A simple and precise aberrant translocation of the rat c-myc gene into the epsilon-heavy chain switch region of the IgE-producing immunocytoma, IR162

We report a simple type of reciprocal chromosomal translocation in the LOU rat IgE-secreting immunocytoma cell line, IR162, involving the c-myc protooncogene and the switch region of the epsilon immunoglobulin heavy chain, c-myc/S epsilon. By cloning and sequencing the translocation-associated and the homologous normal c-myc and S epsilon DNAs, we have identified the position of the translocational junction in both the c-myc 5'-flanking region and the repetitive elements of the S epsilon region. The translocational recombination was precise, and no insertion or N-addition was found in the junctional region, leaving all the c-myc exons, together with two promoter sites, intact. RNase mapping confirmed that the same promoters were utilized in IR162 and normal LOU spleen cells. No point mutation was found in the 5'-flanking region and the 3'-portion of exon 1 of the translocated c-myc gene. However, the putative silencer region was lost with the translocation. It was also noticed that a strikingly AT-rich sequence associated with S epsilon region had translocated to the 5'-flanking region of c-myc gene. We discuss the possibility that a change of DNA topology, perhaps either due to the juxtaposition of an AT-rich sequence of the S epsilon region, or to the loss of the putative silencer element, may contribute to c-myc gene deregulation in IR162.     

Polymorphisms of the immunoglobulin heavy-chain delta gene and association with other constant-region genes.

Polymorphisms have previously been reported for the C mu, C alpha, C epsilon, and C gamma genes of the immunoglobulin heavy-chain (IGH) gene cluster. Here we report polymorphisms of the IGH C delta gene region, observed using the enzymes ApaI, AvaII, TaqI, and XbaI. The TaqI and XbaI polymorphisms were used in an investigation of linkage disequilibrium throughout the cluster of constant-region genes. The TaqI polymorphism, located 5' to the C delta gene, is in linkage disequilibrium with a polymorphism of the C mu switch region. The XbaI polymorphism, which is in the vicinity of the C delta 2 exon, is not strongly associated with any other polymorphisms, including the TaqI polymorphism and the Gm polymorphism of C gamma 3. Although there is a high degree of association between most genes of the IGH region, there is a lack of association between C delta and C gamma 3, which may indicate a hot spot for recombination.     

Evolution of the rat immunoglobulin gamma heavy-chain gene family

The sequences of the four immunoglobulin gamma heavy chains of the rat (gamma 1, gamma 2a, gamma 2b, gamma 2c) have been determined. These sequences reveal that the rat genes have evolved differently from the closely related mouse gamma genes (gamma 1, gamma 2a, gamma 2b, gamma 3): in rat two of the four genes (gamma 2a and gamma 1) are 94% homologous to each other and best resemble the single mouse gamma 1 gene. Rat gamma 2b is equivalent to the mouse gamma 2a/gamma 2b pair as regards both nucleotide sequence and antibody effector functions whilst rat gamma 2c resembles mouse gamma 3. In evolutionary terms this suggests the existence of a set of three common C gamma genes before separation of rat and mouse as individual species. In addition, two independent duplication events must have occurred after species separation affecting different constant regions; this yielded rat gamma 2a and gamma 1 as a recently evolved pair and mouse gamma 2a and gamma 2b as a different pair. Furthermore, the sequence comparisons reveal several other features of interest; rat IgG2b lacks two amino acids in CH1 which are conserved in all other sequenced gamma chains. Residues believed to be essential for monocyte interaction (FcRI) are retained only in rat gamma 2b and not in the other rat gamma genes whilst a particular motif involved in C1q interaction shows a variation in both rat IgG1 and rat IgG2a which has not been observed previously.     

Possible palindromes in immunoglobulin heavy-chain genes: Their role in membrane attachment

An analysis of mRNA structures as deduced from the amino acid sequences of immunoglobulin heavy chains reveals possible base-paired regions within the sequences coding for the 20 C-terminal amino acids of the human and chains. The two regions are similar in structure and contain a palindrome which might serve as an enzyme recognition site. Although other base-paired regions can be predicted in the remainder of the constant regions of these heavy chains, they have no common features. These regions and the palindromes within them may be involved in the regulation of membrane and serum immunoglobulin synthesis. Two possible mechanisms for this are proposed.     

Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody

B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-V_H) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-V_H sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-V_H genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-V_H genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the V_H2 family as well as in specific mini-genes like V_H3–23, V_H 4–34 or V_H 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period.     

Absence of missense mutations in activated c-myc genes in avian leukosis virus-induced B-cell lymphomas.

We have determined the nucleotide sequences of two independent DNA clones which contained the activated c-myc genes from avian leukosis virus-induced B-cell lymphomas. Neither of these c-myc genes contained missense mutations. This strongly supports the notion that the c-myc proto-oncogene in avian leukosis virus-induced B-cell lymphomas can be oncogenically activated by altered expression of the gene without a change in the primary structure of the gene product.     

Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.     

Expressions of the c-Ha-ras and c-myc genes in rat liver tumors

Expressions of the c-Ha-ras and c-myc genes were studied by Northern blotting of total RNA from primary tumors and non-tumorous parts of the liver of rats given diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and from established rat hepatoma cell lines. The expression of the c-Ha-ras gene was found to be high in the primary tumors, non-tumorous parts of 3'-Me-DAB-treated livers and hepatoma cell lines. In contrast, the c-myc gene was expressed at a high level only in primary tumors and hepatoma cell lines. During 3'-Me-DAB treatment, the c-Ha-ras mRNA level in the liver increased by day 5 and then remained high. Increase in expression of the c-Ha-ras gene in regenerating liver was confirmed. These findings suggest that increase in expression of the c-Ha-ras gene is related to proliferation of hepatocytes, whereas expression of the c-myc gene is associated with hepatocarcinogenesis.     

Complex protein binding within the mouse immunoglobulin heavy-chain enhancer.

We have begun to purify and characterize several proteins which bind to the mouse immunoglobulin heavy-chain enhancer to understand the molecular interactions important for enhancer activity. Three proteins which bind to different sites on the immunoglobulin heavy-chain enhancer have been chromatographically separated and partially purified. One protein binds a site which has not been reported previously and does not bind to other reported protein-binding sites on the immunoglobulin heavy-chain enhancer. Binding-site boundaries for the three partially purified proteins have been precisely mapped by methylation interference, DNase I footprinting, and orthophenanthroline/copper chemical nuclease footprinting. We have also characterized these three proteins with respect to dissociation rate constants.     

Immunoglobulin heavy-chain joining genes in the rat: comparison with mouse and human

We have cloned by the polymerase chain reaction a 2.1-kb fragment carrying heavy-chain joining (JH) gene segments and a part of the JH-C mu intron of the rat. Sequencing showed that the rat genome has four functional JH segments and that only a slight divergence has occurred after the separation of rat and mouse. A systematic sequence comparison between the two species and human revealed an additional JH pseudogene in rat and mouse 5' of JH1 which has not been described so far. The implications in evolutionary terms are discussed. In addition, we give an assessment of the misincorporation rate of the Taq polymerase.     

Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.     

Dispersed localization of D segments in the human immunoglobulin heavy-chain locus.

We have studied the organization of the human immunoglobulin heavy-chain genes by pulse field gel electrophoresis as well as by isolation of cosmid clones. The total length of the heavy-chain variable region locus was estimated to be approximately 3000 kb. We found that D segments including a recently isolated D5 segment were dispersed among VH segments. We identified a pseudo V segment 18 kb 3' to the D5 segment in isolated cosmid clones. A 300 kb fragment produced by MluI digestion contained VH, D, JH segments and the distance between VH and D was estimated to be approximately 240 kb. Overlapping cosmid clones containing the human D1, D2, D3, D4, JH, Cmu and C delta genes were isolated. Restriction maps of these regions indicated that the distance between D and JH is about 22 kb. A partial restriction map of the VH locus was constructed using the pulse field gel electrophoresis technique and deletion of VH segments in B cells.