Immunoglobulin Heavy Chain Exclusion in the Shark (original) (raw)
Figure 5
Single-Cell PCR of Nurse Shark Lymphocytes
Diagram: A first PCR round was performed with degenerate primers targeting the leader intron (“GR” series) and JH (JH5) sequences of Group 1–5 genes. Aliquots from the first round were amplified in a second round of PCR employing nested degenerate primers in VH (“VG” series) and in JH (JH6) that also collectively targeted the same genes. To identify the rearranged genes amplified by the VG/JH6 primers, separate second PCR rounds were done with JH6 in combination with five nested primers (Fam1, Int, GR3N2, Fam4, and Fam5) targeting leader intron sequence downstream of GR and specific to each of the five Groups. Top and middle: nurse shark thymocytes depleted of surface L chain-positive cells from shark-PI were picked by hand. After every second thymocyte, a RBC was picked as a check for the purity of isolation and processing. Top: A first PCR round was performed with the GR/JH5 primers targeting all Groups 1–5 genes; the nested round of PCR with VG/JH6 is shown in this panel. The expected band sizes are: 1.6 kb (GL), 1.2 kb (one rearrangement, 1R), 0.8 kb (2R), 0.4 kb (3R). Middle: one of the Group-specific nested reactions (primer pair Int/JH6), that targeting Group 2 genes, is shown in the middle. The DNA fragments of nested Group-specific PCR are expected to be overall about 52 bp longer than those described for the nested VG/JH6 reactions. Rearranged Group 2 products are identified in Figure S4. Bottom: surface L chain-positive peripheral blood leukocytes (i.e., B cells) from shark-GR were picked alternating with RBC for purity controls. PCR reactants and conditions are identical to that described in top panel. Each sIg+ PBL is flanked by a RBC. The names of the cells are shown per lane and correspond to those in Table 3.