Figure 4.

Smaller deletions within the 5′ half of the Ig-μ VDJ exon did not affect its NMD-promoting activity. (A) Alignment of VDJ exon sequences of several different human and mouse Ig-μ and TCR-β genes. ‘Miniμ’ is from the mouse Ig-μ of the hybridoma cell line Sp6 (23). Accession numbers M13832 and M85255.1 are rearranged VDJ regions of mouse and human Ig-μ, respectively. Vβ2 (accession no. X04331), Vβ5.1 (19) and Vβ8.1 (19) are mouse TCR-β sequences. The underlined sequence corresponding to amino acid positions 52–70 in our Ig-μ minigene contains conserved motifs and is deleted in hyb9 and hyb10. (B) Total cellular RNA of HeLa cells transiently transfected with the indicated Ig-μ/Sxl hybrid genes were analyzed 48 h post transfection by real-time RT–PCR. Relative Ig-μ mRNA levels were normalized to neomycin mRNA encoded on the transfected plasmid. Average values of three real-time PCR runs of a typical experiment are shown. Error bars indicate standard deviations. (C) Predicted stem–loop structure located between amino acid positions 19 and 30 of Ig-μ. (D) The 33 bp comprising the predicted stem–loop structure depicted in (C) were deleted from hyb4 and hyb6, giving hyb11 and hyb12, respectively. The 63 bp between the predicted stem–loop and the underlined motif in (A) were deleted from hyb4 and hyb6, giving hyb13 and hyb14, respectively. The 18 bp comprising amino acid positions 71–76 were deleted from hyb4, giving hyb18. Transfection and real-time RT–PCR were as described in (B).