Abstract
Human cloned antigen-specific T lymphocytes were used to characterize DNA rearrangements using a cDNA probe from the T cell receptor (TCR)-alpha chain gene. Rearranged patterns were detected in some T cell clones, confirming that normal mature T cells are rearranged in TCR-alpha locus. Similarly, rearranged DNA patterns were found in T cell clones from the same panel, using a DNA probe (clone K-40, [1]) isolated from chromosome 14 (14q11), where the TCR-alpha locus has been mapped. These results suggest that this genomic DNA clone is located within the TCR-alpha chain locus.
Highlights
Materials and MethodsCloning was performed by micromanipulation, as described elsewhere [12]
In this report we show that DNA rearrangements in T-cell clones are detected by hybridization (a) with a T cell receptor (TCR)-a chain cDNA clone, and (b) with the K40 probe
The BJ T cell clones derived from an in vitro allostimulated IL-2-dependent T cell line, BJL, whereas the SA T cell clones were obtained by cloning cells directly from PBMC treated with rIFN-7 and rIL-2 (Bourge, Degos, and Bensussan, manuscript submitted for publication)
Summary
Cloning was performed by micromanipulation, as described elsewhere [12]. SA-K10, SA-1, and SA-6 clones derived from PBL of a healthy donor incubated with rIFN-3, (Biogen, Cambridge, MA) for 48 h before cloning and recloning, as previously described [13]. Cloning was performed by limiting dilution in Terasaki plates at 0.3 cell/well in the presence of 5% IL-2-conditioned medium, autologous irradiated feeder cells, and PHA (Wellcome, Beckenham, United Kingdom). PHA was used as a mitogenic stimulus because of its ability to provide high cloning efficiency and growth of T cells regardless of antigenic specificity. 3~p labelling of probes by nick-translation and Southern blot hybridization were done as previously described [16] A mixture of two probes corresponding to a 1.3 kb Xba fragment (K40-B) and a 1.5 kb Hind III fragment (K40-C) (see Fig. 2) was used. 3~p labelling of probes by nick-translation and Southern blot hybridization were done as previously described [16]
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