Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, it and v cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4-to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.

We have generated transgenic mouse lines that carry one of three different constructs in which the murine N‐myc gene is expressed under the control of the immunoglobulin heavy chain transcriptional enhancer element (E mu‐N‐myc genes). High‐level expression of the E mu‐N‐myc transgenes occurred in lymphoid tissues; correspondingly, many of these E mu‐N‐myc lines reproducibly developed pre‐B‐ and B‐lymphoid malignancies. The E mu‐N‐myc transgene also appeared to participate in the generation of a T cell malignancy that developed in one E mu‐N‐myc mouse. These tumors and cell lines adapted from them expressed exceptionally high levels of the E mu‐N‐myc transgene; the levels were comparable to those observed in human neuroblastomas with highly amplified N‐myc genes. In contrast, all of the E mu‐N‐myc cell lines had exceptionally low or undetectable levels of the c‐myc RNA sequences, consistent with the possibility that high‐level N‐myc expression can participate in the negative ‘cross‐regulation’ of c‐myc gene expression. Our findings demonstrate that deregulated expression of the N‐myc gene has potent oncogenic potential within the B‐lymphoid lineage despite the fact that the N‐myc gene has never been implicated in naturally occurring B‐lymphoid malignancies. Our results also are discussed in the context of differential myc gene activity in normal and transformed cells.

Among 631 substitutions present in 90 nurse shark immunoglobulin light chain somatic mutants, 338 constitute 2-4 bp stretches of adjacent changes. An absence of mutations in perinatal sequences and the bias for one mutating V gene in adults suggest that the diversification is antigen dependent. The substitutions shared no patterns, and the absence of donor sequences, including from family members, supports the idea that most changes arose from nontemplated mutation. The tandem mutations as a group are distinguished by consistently fewer transition changes and an A bias. We suggest this is one of several pathways of hypermutation diversifying shark antigen-receptor genes--point mutations, tandem mutations, and mutations with a G-C preference--that coevolved with or preceded gene rearrangement.

Using class specific monoclonal antibodies we analyzed the tissue distribution of B cells expressing the three immunoglobulin (Ig) isotypes (IgM, IgX, IgY) in Xenopus. Large numbers of IgM- and IgX-, but not IgY-, positive B cells are located in the gut epithelium of the intestine. In this organ up to 60% of all B cells can be IgX positive, while in the spleen or liver they are hardly detectable. The majority of IgX-producing cells resemble plasma cells. IgY-producing cells are found in the liver and spleen but not in the intestine. In contrast to IgY, the expression of IgM and IgX is thymus independent. Upon systemic immunization, a several-fold increase of specific IgM and IgY, but not IgX, antibodies was detected in the sera. This and its association with the mucosae of the intestine resembles results reported for mammalian IgA; therefore, IgX of Xenopus might be considered an analog of IgA.

Unlike mammals, cold-blooded vertebrates produce antibodies of low heterogeneity that show little increase in binding affinity with time after immunization. In secondary responses, antibody titers and affinities are often little, if any, higher than in primary responses. That is, specificity, diversity, and memory--the hallmarks of the immune system--are rather meager in the humoral immune responses of exothermic vertebrates. As the genetic components of the immunoglobulin (Ig) gene systems in fishes, amphibians or reptiles are not deficient in number or diversity, their responses probably do not stem from restrictions in the primary antibody repertoire. Somatic hypermutation at the Ig locus, which generates diversity and higher affinity antibodies in mammals, is not lacking in the South African frog Xenopus or in the shark. However, the Ig mutants recovered are strongly biased toward alterations at GC pairs, an indication that they have not undergone effective selection. While cells resembling follicular dendritic cells are present in cold-blooded vertebrates, germinal centers do not form. It is suggested that this absence of germinal centers, the site of selection for the mutants with higher affinity receptors and of differentiation into memory B cells in mammals, may explain the principal differences between cold and warm-blooded vertebrates.