Figure 1.

Effects of CSB-containing element on the expression of the CAT gene. (A) Schematic diagram of constructs used for the assays. pA10CAT2 is a fragment containing the partial SV40 promoter, the CAT gene, and the poly(A) site. It was inserted into pBluescript KS(−), and the resultant plasmid (pCT40) was used as the basic CAT vector. Mouse CSB is a CSB-containing element obtained by restriction enzyme digestion of mouse genomic DNA. The solid bar indicates the actual location of CSB in this fragment DNA. C alpha enhancer corresponds to a PvuII–BglII fragment containing the previously identified TCR α enhancer. The line below indicates the position of CSB and TCR α enhancer within the 95 kb of the mouse Cα/Cδ region. (B) Up-regulation of TCR α enhancer activity in αβ-expressing T cells. Constructs were electroporated into EL4 cells. The cells were harvested and samples were normalized with respect to total protein prior to performing the CAT assay. The acetylated chloramphenicol levels were measured qualitatively by autoradiography and quantitatively by a phosphoimager. The quantitative values are given in parenthesis below. Lane 1, assay blank (0); lane 2, E. coli CAT as a positive control (1,350); lane 3, baseline construct (pCT40) (16); lane 4, CSB in front of CAT gene (pCT44B) (12); lane 5, CSB in back of CAT gene (pCT45B) (0); lane 6, α enhancer in front (pCT46B) (176); lane 7, α enhancer in back (pCT47B) (74); lane 8, CSB in front, α enhancer in back (pCT48A) (90); lane 9, α enhancer in front, CSB in back (pCT49A) (333); lane 10, SV40 enhancer in front (138); and lane 11, mock transfection control (0). Chl indicates unacetylated [¹⁴C]chloramphenicol; Ac-Chl indicates acetylated [¹⁴C]chloramphenicol.