Fig. 2. Molecular identification of the let-99 gene. (A) Genetic and physical maps of the region containing let-99 on chromosome IV. (B) Restriction map and rescue data for cosmid C13H6 and fragments. (C) Restriction map and transcribed regions present in the smallest rescuing fragment. Northern blot analysis identified a 2.4 kb transcript that was expressed at high levels in the germline (let-99, open boxes) and two other transcripts (arrows), the positions of which were determined by the Genome Consortium (Genome Consortium, 1998). Injection of RNA corresponding to the 2.4 kb transcript into wild type resulted in a phenocopy of the let-99 mutant phenotype in progeny embryos. (D) The predicted LET-99 and LRG-1 proteins, showing the positions of let-99 mutations and the corresponding amino acids change. The DEP domain is boxed; shaded box indicates a block of amino acids (153-212) present in LET-99 but not LRG-1; LRG-1 is 86% identical to the N-terminal region of LET-99. The Genbank Accession Number for let-99 (K08E7.3) is Z77666 and for lrg-1(F55H2.4) is NP_499092. (E) Western blot of wild-type (N2) and let-99(or81) mutant embryos probed with affinity-purified anti-LET-99 antibodies and reprobed with anti-tubulin as a loading control.