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Restricted immunoglobulin variable region (Ig V) gene expression accompanies secondary rearrangements of light chain Ig V genes in mouse plasmacytomas.

Diaw L, Siwarski D, Coleman A, Kim J, Jones GM, Dighiero G, Huppi K - J. Exp. Med. (1999)

Bottom Line: The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl.Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody.These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

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JH rearrangements in SIPC tumors. Genomic DNAs from SIPC tumors and BALB/c liver were digested with EcoR1 and hybridized with the JH probe: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. An arrow (left) depicts the germline (nonrearranged) JH fragment, whereas the 3.5-kb EcoR1 rearrangement seen in several tumors is also highlighted.
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Figure 3: JH rearrangements in SIPC tumors. Genomic DNAs from SIPC tumors and BALB/c liver were digested with EcoR1 and hybridized with the JH probe: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. An arrow (left) depicts the germline (nonrearranged) JH fragment, whereas the 3.5-kb EcoR1 rearrangement seen in several tumors is also highlighted.

Mentions: Initially, Ig heavy chain–specific rearrangements were identified in each tumor by Southern blot analysis using a JH probe (Fig. 3). Heavy chain-specific rearrangements were found in all tumors, including apparent rearrangements of both alleles in SIPC3385, SIPC3282, and SIPC3301. We also found shared rearrangements with both BamH1 (not shown) and EcoR1 digestions in SIPC3308, SIPC3336, and SIPC3282, suggesting the same VH gene may be expressed in these tumors. We cloned and sequenced the 3.5-kb EcoR1 fragment from SIPC3336, and established that the rearrangement consists of a member of the VH-J558 family (H13-3; reference 12) rearranged to DHSP2-9 and JH3. To more specifically determine VH gene usage in the SIPC tumors, we examined the expressed sequences by reverse transcription (RT)-PCR (Fig. 4). Indeed, three of the SIPC tumors (SIPC3282, SIPC3336, and SIPC3308) that share the 3.5-kb EcoR1 rearrangement all use the same H13-3 gene from the VH-J558 family. We verified that this sequence was germline (nonmutated) by sequencing eight clones derived from the PCR product of BALB/c genomic DNA which was amplified by primer pairs specific for H13-3 (see Table ). Interestingly, these same three tumors have also rearranged to JH3, and use the same DH region (DHSP2-9) encoding the amino acid residues Trp-Phe. Although rearranged to JH3 and DHSP2-9 as well, SIPC3301 has a longer nucleotide addition sequence, and is in fact encoded by another member (26.4.1-α; reference 13) of the VH-J558 family. The RT-PCR product of SIPC3385 was also found to use a member of the VH-J558 family, but in this case the nitrophenyl-binding 4m3 VH gene 14. In SIPC3385, the rearrangement involves JH4 and another member of the DHSP2 family with N sequence additions. Analysis of the VH region sequences reveals somatic mutational activity with high replacement to silent (R/S) ratios, since all the SIPCs that express H13-3 exhibit four to six replacement changes with no accompanying silent base changes (Fig. 4). In the case of SIPC3385, we only find a single replacement in VH.


Restricted immunoglobulin variable region (Ig V) gene expression accompanies secondary rearrangements of light chain Ig V genes in mouse plasmacytomas.

Diaw L, Siwarski D, Coleman A, Kim J, Jones GM, Dighiero G, Huppi K - J. Exp. Med. (1999)

JH rearrangements in SIPC tumors. Genomic DNAs from SIPC tumors and BALB/c liver were digested with EcoR1 and hybridized with the JH probe: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. An arrow (left) depicts the germline (nonrearranged) JH fragment, whereas the 3.5-kb EcoR1 rearrangement seen in several tumors is also highlighted.
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Related In: Results  -  Collection

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Figure 3: JH rearrangements in SIPC tumors. Genomic DNAs from SIPC tumors and BALB/c liver were digested with EcoR1 and hybridized with the JH probe: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. An arrow (left) depicts the germline (nonrearranged) JH fragment, whereas the 3.5-kb EcoR1 rearrangement seen in several tumors is also highlighted.
Mentions: Initially, Ig heavy chain–specific rearrangements were identified in each tumor by Southern blot analysis using a JH probe (Fig. 3). Heavy chain-specific rearrangements were found in all tumors, including apparent rearrangements of both alleles in SIPC3385, SIPC3282, and SIPC3301. We also found shared rearrangements with both BamH1 (not shown) and EcoR1 digestions in SIPC3308, SIPC3336, and SIPC3282, suggesting the same VH gene may be expressed in these tumors. We cloned and sequenced the 3.5-kb EcoR1 fragment from SIPC3336, and established that the rearrangement consists of a member of the VH-J558 family (H13-3; reference 12) rearranged to DHSP2-9 and JH3. To more specifically determine VH gene usage in the SIPC tumors, we examined the expressed sequences by reverse transcription (RT)-PCR (Fig. 4). Indeed, three of the SIPC tumors (SIPC3282, SIPC3336, and SIPC3308) that share the 3.5-kb EcoR1 rearrangement all use the same H13-3 gene from the VH-J558 family. We verified that this sequence was germline (nonmutated) by sequencing eight clones derived from the PCR product of BALB/c genomic DNA which was amplified by primer pairs specific for H13-3 (see Table ). Interestingly, these same three tumors have also rearranged to JH3, and use the same DH region (DHSP2-9) encoding the amino acid residues Trp-Phe. Although rearranged to JH3 and DHSP2-9 as well, SIPC3301 has a longer nucleotide addition sequence, and is in fact encoded by another member (26.4.1-α; reference 13) of the VH-J558 family. The RT-PCR product of SIPC3385 was also found to use a member of the VH-J558 family, but in this case the nitrophenyl-binding 4m3 VH gene 14. In SIPC3385, the rearrangement involves JH4 and another member of the DHSP2 family with N sequence additions. Analysis of the VH region sequences reveals somatic mutational activity with high replacement to silent (R/S) ratios, since all the SIPCs that express H13-3 exhibit four to six replacement changes with no accompanying silent base changes (Fig. 4). In the case of SIPC3385, we only find a single replacement in VH.

Bottom Line: The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl.Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody.These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

Show MeSH
Related in: MedlinePlus