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A structural and functional characterization of the alpha IIb gene locus.
- Format:
- Book
- Thesis/Dissertation
- Author/Creator:
- Thornton, Michael Anthony.
- Language:
- English
- Subjects (All):
- Cytology.
- Genetics.
- 0369.
- 0379.
- Penn dissertations--Cell and molecular biology.
- Cell and molecular biology--Penn dissertations.
- Local Subjects:
- Penn dissertations--Cell and molecular biology.
- Cell and molecular biology--Penn dissertations.
- 0369.
- 0379.
- Physical Description:
- 359 pages
- Contained In:
- Dissertation Abstracts International 62-02B.
- System Details:
- Mode of access: World Wide Web.
- text file
- Summary:
- The alphaIIb gene codes for a fibrinogen receptor required for normal hemostasis and is expressed preferentially in platelets and their precursors megakaryocytes. We have conducted a detailed analysis of both the structure and function of this gene locus. Our structural studies revealed that the halphaIIb and malphaIIb genes were surrounded by two conserved non-megakaryocytic, ubiquitously expressed genes, the Granulin gene ∼18 kb downstream to alphaIIb, and the KIAA 0553 gene ∼5.7 kb upstream. Homology plots between the human (h) and murine ( m) alphaIIb gene loci revealed conserved DNA sequences in the intergenic region between the alphaIIb gene and KIAA0553. DNase I hypersensitivity mapping of this same region localized four DNase I hypersensitivity sites (HS), which overlap with these conserved sequences. Two of the HS were tissue specific, being detected in the megakaryocyte cell lines, HEL and CHRF but not in epithelial HeLa cells. We additionally made transgenic mouse lines containing various lengths of the human alphaIIb gene locus. The initial construct, containing 2.5 kb of 5'-flanking region, all the halphaIIb exons/introns and 2.0 kb of 3' -flanking sequence, demonstrated low halphaIIb expression in RT-PCR studies, being 1.5 x 103-fold lower than endogenous malphaIIb expression and not copy number-dependent. However, expression was limited to bone marrow and platelets. Expression for constructs containing an additional 4.6 kb sequence upstream of the halphaIIb gene, was platelet-restricted, copy-number dependent and position-independent. However, this construct had a further decrease in expression, being 6 x 103-fold lower than endogenous malphaIIb expression. A third transgenic construct with an additional 5 kb of downstream sequence starting at alphaIIbs stop codon, expressed 3-fold higher on average than native malphaIIb and the halphaIIb protein could be detected by Western blot and by flow cytometric analysis. Functionally, it appears the 5' region between KIAA0553 and the alphaIIb gene is subdivided into two domains, the immediate 5'-promoter region defines tissue-specificity, but drives extremely low levels of expression; further upstream is an additional element that leads to copy number dependent- and position-independent expression. Whereas, the region downstream between alphaIIb and the Granulin gene, apparently contains a region analogous to an enhancer element required for high levels of expression in vivo.
- Notes:
- Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2001.
- Source: Dissertation Abstracts International, Volume: 62-02, Section: B, page: 0670.
- Supervisor: Mortimer Poncz.
- Local Notes:
- School code: 0175.
- ISBN:
- 9780493134512
- Access Restriction:
- Restricted for use by site license.
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