Faculty Bibliography

Amplification of the N-myc gene is a significant adverse prognostic factor in neuroblastoma, a common childhood tumor. In non-transformed cells, myc expression is controlled through an autoregulatory circuit, through which elevated Myc protein levels lead to down-regulation of myc transcription. The precise mechanism of myc gene autoregulation is unknown. Loss of c-myc autoregulation has been documented in transformed cells from a number of different lineages, but N-myc autoregulation has not yet been investigated. In neuroblastoma, the increased N-Myc protein produced by amplified tumors would be expected to silence N-myc transcription if the autoregulatory loop were intact. To determine whether N-myc autoregulation is operative in human neuroblastoma, and to localize cis-acting elements which mediate N-myc autosuppression, we transfected a series of N-myc 5' promoter constructs into a panel of human neuroblastoma cell lines carrying one or multiple copies of N-myc. The transfected promoter was equally active in single-copy and amplified lines. Significant promoter activity in the presence of abundant Myc protein in amplified neuroblastoma lines indicates that autoregulation is disabled in this subset of tumors. To investigate whether single-copy lines produce insufficient N-Myc protein to trigger autosuppression yet retain an intact autoregulatory circuit, we transfected neuroblastoma lines with 5' promoter constructs in the presence of a c- or N-myc expression vector. Overexpression of c- or N-Myc resulted in diminution of activity of both the transfected promoter and the endogenous N-myc gene in single-copy, but not amplified lines. Using a series of 5' promoter-deletion minigenes, we localized a cis-acting element required for autoregulation close to the transcription start sites. While the precise mechanism of autosuppression remains unknown, we demonstrated that Myc is incapable of silencing the adenovirus major late promoter (AdMLP) in neuroblastoma cells, indicating that Myc suppression of its own promoter and the AdMLP involve distinct components. These studies provide the first systematic investigation of autoregulation in neuroblastoma, and indicate that single-copy neuroblastoma lines produce insufficient N-Myc protein to activate downstream effector(s) of autosuppression; the autoregulatory circuit is otherwise intact. Amplified lines, in contrast, have lost autoregulation

Interleukin-10 (IL-10) downmodulates phagocytic immune responses and accentuates humoral responses. Human neonates exhibit broad immune deficits that parallel actions of IL-10. We postulated that IL-10 production would be diminished in neonatal blood cells. We found that IL-10 production by lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMNCs) in vitro was greater by adult cells than by term cells and preterm cells. Additional studies were undertaken to identify mechanisms responsible for the developmental differences in IL-10 gene expression. IL-10 transcription was present in freshly isolated adult and neonatal cells in the absence of detectable levels of transcript. Transcription rates were not different between adult and neonatal cells. IL-10 transcripts were approximately 40% more abundant in adult cells than in term cells and were consistent with differences in secreted protein; however, no differences were noted in mRNA stability. IL-10 half-life was 60 minutes for both adult and term PBMNCs. We conclude that up-regulation of IL-10 gene expression in PBMNCs is modulated at the post-transcriptional level, that IL-10 protein production and mRNA content are greater in activated cells from adults compared with those from neonates, and that maturational differences in IL-10 expression are not due to differences in transcription rate or mRNA stability. Maturational differences in IL-10 expression might be due to differences in subpopulations of cytokine-producing cells or differences in nucleo-cytoplasmic transport

Somatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis-acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti-NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5' IgH sequences, a rearranged VDJ region, the 5' matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3' IgH sequences (an alpha constant region and the 3' enhancer) and either the original VDJ segment or a substituted T cell receptor beta segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3' enhancer

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