Identification and classification of genes that act antagonistically to let-60 Ras signaling in Caenorhabditis elegans vulval development

Abstract

The synthetic multivulva (synMuv) genes negatively regulate Ras-mediated vulval induction in the nematode Caenorhabditis elegans. The synMuv genes define three classes, A, B, and C, such that double mutants carrying mutations in genes of any two classes are multivulva. The class B synMuv genes include lin-35, a homolog of the retinoblastoma (Rb) tumor suppressor gene, as well as homologs of genes that function with Rb in transcriptional regulation. We screened for additional synMuv mutations using a strategy different from that of previous synMuv genetic screens. Some of the mutations we recovered affect new synMuv genes. We present criteria for assigning synMuv mutations into different genetic classes. We also describe the molecular characterization of the class B synMuv gene lin-65.

Figure 1.

Molecular cloning of lin-65. (A) The genetic map location of lin-65 on linkage group I (top) and the physical map interval between the C. elegans strain CB4856 polymorphisms Y71G12B.17 and Y71G12B.18 and including lin-65 (bottom). (B) lin-65 gene structure as derived from cDNA and genomic sequences. Shaded boxes indicate coding sequence and an open box indicates the 3′-untranslated region (lin-65 transcripts also contain a 5′-untranslated region that is too small to be viewed in this representation). Predicted translation initiation and termination codons and the poly(A) tail are shown. Arrows above indicate the positions of the lin-65(n3441), lin-65(n3541), and lin-65(n3543) mutations. The fourth exon of the cDNA yk1279h11 is smaller than that of the other five lin-65 cDNAs (the end of the yk1279h11 fourth exon is indicated by a dashed line). The use of an alternative splice donor may have created this shorter fourth exon. However, if the end of the yk1279h11 fourth exon were the site of alternative splicing, a CA and not the typical GT splice donor would have been used. In addition, the end of the yk1279h11-specific fourth exon and the beginning of the fifth exon encode multiple glutamine residues and are highly similar in DNA sequence (see Figure 2). The intervening sequence between two regions of highly similar sequence can be lost because of recombination in bacteria (Robinett et al. 1996). For these reasons we speculate that the apparent alternative splice site at the end of the fourth exon in yk1279h11 may be artifactual and have resulted from an error during the generation or maintenance of this cDNA clone. In support of this possibility: (1) We failed to amplify a shorter-than-wild-type yk1279h11 product in a high-stringency RT–PCR using oligonucleotide primers that flank the putative alternative splice junction, and (2) we failed to amplify any RT–PCR products when an oligonucleotide spanning the putative yk1279h11 alternative splice junction was used in a PCR.

Figure 2.

lin-65 cDNA sequence indicating differences among individual lin-65 cDNAs. SL1 and SL2 splice-leader sequences are italicized. The SL1 leader, as observed with one cDNA, is spliced two nucleotides downstream of the site at which the SL2 leader is spliced, as observed with two independently derived cDNAs. Intron positions are indicated by carats. The translation termination codon is underlined. Sites of alternative polyadenylation are indicated with solid arrowheads. The predicted LIN-65 protein is shown beneath. The SL2-spliced cDNAs are predicted to encode a 728-amino-acid protein. The SL1-spliced cDNA cannot encode the predicted initiator methionine of the 728-amino-acid protein; it may use the underlined methionine codon to initiate synthesis of a 691-amino-acid protein. The alternatively spliced cDNA yk1279h11 is predicted to encode a protein lacking amino acids 421–481 of the 728-amino-acid protein, although, as described in the Figure 1 legend, the alternative splicing of yk1279h11 is likely to be artifactual. The site at which the putative fourth exon in yk1279h11 ends is indicated with an open arrowhead. This end is juxtaposed to the beginning of the fifth exon to give a CAGCAACAA/CAACAAAAT junction sequence.