Figure 7.

Derivation of the joints in NS4 germline-rearranged genes. The V and J flanks of split genes are shown without RSS. The possible processing events producing the joints of germline-joined R7, R4, RE18, R6, R19, and R18 are shown aligned with the most similar nonjoined NS4 coding ends isolated in these studies. Assuming that the split ancestor of R7 resembled S10, the R7 joint could have been produced by (a) cleavage at the RSS and relegation of blunt ends, or (b) cleavage at the RSS, formation of hairpin at V and J ends, single-stranded nick occurring two or more nucleotides from end, processing resulting on P region (underlined), and deletion of two bases from the J flank. Pathway R7 (a) does not preclude hairpin formation, but there is no processing evidence for it. Under cDNA, germline split S10 is compared with cDNA sequences obtained by RT-PCR from shark Y that share similar CDR3. The analysis of the cDNA joints suggest P region in both sequences. The sequences in the cDNA joints that do not appear to belong to either V or J may be N region. R4 is similar to R7. RE18 joint could have resulted from deletion in the V coding end (a), or J coding end (b), or both (c). The flanks of nonjoined ancestor of R6, R19, and R18 could have resembled S32, in which case deletion only from J end occurred during recombination (a). In all other possibilities, nucleotides were removed from either end. Examples of coding flanks from NS4 split genes are included for comparison; the RSS resemble those from other vertebrates. Clade I refers to the cluster of genes including R4, R7, and R18, clade II to that including R6, R19, and RE18 (see Fig. 6); clade III genes are characterized by divergent FR3 and CDR3 of 24 bp; complete sequences are not shown. Asterisk indicates split gene is pseudogene. S32 is shown in Fig. 3 and Fig. 4; S15 has TGA in FR2; S5 sequence is not shown but carries TGA in FR3, a GT–AT splice signal combination in leader intron, and noncanonical RSS heptamer sequences.