- Collection:
- Atlanta University and Clark Atlanta University Theses and Dissertations
- Title:
- Isolation and characterization of cDNAs differentially expressed between Cd8+ and Cd4+ T cell lines, 2002
- Creator:
- Adon, Arsene M.
- Date of Original:
- 2002-05-01
- Subject:
- Degrees, Academic
Dissertations, Academic - Location:
- United States, Georgia, Fulton County, Atlanta, 33.749, -84.38798
- Medium:
- theses
- Type:
- Text
- Format:
- application/pdf
- Description:
- To detect novel molecules involved in immune functions, a subtracted cDNA library between two closely related murine lymphoid cell lines was constructed using the suppression subtractive hybridization (SSH) technique. Both cells are cytotoxic T lymphocytes, however the 2C is a CD8+ cytotoxic T cell line and the 5.9 is an inflammatory CD4+T cell line (Thl). They can both secrete the cytolysin or perforin and are significantly resistant to cell-mediated lysis. When these two cell lines were subjected to prolonged exposure to reagents that deplete cells of ATP (2-deoxyglucose, sodium azide, and potassium cyanide), the 5.9 cell line became substantially susceptible, but the 2C and other CD8+ cell line still stood out as being strikingly resistant to granule- mediated lysis. A modified differential screening of colonies randomly picked from the constructed subtractive cDNA library and RNA blot analysis were used to identify cDNA clones expressed in 2C and/or 5.9 cytotoxic T cell lines. Thirteen cDNA clones were isolated, from which seven were expressed in both cell lines, and only two were expressed in 2C but not in 5.9 cell line. All the isolated cDNA clones were sequenced. A search of the Gene Bank database with the BLAST X program revealed no extensive homology of IB and 3F cDNA clones to known genes. Limited homology respectively to a zinc finger motif and the transcription factor MTF-1 were observed. Also RT-PCR analysis of cells purified from primary mixed lymphocyte reaction, demonstrated that both IB and 3F cDNA clones were expressed only in CD8+ but not in CD4+ purified cells. This approach of employing subtraction coupled with partial cDNA sequence determination can be useful for a first selection of putative functionally relevant molecules.
- External Identifiers:
- Metadata URL:
- http://hdl.handle.net/20.500.12322/cau.td:2002_adon_arsene_m
- Rights Holder:
- Clark Atlanta University
- Holding Institution:
- Atlanta University Center Robert W. Woodruff Library
- Rights:
-
