Biomedical subjects

G Siebenkotten

Publications and source records attributed to G Siebenkotten.

6 recordsLinked to original sources

PubMed1999

Sensitive analysis of recombination activity using integrated cell surface reporter substrates.

BACKGROUND: Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures. METHODS: Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. RESULTS: Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch recombination and permitted the detection of recombination events down to 10(-5) per cell and generation. CONCLUSIONS: The high sensitivity of this system allows precise detection of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.

Animals↗

PubMed1999

An extrachromosomal switch recombination substrate reveals kinetics and substrate requirements of switch recombination in primary murine B cells.

Ig class switch recombination occurs in B lymphocytes upon activation, and is targeted to distinct switch (S) regions by cytokine-mediated induction of switch transcripts spanning the entire S region and the adjacent constant region gene segments. Using a novel type of switch recombination substrate, constructed according to the intron-exon structure of the IgH locus, but with heterologous elements, we here have tested the structural requirements for targeting and the kinetics of switch recombination in activated primary murine B cells. When transfected at various times after activation, up to 10% of the transfected B cells perform recombination of the substrate within 12 h. Switch recombination in primary B cells is restricted to the first 72 h after onset of activation, then rapidly decreases to background levels, as obtained in plasmacytoma cells or with substrates carrying no S region sequences. In terms of structural requirements, switch recombination is targeted to any transcription unit that contains an intronic S region and depends on processing of the primary transcript by splicing.

Animals↗

PubMed1998

Processing of switch transcripts is required for targeting of antibody class switch recombination.

Antibody class switching is mediated by somatic recombination between switch regions of the immunoglobulin heavy chain gene locus. Targeting of recombination to particular switch regions is strictly regulated by cytokines through the induction of switch transcripts starting 5' of the repetitive switch regions. However, switch transcription as such is not sufficient to target switch recombination. This has been shown in mutant mice, in which the I-exon and its promoter upstream of the switch region were replaced with heterologous promoters. Here we show that, in the murine germline targeted replacement of the endogenous gamma1 promoter, I-exon, and I-exon splice donor site by heterologous promoter and splice donor sites directs switch recombination in activated B lymphocytes constitutively to the gamma1 switch region. In contrast, switch recombination to IgG1 is inhibited in mutant mice, in which the replacement does not include the heterologous splice donor site. Our data unequivocally demonstrate that targeting of switch recombination to IgG1 in vivo requires processing of the Igamma1 switch transcripts. Either the processing machinery or the processed transcripts are involved in class switch recombination.

Adenoviridae↗

PubMed1997

Organization of the equine immunoglobulin constant heavy chain genes. I. c epsilon and c alpha genes.

We provide a restriction map of the equine c epsilon and c alpha genes as a molecular basis for isotype classification. Human and murine DNA probes were used for identification of homologous equine DNA sequences and for isolation of the equine c epsilon and c alpha genes from a genomic DNA library. A detailed map of the equine 5'-s epsilon/c epsilon-s alpha/c alpha-3' gene region was obtained. Equine c epsilon and c alpha DNA probes were prepared and used for restriction analysis of immunoglobulin heavy chain gene loci from different horses. This analysis indicated the presence of only one equine c epsilon and one c alpha gene in the haploid equine genome. In addition, for the equine c alpha gene, four haplotypes were identified according to BamHI restriction fragment length polymorphism (RFLP) of genomic DNA. The relative location of the c epsilon and c alpha genes 3' of the equine c mu and c gamma genes was determined by restriction analysis of equi-murine heterohybridomas.

Animals↗

PubMed1994

Frequency of immunoglobulin E class switching is autonomously determined and independent of prior switching to other classes.

Both, in humans and in mice, a major fraction of immunoglobulin E (IgE)-expressing B lymphocytes develops by sequential Ig class switching from IgM via IgG to IgE. This sequential class switch might have functional implications for the frequency and repertoire of IgE+ cells. Here we show that in mutant mice, in which sequential switching to IgE via IgG1 is blocked, the frequency of cells switching to IgE is not affected. Thus, sequential class switching to IgE merely reflects the simultaneous accessibility of two acceptor switch regions for switch recombination, induced by one cytokine, but with markedly distinct efficiency. Analysis of switch recombination on both IgH alleles of switched cells shows that the low frequency of switching to IgE is an inherent feature of the S epsilon switch region and its control elements.

Animals↗

PubMed1992

The murine IgG1/IgE class switch program.

Immunoglobulin class switching is controlled by cytokines. Thus, interleukin-4 (IL-4) directs class switching to both IgG1 and IgE. Consistent with this are the results reported here on restriction endonuclease analysis of active and inactive alleles of the IgH locus in IgE-producing cells. In cells that were stimulated in vitro by lipopolysaccharide and IL-4 the silent alleles preferentially switched to gamma 1, whereas in cells that were stimulated by antigen in vivo both active and inactive alleles switched to epsilon. Thirty percent of the recombined switch regions (S mu/S epsilon) contain S gamma 1 sequences, which we interpret as footprints of a previous switch to gamma 1. Since this percentage is a minimum estimate, between 30% and 100% of switching to epsilon must occur sequentially via gamma 1.

Animals↗