Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products.

Abstract

We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The Si nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect.Immunoglobulin genes are assembled from germ line variable-region and constant-region gene segments in developing B cells. Despite the fact that germline variable-region genes possess functional promoters (5), they are not expressed in myeloma cells (46,79). Conversely, germ line constant-region genes that lack an immunoglobulin promoter are transcribed from secondary promoters in the 5' flanking region (54,73). Recently, positive regulatory sequences, or enhancers, have been identified in the J-C intron of both mu and kappa genes (3,29,57). The activation of immunoglobulin genes may be accomplished by transposing the variable region with its immunoglobulin promoter to the vicinity of the constant-region enhancer.Although immunoglobulin enhancers are required for the expression of linked genes introduced into lymphoid cells by transformation, the enhancer may not be required for the maintenance of high-level immunoglobulin expression. Recently, several myeloma mutants have been described in which the heavy-chain enhancer has been deleted (23,42). Despite this deletion, normal levels of heavy-chain mRNA and protein are made by the cell. When the allele with the enhancer deletion is cloned from the genome and introduced into myeloma cells, it is transcriptionally inactive (80).We chose to investigate immunoglobulin gene regulation by ...

Transcription of a Productively Rearranged Ig VDJCα Does Not Require the Presence of HS4 in the Igh 3′ Regulatory Region

“…Reciprocal loss of the 3Ј IgH enhancers, however, had a dramatic effect on Ig transcription in an Ig␣-secreting cell line (LP1.2). Ig␣ transcription fell to 10% of wild-type levels even though E remained within the Ig␣ transcription unit (16,17). These two findings led tocould be supplanted by the 3Ј IgH enhancers.…”

Regulation of the Replication of the Murine Immunoglobulin Heavy Chain Gene Locus: Evaluation of the Role of the 3′ Regulatory Region

“…To test whether the Igh 3Ј regulatory region might be responsible for directing early replication of the Igh locus in B-lymphoid lines, we took advantage of the mutant myeloma cell line LP1.2 (low producing). The LP1.2 line has a reduction in C␣ Ig expression compared to its parent, W3129, accompanied by a ϳ34-kb deletion in the expressed allele 3Ј of C␣, which encompasses all four enhancers identified in the 3Ј regulatory region (17,27). These data implied that 3Ј regulatory sequences were required for high levels of Igh expression in plasmacytoma cells.…”

“…The following probes from the region 3Ј of mouse Igh C␣ were used in these studies. The probe PstI-2.3 (17,27) is a 2.3-kb PstI fragment, located ϳ3 kb downstream of the Igh 3Ј regulatory region, that was isolated from an LP1.2-derived clone. A 0.7-kb HincII-EcoRI fragment (0.7 Hc/E) (GenBank accession no.…”

“…LP1.2 cells (17) were grown in Iscove's modified Dulbecco's medium (Biowhittaker, Walkersville, Md. ), supplemented with 15% heat-inactivated fetal bovine serum (Life Technologies, Bethesda, Md.…”

“…The ϳ35-kb deletion in the mutant myeloma cell line LP1.2 includes all elements in the 3Ј regulatory region (17,27), as indicated by the arrows. The phage clone from which C␣(Ϫ54) was derived (see Materials and Methods) is shown.…”