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Title: IgH allelic exclusion at the level of IgH transcription
Author: Daly, J. N.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2005
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Expression of a productivity rearranged immunoglobulin heavy (μH) chain from a first in-frame VDJH-rearranged allele prevents further recombinant of the second, DJH-rearranged, allele, termed heavy chain allelic exclusion. I investigated at the single cell level the transcriptional status of both IgH alleles in ex vivo cells using RNA fluorescent in situ hybridisation (FISH). Prior to exclusion, in predominantly DJH /DJH-rearranged pro/pre-B1 cells, both IgH alleles are transcribed and detected by RNA-FISH. In contrast, following productive VDJH-recombination and μH expression i.e. in allelically excluded pre-BII cells, mono-allele transcription was detected in the 40% and bi-allelic in 60% of cells. This 40:60 ratio was maintained throughout subsequent B cell developmental stages. A VH-specific transcription assay revealed a VH (J558) transcription co-localising with mono- and bi-allelic μ transcription sites, strongly indicating transcription from VDJH-rearranged alleles in allelically excluded B cells. Combined RNA/DNA FISH showed detection of the VH-DH intergenic regions in cells with mono-allelic μ-transcription. Within these nuclei the μ-transcription site and VH-DH intergenic region were clearly separate and thus do not originate from the same allele, thus suggesting a VDJH/DJH-rearrangement status. In the majority (>80%) of nuclei with two μ-transcription sites no VH-DH-intergenic regions were observed, suggesting VDJH/VDJH-recombination. Semi-quantitative RT-PCR revealed steady-state DJH-Cμ transcripts in splenic B cells, which have been allelically excluded at levels 2-3 fold less than observed in a developmental stage prior to exclusion. Thus the DJH-recombined allele is transcribed after exclusion. Our data show that the process of allelic exclusion results in diminished ability for RNA-FISH to detect the transcription of the DJH- but not the VDJH-rearranged alleles, indicating that the signal for allelic exclusion is received only by the DHJ-recombined alleles. Therefore, in allelically excluded cells, the DJH-but not VDJH-rearranged alleles alter their transcriptional activity and thus act as part of the mechanism for heavy chain allelic exclusion.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available