Two rearranged immunoglobulin kappa light chain genes in one mouse myeloma.

The organization of immunoglobulin gene segments coding for kappa light chains has been studied in uncloned and cloned DNA from mouse liver and a mouse myeloma. It is known that the C (constant, ref. 2) gene segment is present in the tumor DNA on two EcoRI fragments of 14 and 20 kb and in liver DNA on a 15 kb fragment. The 14 kb myeloma and the 15 kb liver fragment have been cloned previously. Here we report on the cloning of the 20 kb myeloma fragment and present detailed restriction maps covering about 22 kb of DNA surrounding the C gene segment in liver and tumor DNA. The region on the 20 kb fragment has been localized where a DNA rearrangement had occurred. The presence of two rearranged kappa light chain genes in one tumor is discussed in regard to the molecular basis of allelic exclusion.     

Differences in the nuclease sensitivity between the two alleles of the immunoglobulin kappa light chain genes in mouse liver and myeloma nuclei.

In mouse myeloma T the productive kappa light chain gene differs from its aberrantly rearranged allele in the patterns of DNAase I hypersensitive sites. In the region of the alleles where they are identical in sequence they have one site in common which lies 0.8 kb downstream of the coding region; but two sites upstream of and within the C gene segment (2) are found only on the non-productive allele. Within the region of different sequences both alleles have analogously located DNAase I hypersensitive sites; they lie 0.15 kb upstream of the respective leader segments and cover putative promoter sequences. Only one of the six DNAase I hypersensitive sites is also very sensitive towards micrococcal nuclease due to its particular DNA sequence. The non-rearranged gene studied in liver nuclei has no DNAase I hypersensitive sites but is preferentially cleaved in A/T rich regions.     

DNA sequence comparisons of the human, mouse, and rabbit immunoglobulin kappa gene

A comparative analysis between human, mouse, and rabbit immunoglobulin (Ig) kappa-gene DNA sequences is presented. New formulas for determining the expected length and variance of the longest block identity (a succession of matching nucleotides) between multiple random sequences are given and are used to establish statistical criteria for ascertaining the significance of block identities shared in r out of s sequences. The statistically significant block identities within and between the Ig-kappa-gene sequences are ascertained, and alignment maps based on these similarities are constructed. The human and rabbit sequences (especially in the noncoding regions) and the human and mouse sequences (on the coding regions) show a similarity much stronger than that between the mouse and rabbit sequences. The existence of several highly significant shared oligonucleotides occurring in alignment with each other or with respect to the J- and C-gene segments suggests a configuration of multiple control sites. Discussion and interpretations of the form and distribution of the block identities are given.      

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions.

The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer.     

Synthesis of immunoglobulin and nuclear protein in synchronized mouse myeloma cells

The synthesis of immunoglobulin and of nuclear proteins has been studied in synchronized mouse myeloma cells of the C1 line. Synchronization has been obtained by a double thymidine block. C1 cells synthesize immunoglobulin at a relatively constant rate throughout the cell cycle except for mitosis, when a decrease in the rate of synthesis of total protein and of immunoglobulin is observed. Cell synchrony around mitosis is not sufficiently good to determine whether immunoglobulin is synthesized at all. Nuclear protein and in particular histones appear to be synthesized synchronously with DNA during the S phase of the cell cycle.     

Synthesis of immunoglobulin by substrate attached mouse myeloma cells

Cultured mouse myeloma cells grow in suspension and synthesize and secrete large amounts of immunoglobulin. Mouse myeloma cells which attach to a plastic substratum have been obtained by mutagenesis and subsequent selection. Normal mouse myeloma cells will also attach to plastic tissue culture dishes pre-treated with poly-L-lysine. The attached cells synthesize and secrete the same large amounts of immunoglobulin as the suspended cells.     

Identification and partial purification of a protein binding to the human immunoglobulin kappa enhancer kappa E2 site.

We previously described a domain in the 5'' half of the human immunoglobulin kappa enhancer which could bind nuclear proteins in vitro, as detected by a lambda exonuclease protection assay. A second more 3'' binding domain in the enhancer has now been detected by a similar assay employing a different exonuclease, the T7 gene 6 exonuclease. Using this assay and starting with a pig spleen nuclear extract, we have purified 5000-fold a protein that binds to the 3'' domain. In a DNase I footprint experiment the partially purified protein protects a 27 bp segment in the enhancer centered around the sequence CAGGTGGC, which corresponds to the kappa E2 sequence motif described in the mouse kappa enhancer. The protein, designated NF-kappa E2, also appears to bind at a position downstream of kappa E2, at or near the kappa E3 site. Proteins capable of binding at kappa E2 are found in several mammalian species and are expressed in both lymphoid and non-lymphoid tissues.