المؤلفون: Jean-Marie Buerstedde, Vera Batrak, Artem Blagodatski

المصدر: Methods in Molecular Biology ISBN: 9781617791284

مصطلحات موضوعية: Affinity maturation, Genetics, Reporter gene, Point mutation, Somatic hypermutation, Locus (genetics), Biology, Immunoglobulin light chain, Gene, Green fluorescent protein

الوصف: The immunoglobulin (Ig) genes of B cells are diversified at high rate by point mutations whereas the non-Ig genes of B cells accumulate no or significantly fewer mutations. Ig hypermutations are critical for the affinity maturation of antibodies for most of jawed vertebrates and also contribute to the primary Ig diversity repertoire formation in some species. How the hypermutation activity is specifically targeted to the Ig loci is a long-standing debate. Here we describe a new experimental approach to investigate the locus specificity of Ig hypermutation using the chicken B-cell line DT40. One feature is the use of a green fluorescent protein (GFP) gene as a mutation reporter. Some nucleotide changes produced by somatic hypermutation can cripple the GFP gene which leads to a decrease or loss of the green fluorescence. Therefore such changes can be easily quantified by fluorescence-activated cell sorting (FACS). Another advantage of this approach is the targeted integration of the mutation reporter into a defined chromosomal position. This system allowed us to identify a 10 kb sequence within the Ig light chain (IgL) locus, which is both necessary and sufficient to activate hypermutation in the neighboring reporter gene. We have called this sequence Diversification Activator (DIVAC) and postulated that similar cis-acting sequences exist in the heavy and light chain Ig loci of all jawed vertebrate species. Our experimental system promises further insight into the molecular mechanism of Ig hypermutation. For example, it may be possible to identify smaller functional motifs within DIVAC and address the role of putative transacting binding factors by gene knock-outs.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::993d43407b78dfcb187bc0810a1156c3Test
https://doi.org/10.1007/978-1-61779-129-1_18Test

المؤلفون: Artem Blagodatski, Jean-Marie Buerstedde, Sabine Schmidl, Hiroshi Arakawa, Ulrike Schoetz, Vera Batrak, Randolph B. Caldwell

المصدر: PLoS Genetics
PLoS Genetics, Vol 5, Iss 1, p e1000332 (2009)
PLoS Genet. 5:e1000332 (2009)

مصطلحات موضوعية: Cancer Research, lcsh:QH426-470, Green Fluorescent Proteins, Somatic hypermutation, Locus (genetics), Regulatory Sequences, Nucleic Acid, Transfection, Transcription (biology), Genes, Reporter, Cytidine Deaminase, Genetics, Activation-induced (cytidine) deaminase, Animals, Insertion, Gene, Molecular Biology, Genetics (clinical), Ecology, Evolution, Behavior and Systematics, AID, B cell, DT40, enhancer, gene conversion, immunoglobulin, somatic hypermutation, transcription, B-Lymphocytes, biology, Activator (genetics), Chromosome Mapping, Genetic Variation, Cytidine deaminase, Sequence Analysis, DNA, Cell Biology, Flow Cytometry, lcsh:Genetics, Gene Expression Regulation, Mutation, biology.protein, Genetics and Genomics/Genetics of the Immune System, Genes, Immunoglobulin Light Chain, Somatic Hypermutation, Immunoglobulin, Immunology/Genetics of the Immune System, Chickens, Gene Deletion, Research Article, Biotechnology

الوصف: Hypermutation of the immunoglobulin (Ig) genes requires Activation Induced cytidine Deaminase (AID) and transcription, but it remains unclear why other transcribed genes of B cells do not mutate. We describe a reporter transgene crippled by hypermutation when inserted into or near the Ig light chain (IgL) locus of the DT40 B cell line yet stably expressed when inserted into other chromosomal positions. Step-wise deletions of the IgL locus revealed that a sequence extending for 9.8 kilobases downstream of the IgL transcription start site confers the hypermutation activity. This sequence, named DIVAC for diversification activator, efficiently activates hypermutation when inserted at non-Ig loci. The results significantly extend previously reported findings on AID-mediated gene diversification. They show by both deletion and insertion analyses that cis-acting sequences predispose neighboring transcription units to hypermutation.
Author Summary It remains an open question how AID-mediated gene diversification is targeted to the immunoglobulin loci. Here we define a cis-acting sequence, named DIVAC for diversification activator, which is required for hypermutation of the Ig light chain gene and sufficient to activate hypermutation at various non-Ig loci in the DT40 B cell line. DIVAC is composed of multiple interacting sequences and able to work over considerable distances both upstream and downstream of its target gene. This work provides the first conclusive evidence that AID-mediated gene diversification is targeted to the Ig loci by cis-acting sequences. The conservation of AID-mediated Ig gene diversification during vertebrate evolution suggests that DIVACs also play a role in gene conversion, hypermutation, and switch recombination in mammalian B cells. The findings should be of general interest not only for molecular immunology and the pathogenesis of B cell lymphomas but also the whole field of biology as a unique example of how locus-specific gene diversification is controlled. The described experimental system offers unique advantages to further clarify the molecular mechanism of DIVAC.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3c9303381e5327b68fc6465265d13502Test
https://pubmed.ncbi.nlm.nih.gov/19132090Test