Faculty Bibliography

PURPOSE: Burkitt's lymphoma is a malignancy of mature, immunoglobulin (Ig)-bearing B cells characterized by translocation between c-myc and Ig gene loci. A role for the juxtaposed Ig genes in the mutation and deregulation of c-myc expression typical of endemic Burkitt's lymphoma (eBL) has been proposed, but never proven. Our objective was to determine whether Ig gene hypermutation is ongoing in eBL. METHODS: We isolated Ig heavy-chain sequences from K962 eBL tumor cells using reverse transcription and polymerase chain reaction (PCR) amplification. The PCR product was ligated into Bluescript II vectors. Multiple subclones were sequenced and the variable regions were compared for evidence of ongoing Ig hypermutation. RESULTS: Six total single base substitutions were observed within four of the nine subclones studied. Four substitutions resulted in amino acid changes and two were silent. There was no clustering of mutations in hypervariable regions, or a high incidence of amino acid replacement or link substitutions, all of which are characteristic of Ig hypermutation. The observed mutations occurred at a rate consistent with Taq polymerase error. CONCLUSIONS: Our data indicate that in the eBL tumor sample K962, the mechanism underlying c-myc mutation is distinct from that which gives rise to Ig hypermutation

Cross-reactivity of anti-streptococcal Abs with human cardiac myosin may result in sequelae following group A streptococcal infections. Molecular mimicry between group A streptococcal M protein and cardiac myosin may be the basis for the immunologic cross-reactivity. In this study, a cross-reactive human anti-streptococcal/anti-myosin mAb (10.2.3) was characterized, and the myosin epitopes were recognized by the Ab identified. mAb 10.2.3 reacted with four peptides from the light meromyosin (LMM) tail fragment of human cardiac myosin, including LMM-10 (1411-1428), LMM-23 (1580-1597), LMM-27 (1632-1649), and LMM-30 (1671-1687). Only LMM-30 inhibited binding of mAb 10.2.3 to streptococcal M protein and human cardiac myosin. Human mAb 10.2.3 labeled cytoskeletal structures within rat heart cells in indirect immunofluorescence, and reacted with group A streptococci expressing various M protein serotypes, PepM5, and recombinant M protein. The nucleotide sequence of gene segments encoding the Ig heavy and light chain V region of mAb 10.2.3 was determined. The light chain V segment was encoded by a V kappa 1 gene segment that was 98.5% identical with germ-line gene humig kappa Vi5. The V segment of the heavy chain was encoded by a VH3a gene segment that differed from the VH26 germ-line gene by a single base change. VH26 is expressed preferentially in early development and encodes autoantibodies with anti-DNA and rheumatoid factor specificities. Anti-streptococcal mAb 10.2.3 is an autoantibody encoded by VH and VL genes, with little or no somatic mutation

The B cell repertoire was studied in intestinal mononuclear cells from normal individuals and patients with inflammatory bowel disease (IBD) by examining Ig heavy chain variable gene (VH) usage. Using reverse transcription of intestinal mucosal RNA followed by polymerase chain reaction with primers specific for each VH family and a housekeeping gene, a semiquantitative assay of VH family content in RNA samples was developed. While all VH family members were expressed, differences in VH usage in lamina propria intestinal B cells were noted between Crohn's disease, ulcerative colitis, and normal individuals. mRNA transcripts for VH4 were present at seemingly higher levels than their genomic representation and transcripts for VH1 and VH4 appeared to have higher levels in active, compared to inactive, IBD. Thus, within the massive polyclonal intestinal B cell response, there is a skewed VH usage which may be relevant to the antigenic and/or autoimmune response noted in IBD

Recombinant-DNA technology is now commonly used in virtually every aspect of the biological sciences. The purpose of this brief exposition is to provide an outline of the approaches used to identify genes, to isolate the gene of interest, to amplify the gene if necessary, and to clone genes. A short introduction to the principles of separating very large genes is provided, along with a description of an approach to propagating and cloning these large genes

We have previously shown that human antibody (Ab) directed against the capsular polysaccharide of the important bacterial pathogen, Haemophilus influenzae type b (Hib) is encoded by a small group of VH3 gene family members. The majority of anti-Hib PS Ab use members of the smaller VH3b subfamily. To examine directly the available human VH3 repertoire, we have used PCR to amplify and clone candidate germ-line VH3b H chain V region genes from two unrelated subjects from whom anti-Hib polysaccharide mAb had been previously obtained. A single functional VH3b germ-line gene was obtained from one subject. This gene is identical throughout the coding region to the previously identified gene 9.1. Twelve distinct VH3b germ-line sequences, 87.6-99.8% homologous to one another, were obtained from the second subject. One of these genes, LSG1.1, is also identical to the 9.1 germ-line gene, and a second, LSG6.1, is identical to a previously reported cDNA, M85. These germ-line VH3b genes are 82.7-94.1% homologous to rearranged anti-Hib PS VH3b segments obtained from these subjects. Our findings further demonstrate that considerable polymorphism of VH segments exists in the human population. Despite the presence of very highly homologous VH elements in the germ line, particular genes are highly conserved within the outbred human population

To examine the human antibody repertoire generated against a biologically significant antigen we have obtained sequences of heavy chain variable region genes (IgVH) from 15 monoclonal antibodies specific for the capsular polysaccharide of Haemophilus influenzae type b (Hib PS). All VH segments are members of the VH3 family and 9 of 15 are members of the smaller VH3b subfamily. Restriction is evident by the shared use of certain VDJ joints in independent hybridomas from different subjects. Two hybridomas generated from the same subject demonstrate identical heavy chain variable region gene sequences but differ in isotype and rearrange alternative light chain variable region genes (IgVL), suggesting that in a normal immune response, a single pre-B cell clone may use different light chain rearrangements and give rise to progeny capable of reacting with antigen. Using a polymerase chain reaction assay optimized to detect base pair differences among VH genes we demonstrate that at least a portion of expressed anti-Hib PS VH genes have undergone somatic mutation. Anti-Hib PS heavy chain genes are homologous to VH segments encoding autoantibodies and two hybridomas secrete anti-Hib PS antibody that cross-reacts with self antigens (double-stranded DNA and single-stranded DNA). Comparison of VH regions of self-reactive and monospecific anti-Hib PS Ab demonstrates no consistent structural feature correlating with fine antigen specificity. These data demonstrate significant restriction in VH usage and VDJ recombination in the anti-Hib PS response and confirm that autoantibodies may be elicited during normal immune responses

The response to the capsular polysaccharide of Haemophilus influenzae type b (Hib PS) has been used to determine the molecular basis of antibody gene diversity in humans. In contrast to the relatively restricted nature of anti-Hib PS heavy-chain variable region gene expression, a variety of light-chain variable region genes may encode this antibody (Ab) response. Light-chain variable region gene usage appears to determine the expression of certain Ab idiotypes and fine antigen specificity. To further define the role of light-chain variable region gene usage in important anti-Hib PS Ab subgroups, we have cloned and sequenced a number of immunoglobulin light-chain variable region genes (IgVL) from human monoclonal IgA anti-Hib PS Ab generated in response to Hib PS-protein conjugate vaccines. Three of these Ab are encoded by unusual variable segments. One kappa-Ab is encoded by the 'predominant' V kappa II A2 germline gene but, in contrast to a previously reported A2-encoded IgVL sequence, differs from the A2 germline sequence. The IgVL sequence of a second Ab is the only sequence of a kappa-Ab that cross-reacts with the structurally related antigen Escherichia coli K100 polysaccharide reported to date. This IgVL is encoded by a V kappa III-segment most closely homologous to the Humhv328/L16 germline gene, whereas previous reports suggested V kappa III-encoded anti-Hib PS Ab might be exclusively encoded by the germline gene Humhv325/A27.(ABSTRACT TRUNCATED AT 250 WORDS)

Translocation between the c-myc protooncogene and one of the three immunoglobulin loci is a cytogenetic hallmark of the B cell tumor, Burkitt's lymphoma. The resulting deregulation of c-myc expression is a critical step in tumorigenesis. The translocation breakpoint may lie within c-myc proper, in which case deregulation is due, in part, to dissociation of key 5' regulatory sequences from the protein-coding portions of the gene. Alternatively, the breakpoint may flank c-myc, leaving the gene grossly intact. In these latter cases, mutation, which may be extensive, is usually seen within c-myc, specifically at or near the same key regulatory sequences. The precise contribution of these mutations to c-myc deregulation is gradually being clarified. The mechanisms underlying c-myc mutations are not known. Hypermutation in c-myc may reflect the influence of the juxtaposed immunoglobulin gene, which is subject to hypermutation during an intermediate stage of normal B lymphoid development. This relationship, however, has not been firmly established