Functional Analysis of Iα Promoter Regions of Multiple IgA Heavy Chain Genes

Abstract

The 13 nonallelic IgA H chain genes of rabbit are differentially expressed in vivo. They can be grouped into those expressed at high levels (Cα4, Cα5, Cα6, Cα9, Cα10, Cα12, and Cα13), those expressed at low levels (Cα1, Cα2, Cα7, and Cα11), and those that are not expressed (Cα3 and Cα8). We tested whether the differential in vivo expression is due to differential responses of the Iα promoters to TGF-β stimulation. We stimulated the rabbit B cell line 55D1 with TGF-β and, using single-cell RT-PCR, found that expression of germline (GL) transcripts of α3 and α8 could not be induced. By luciferase reporter gene assay and EMSA we found that the promoters of the unexpressed isotypes Cα3 and Cα8 are defective, thereby explaining the absence of IgA3 and IgA8 in vivo. When comparing the promoter activities of the other isotypes we found that the activities did not reflect the degree of in vivo expression. Instead, the promoters of the isotypes expressed at high or low levels promoted expression of the luciferase gene to a similar degree, except for the Iα4 promoter, which had much higher activity. Also the degree to which TGF-β induced GL expression of the various isotypes in 55D1 B cells did not reflect in vivo expression. However, most of the TGF-β-stimulated cells expressed GL mRNA of multiple isotypes; no isotype was expressed preferentially. These results suggest that the final switch to a single isotype is regulated in a step subsequent to GL transcription, rather than by induction of GL transcripts by the Iα promoter.

B Lymphocyte Development in Rabbit: Progenitor B Cells and Waning of B Lymphopoiesis

“…For RT-PCR amplification of VpreB and 5, cDNA was prepared as described previously (27) from groups of FACS-sorted proB and preB cells (MHC class II ϩ , surf-Ϫ ). Two rounds of 40 cycles of PCR amplification were performed on the cDNA using the following sets of nested primers: VpreB primers were 5Ј-TTGTGCTGTGTGGCTAGGAA-3Ј (sense outside primer), 5Ј-ATGTCCTGGATTCCACTTTG-3Ј (sense inside primer), 5Ј-GCACCGATCCTGGTGTCTCCA-3Ј (antisense outside primer), and 5Ј-CCTCAAGGCGCGCTGTCCTT-3Ј (antisense inside primer); 5 primers were 5Ј-TATCTCAAGTCTGGAAGGA-3Ј (sense outside primer), 5Ј-ATGAGGCCCCGGAGTGGCCT-3Ј (sense inside primer), 5Ј-GGGTGG GTGAGAGTTGTT-3Ј (antisense outside primer), and 5Ј-GAGCTGGG TCCCACTGCCAA-3Ј (antisense inside primer), and VDJ primers were 5Ј-CACTCACCATGGAGACT-3Ј (sense outside primer), 5Ј-GGACACG GCCACCTATTTCT-3Ј (sense inside primer), 5Ј-ACGGTCCGGCTGCT GATCTC-3Ј (antisense outside primer), and 5Ј-GTTGTTCTTGAAGCTC CAG-3Ј (antisense inside primer).…”

Phylogeny and Comparative Physiology of Mucosal Immunoglobulins

“…Molecular cloning of portions of the rabbit IGH locus revealed a linear arrangement of the C α genes downstream of single C γ and C ε genes, with the C αμ locus in the most 5′-proximal position (Spieker-Polet et al, 1993). Functional analysis of I α promoters upstream of the C α genes was consistent with a mechanism of cytokine-activated CSR for switching to the different IgA subclasses, and the finding of nonfunctional I α promoters upstream of the C α3 and C α8 genes explained the lack of expression of IgA3 and IgA8 (Spieker-Polet et al, 2002). Subsequent mapping of the IGH locus in rabbits revealed single copies of C μ , C γ , and C ε genes and the absence of a C μ gene (Lanning et al, 2003;Ros et al, 2004).…”

DNP-KLH Yields Changes in Leukocyte Populations and Immunoglobulin Isotype Use with Different Immunization Routes in Zebrafish

“…Whereas others have found differential expression of the two IgZ subclasses, including more IgZ2 up-regulation in spleen with LPS stimulation ( 25 ), we found parallel expression in our study. It remains to be seen whether the significant primary amino acid differences in the constant regions of these two antigen receptors translate into physiological differences, as constant regions of other vertebrate antigen receptors have been found with significant diversity due to polygeny [e.g., 13 rabbit IgA genes ( 44 )] or polymorphism [e.g., high allelic polymorphism at teleost α and β T cell receptor constant domain genes ( 45 , 46 )] without clear functional distinction. More work is needed to determine if IgZ2 functions on a distinct set of B cells, and if so where they are activated.…”