Supplemental Material

This article contains the following supporting material:

• Supplement 1 - Consensus translation of rendezvin orthologs. (A) Schematic diagram of S. purpuratus RDZ gene based on sequence alignments between the cDNA and genome assemblies and traces (see Supplement 4). Note two exons duplications of exons 31 and 32 found between exons 33 and 34. Exons represented in the cDNA are shown in grey boxes with spacers for presumptive introns (incomplete). Splice donor and acceptors are assumed to be 3’-A to 5’-G (box), unless otherwise indicated. The S. purpuratus RDZ open reading frame is mapped below, including the transmembrane region and signal sequence (shaded box), predicted cleavage site that releases the amino-terminal 60-kDa fragment, based on amino-terminal microsequencing (arrow), CUBs (dashed boxes), and the PYQ repeat motif (solid box). (B) Alignment of the open reading frame of RDZ orthologs from S. purpuratus (top line) and L. variegatus (bottom line). Dots indicate amino acid identity while dashes indicated gaps. The transmembrane region and signal sequence (shaded box), predicted cleavage site (arrow) for S. purpuratus that generates separates RDZ⁶⁰ from RDZ⁹⁰ and RDZ¹²⁰ isoforms, based on amino-terminus microsequencing (underlined), and CUBs (dashed boxes) are indicated. One hundred residues are shown per line. The GenBank accession numbers for rdz in S. purpuratus and L. variegates are EF071483 and EF071484, respectively.' (rdz, S. purpuratus, and L. variegates are all italicized).
• Supplement 2 - Identification of rendezvin splice-variant transcripts by reverse transcriptase PCR amplification. (A) Schematic diagram identifying position of nested primer sets designed to flank predicted splice donor and acceptor sites within rendezvin. Motifs and symbols are as in FIG 1 and SUPP 1. Smaller arrow represents the first, outer nested primer pair used; larger arrow represents the second, inner primer pair used. (B) Ethidium bromide-stained 2% agarose gels with paired, nested amplification reactions from 1 µg of either S. purpuratus (top) or L. variegatus (bottom) total ovary RNA. Asterisks indicate amplifications that are smaller than expected from the primer sets used, indicative of RNA splicing. Nucleotide sequences of smaller bands are 100% identical to select regions of respective cDNA contigs. Size markers are indicated in flanking lanes.
• Supplement 3 - Table of primers. (A) Primers used for detection of splice sites. (B) Primers used for multiplex PCR.
• Supplement 4 - Insect cell expression of recombinants. (A) Expression vector map constructed and utilized for the secreted expression of various fertilization envelope constructs from embryonic Drosophila Schneider S2 cells via a copper-inducible promoter. This vector was created by first mutating one of two PvuII sites (2470) of pCoHygro. Primers containing a PvuII overhang were then used to amplify the pMT promoter, MCS, V5-epitope, 6x-His epitope, and poly-A tail from the parent pMT/BiP/V5 vectors. This fragment was subcloned into the retained PvuII site (308) in the mutated pCoHygro vector. Clones containing the pMT promoter in an orientation opposite the hygromycin B phosphotransferase promoter were selected for further subcloning. This new parent vector was designated pcoHyg/MT. (B) Anti-V5 immunoblots of antisera pull-downs using 5 µg V5-reactive -equivalent masses of recombinants as target (input). Affinity-purified anti-proteoliaisin (PLN) and anti-rendezvin (RDZ) IgGs used for initial pull-downs from nickel-purified proteins isolated from dialyzed culture media. GFP-V5 included as a control for anti-V5 immunoblotting.