Specific metaphase and interphase detection of the breakpoint region in 8q24 of burkitt lymphoma cells by triple-color fluorescence in situ hybridization

Triple fluorescence in situ hybridization with a plasmid DNA library from sorted human chromosomes 8 in combination with bacteriophage clones flanking the breakpoint in 8q24 of the Burkitt lymphoma cell line Jl was used for the specific delineation of this breakpoint in individual tumor cells. With this approach, tumor-specific breakpoints in translocation chromosomes can be detected at all stages of the cell cycle with high specificity

Localization, analysis and evolution of transposed human immunoglobulin VK genes

The localization of Vκ gene regions to chromosome 2, on which the κ locus is located, and to other chromosomes is described. The Vκ genes that have been transposed to other chromosomes are called orphons. The finding of two new Vκ genes on chromosome 22 is reported. A Vκ II gene of this region and two Vκ I genes of the Chr 1 and the cos 118 regions were sequenced. The two Vκ I orphon sequences and two others that had been determined previously were 97.5% identical, indicating that they may have evolved from a common ancestor by amplification. A model of the evolution of the human Vκ orphons is discussed. Author Keywords: Human-rodent cell hybrids; cosmids; restriction maps; ligation artifacts; orphon; recombinant DNA Abbreviations: aa, amino acid(s); bp, base pair(s); Chr1, Vκ gene-containing regions of chromosomes 1; Chr22, Vκ gene-containing regions of chromosomes 22; FR, framework regions; CDR, complementary determining regions; kb, kilo-base(s) or 1000 bp; L, L′, parts of a leader gene segment; m219-1, the first subclone of the cosmid clone cos 219; orphon, Vκ gene outside the κ locus on chromosome 2pl2; SSC, 0.15 M NaCl, 0.015 M Na3-citrate, pH 7.6; V, variable gene segments; J, joining gene segments; C, constant gene segments; Vκ I to Vκ IV, variable gene segments of immunoglobulin light chains of the κ type belonging to subgroups I to IV; for reasons of simplicity Vκ gene segments are generally called Vκ gene

AID-Targeting and Hypermutation of Non-Immunoglobulin Genes Does Not Correlate with Proximity to Immunoglobulin Genes in Germinal Center B Cells

Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this “collateral damage” model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination