Figure 3. One step RT-PCR amplification of immunoglobulin gene transcripts from B cell cloning cultures.

Legend: (A) Samples containing B cells from cell cloning cultures, 0.66 cells/well, in the presence of S17 stromal cells (3000 cells/well), that scored positive for the secretion of IgM were subjected to one step RT-PCR for the amplification of V_H transcripts using a promiscuous V_H primer. The final RT-PCR products (~ 400 bp, lanes c-m) were identified by electrophoresis and are indicated in the figure with an arrow. In the gel shown, transcripts from 7 out of 11 IgM+ cultures were amplified (64 % efficiency): lane “a”, DNA ladder; lane “b”, culture with S17 alone. (B) mRNA from samples containing B cells from cloning cultures that scored positive for the recognition of the 190 kD antigen in mouse brain were subjected to one step RT-PCR for the amplification of transcripts of immunoglobulin V genes using a promiscuous primer as described. cDNA sequences: the V_H family and the sequence of the heavy chain CDR3 (CDR-H3) are shown with the respective V_L and J_L genes for each clonotype. Clone ID, clone identifier. V_H family, name according to the IMGT classification. 3′V_H region, the most 3′ nucleotides of the V_H (not including Cys = TGT). P-N-P, palindromic (P) and N region sequence. D region, D_H gene sequence. 5′J-Region, the most 5′ nucleotides of the J_H (not including Trp = TGG); underlined sequences can be from either of two germline genes. Dots represent nucleotides lost from the germline D_H and J_H gene segments. J_H gene indicates the number of the J_H element. D_H gene, the D_H element used; RF, the D_H reading frame used. Light Chain, the Vκ Family and the Jκ usage of the light chain paired with the respective heavy chain is indicated. Predicted amino acid sequences derived from the cDNA sequences: Base, the predicted amino acid sequence of the CDR-H3 base. Loop, the predicted amino acid sequence of the CDR-H3 loop. Hydrophobicity, the average, normalized Kyte-Doolittle hydrophobicity of the CDR-H3 loop. CDR-L3, the predicted amino acid sequence of the light chain CDR-3. (C) A parallel set of cultures were done using rat thymocytes as feeder cells. Samples containing B cells from cell cloning cultures, 0.66 cells/well, that had scored positive for the secretion of IgM were submitted to one step RT-PCR as above. No RT-PCR amplification product could be detected (results representative of 3 different experiments).