) animals were co-injected with LN/B3-SB (50 to 100 μg/ml) and JM24 (lin-15; 50 μg/ml). Transgenic animals (selected by rescue of the Lin-15 phenotype at 20°C) were scored for rescue of flp-1 phenotypes as described.
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We thank R. H. A. Plasterk for generously screening for and providing the flp-1::Tc1 insertional strain NL242 (pk41); L. Brundage, J. Mendel, P. Sternberg, J. Kaplan, L. Avery, and R. Korswagen for providing G protein-subunit mutants, advice on assays, and communication of unpublished results; H. R. Horvitz for providing mapping strains; A. Fire for providing microinjection vectors; J. Cherry and E. Widmaier for patiently providing help with the statistical analyses; S. Craven, H. Field, G. Jang, Y. Honzano, and C. Milliken for graciously providing technical assistance; T. Gilmore for comments on the manuscript; and members of the lab for helpful discussions. Supported in part by grants from NSF (IBN9320262) and NIH (AG00708) to C.L.
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