Start of mu-chain production by the further two-step rearrangements of immunoglobulin heavy chain genes on one chromosome from a DJH/DJH configuration in an Abelson virus-transformed cell line: evidence of secondary DJH complex formation

A variant was selected from an Abelson virus-transformed cell line with identifiable and distinguishable DJH rearrangements on both chromosomes. Southern blotting experiments suggested that this variant resulted from a secondary DJH complex formation accompanied with the deletion of one of the DJH rearrangements on the same chromosome. Furthermore, this variant started making mu-chains by the further functional join of a VH gene to the secondarily formed DJH complex. Thus, we describe here the first evidence of a secondary DJH complex formation.     

Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus.

Sequences of immunoglobulin (Ig) cDNA clones of Xenopus laevis show that at least three different VH families are expressed in association with different JH elements and different isotypes of Ig constant regions. In genomic Southern blot analysis, the VH probes for each family hybridize to a distinct set of multiple DNA fragments. In contrast, the genomic JH elements and the IgM constant region gene are localized in a single DNA fragment of approximately 15 kb. Genomic VH elements contain regulatory sequences similar to those in VH genes of shark, fish and mammals and have a leader peptide sequence that contains an intron; they encode the VH region until residue 95 and have heptamer--23-bp--nonamer motifs similar to the rearrangement signal sequences (RSS) in all other vertebrate VH elements. The six genomic JH elements so far sequenced have a nonamer--23-bp--heptamer motif at their 5' end. These RSS motifs imply the existence of DH elements. The comparison of cDNA clones that contain similar constant regions but different VH regions or JH elements suggest rearrangement events. This is shown by Southern blot analysis of erythrocyte and B cell DNA with a JH probe. Thus, the overall organization of the Xenopus Ig gene locus is similar to that of mammals but strikingly different from shark.     

The variability, arrangement, and rearrangement of immunoglobulin genes

The multiplicity of heavy-chain variable-region (VH) genes in mouse and human DNA has been estimated using a mouse heavy-(H) chain cDNA clone. We found about 10 hybridization components in mouse DNA and about 20 components in human DNA. Cross-hybridization studies of variable region (V) genes indicate that these components represent the numbers of genes within the VH subgroups of each of these species. The arrangement and rearrangement of the H-chain gamma subclasses have been studied in order to assess possible mechanisms of the H-chain switch. Evidence has been found for rearrangement events involving the gamma 2a and gamma 2b constant-region (CH) genes in DNA from cells making IgG2a and IgG2b respectively. In addition we found that cells making IgG2a lack detectable genes for gamma1 and gamma 2b. Both sets of observations are discussed in relation to H-chain diversity and the switch.     

Enumeration and characterization of DJH structures in mouse fetal liver.

The primary immunoglobulin (Ig) repertoire in the mouse develops during fetal life in the liver. The first Ig gene rearrangement--the joining of a DH to a JH gene segment--contributes largely to the diversity found in CDR3, as well as potentially encoding the D mu protein which is believed to function in the development of a B cell. In this report, the number of DJH joins in two mouse strains, C57BL/6 and BALB/c, were enumerated from days 12 to 16 of fetal development. It was found that the number of DJH structures increased from less than 300 per liver on day 12 to greater than 700,000 (C57BL/6) and 300,000 (BALB/c) on day 16. Each JH gene segment was used approximately equally on each day examined. When the DJH structures were examined by cloning and sequencing it was found that the DJH reading frame (RF) usage (with respect to JH) was not random--RF1 was used 70% of the time. Moreover, a single D gene segment, DFL16.1, was used in greater than 50% of all joins reinforcing the notion that the fetal repertoire is restricted in its antigen binding potential.     

Ordered rearrangement of immunoglobulin heavy chain variable region segments

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.     

Analysis of immunoglobulin and T-cell receptor gene rearrangement in cytologic specimens

Recombinant DNA techniques to detect the rearrangement of genes encoding immunoglobulins and T-cell-antigen receptors have been used to identify clonality in lymphoid lesions. To determine the utility of such techniques in cytologic specimens, DNA was analyzed in 24 effusions and 6 fine needle aspirates. Immunophenotypic studies were also performed on the 19 specimens with suspected hematopoietic malignancies. Sufficient material for DNA analysis was present in 28 of the 30 specimens. Immunoglobulin or T-cell-receptor gene rearrangement was present in 13 specimens with atypical cytologic findings; DNA studies provided more information than did the immunologic studies in 3 cases. One T-cell malignancy showed T-cell receptor and heavy-chain gene rearrangement, and one B-cell malignancy showed immunoglobulin and T-cell receptor gene rearrangement. In all patients except one with no evidence of gene rearrangement, the morphologic and immunologic studies also favored a reactive process. Control specimens showed a germline configuration. This study demonstrated that DNA gene rearrangement studies are feasible on many cytologic specimens and may be useful in diagnostically difficult cases.     

Assessing the pathogenic potential of the V(D)J recombinase by interlocus immunoglobulin light-chain gene rearrangement.

Chromosomal translocations involving antigen receptor genes and oncogenes have been observed in several forms of lymphoid malignancy. Observations of their lymphocyte-restricted occurrence and a molecular analysis of some translocation breakpoints have suggested that some of these rearrangements are generated by V(D)J recombinase activity. However, a direct correlation between this activity and the generation of such rearrangements has never been established. In addition, because these aberrant rearrangements are usually detected only after a tumor has been formed, the frequency with which the recombinase machinery generates translocations has never been assessed directly. To approach these issues, immunoglobulin light-chain gene rearrangements were induced in pre-B cells transformed by temperature-sensitive mutants of Abelson murine leukemia virus and PCR was used to identify interlocus recombinants. Vlambda Jkappa and Vkappa Jlambda rearrangements as well as signal joints resulting from the recombination of Vlambda and Jkappa coding elements were recovered and were found to be similar in structure to conventional intrachromosomal joints. Because these products were detected only when the cells were undergoing active intralocus rearrangement, they provide direct evidence that translocations can be generated by the V(D)J recombinase machinery. Dilution analyses revealed that interlocus rearrangements occur about 1,000 times less frequently than conventional intralocus rearrangements. Considering the large numbers of lymphocytes generated throughout life, aberrant rearrangements generated by the V(D)J recombinase may be relatively common.