SummaryWe have examined the nature and localization of somatic mutations in three tc transgenes cloned from IgG-secreting hybridomas. All of the mutations identified were single base substitutions. Mutations were localized to the variable (V) region and its flanking sequences. In every case, the nuclear matrix association region, K enhancer, and C gene were spared. These data indicate that the rearranged « gene contains the necessary sequences for targeting of the mutation process, and suggest that the observed localization of mutations to the V region reflects the inherent specificity of this mutation process.

The rearrangement of Ig genes is known to be regulated by the production of H and kappa L chains. To determine whether lambda L chains have a similar effect, transgenic mice were produced with a lambda 2 gene. It was necessary to include the H chain enhancer, since a lambda gene without the added enhancer did not result in transgene expression. The lambda 2 transgene with the H enhancer was expressed in lymphoid cells only. The majority of the B cells of newborn transgenic mice produced lambda, whereas kappa + cells were reduced. Concomitantly, serum levels of kappa and kappa mRNA were diminished. By 2 wk after birth the proportion of kappa-expressing cells was dramatically increased. Adults had reduced proportions of B cells that produced lambda only, but the levels of lambda were still higher than in normal littermates. Also, kappa + cells were still lower than in normal mice. Analysis of hybridomas revealed that reduction of kappa gene rearrangement was the basis for the decreased frequency of kappa + cells. Furthermore, many cells also contained an unrearranged H chain allele. It was concluded that feedback inhibition by the lambda 2 together with endogenous H protein may have inhibited recombinase activity in early pre-B cells, leading to inhibition of both H chain and kappa gene rearrangement. Thus, lambda 2 can replace kappa in a feedback complex. The levels of serum lambda 1 and, to a lesser degree, of spleen lambda 1 mRNA were reduced in the lambda 2 transgenic mice. However, the proportion of hybridomas with endogenous lambda gene rearrangement was at least as high as in normal mice. It was therefore concluded that the suppression of functional lambda 1 may be a consequence of decreased selection of endogenous lambda-producing cells because of the excess of transgenic lambda. The escape of kappa-producing cells from feedback inhibition may be the result of several mechanisms that operate to varying degrees, among them: (a) kappa rearrangement during a period in which the recombinase is still active after appearance of a lambda 2/mu stop signal; (b) a B cell lineage that is not feedback inhibited at the pre-B cell stage; (c) subthreshold levels of transgenic lambda 2 in some pre-B cells; and (d) loss of the lambda 2 transgenes in rare pre-B cells.

SummaryIn an effort to identify c/s-acting elements required for targeting of the somatic hypermutation process in mice, we examined whether a T cell receptor (TCR) transgene under the control of the immunoglobulin (Ig) heavy (H) chain intron enhancer would be mutated in antigenstimulated B cells. Hybridomas were established from splenic B cells of mice carrying two copies of the TCR transgene after hyperimmunization with phosphorylcholine keyhole limpet hemocyanin. Northern analysis revealed that all of the transgene-containing hybridomas expressed the TCR mRNA. Multiple somatic point mutations were found in seven of eight endogenous Ig VH genes examined. In contrast, 29 of 32 TCR genes examined contained no mutations. One potential mutation was seen in each of the three other TCR genes. Our data indicate that although the TCR transgene is expressed in B cells, it is not efficiently targeted by the mutator mechanism. Furthermore, the presence of an Ig H chain enhancer is itself not sufficient for targeting of the somatic hypermutation mechanism.

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Xenotropic MLV-Related Virus (XMRV) was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS). Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS) provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs) and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA). Although 9 of 332 (2.7%) samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.