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    DNA Sequences Involved in Immunoglobulin Germ-line C [alpha] Gene Transcription: a Thesis

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    Authors
    Lin, Yi-chaung A.
    UMass Chan Affiliations
    Graduate School of Biomedical Sciences, Immunology
    Document Type
    Doctoral Dissertation
    Publication Date
    1992-06-01
    Keywords
    Base Sequence
    DNA
    Transcription, Genetic
    Academic Dissertations
    Life Sciences
    Medicine and Health Sciences
    
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    Abstract
    Expression of germ-line α transcripts precedes class switching to IgA, and therefore study of the regulation of germ-line α RNA transcription is important for understanding the class switching process. Transforming growth factor β1 (TGFβ1) increases the transcription of the Ig constant region a gene and class switching to IgA in normal B cells and in the I.29μ B lymphoma cell line. The structure of germ-line α transcripts in I.29μ cells was analyzed by RNase protection and primer extension assays. Two initiation sites for germ-line α transcripts were identified 2 kb upstream to the α switch region. No TATA or Sp1 elements are found near the RNA initiation sites. The DNA segment located 5' to the initiation sites of germ-line α RNA can drive expression of a luciferase reporter gene when transiently transfected into I.29μ (subclone 22D) and A20.3 cell lines. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. In deletion and substitution mutation studies, an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line α promoter, but mutation of this motif does not diminish TGFβ1 inducibility. Induction by TGFβ1 requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemlyrepeated sequence 5' CACAG(G) CCAGAC 3' (termed Igα TGFβ-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats could confer TGFβ1 inducibility to a heterologous promoter. An additional copy of the TGFβ-RE was identified at -41/-30 and its deletion reduced the TGFβ1 response. Thus, tandem repeats of a novel TGFβ-RE are the positive regulatory elements for the TGFβ1 response. Gel mobility shift assays demonstrated specific binding to the TGFβ-RE by nuclear factors but the binding activity was not enhanced by TGFβ1. This study supports previously published evidence that TGFβ1 directs class switching to IgA through induction of germ-line Cα gene transcription.
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/31551
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