CIS-ACTING DNA ELEMENTS AND CELL-TYPE-SPECIFIC NUCLEAR PROTEINS WHICHMAY PLAY A ROLE IN REGULATION OF MOUSE CD8-ALPHA (LYT-2) GENE-TRANSCRIPTION

Fusion of mouse CD8(+) class I MHC-restricted T cells with the BW5147 thymoma invariably yields CD8(-) hybridomas in which RNA transcribed f rom the CD8 alpha (Lyt-2) gene is undetectable. To determine whether c is-acting DNA sequences may negatively regulate transcription of the L yt-2 gene in BW5147 cells, one possible explanation for the above obse rvation, BW5147 cells were stably transfected with the Lyt-2 gene cont aining 1-11,000 nucleotides of 5' flanking DNA and surface expression of Lyt-2 was monitored by flow microfluorometry. Initial results sugge sted the presence of a negative element between 1400 and 5000 nucleoti des upstream of the site of transcription initiation. Further studies suggested the presence of two potential negative regulatory elements i n this region, one of which includes a 269 nucleotide Accl-Sstl fragme nt comprised of nucleotides -4700 to -4431 which bound nuclear protein s from CD8(+) and CD8(-) cell lines in electrophoretic mobility shift assays (EMSA). EMSA studies performed using nuclear extracts from a va riety of cell lines and tissues demonstrated that unique retarded comp lexes, called bands 1 and 2, correlated significantly with expression or non-expression of Lyt-2 respectively. EMSA analysis of proteins fra ctionated by SDS - PAGE from nuclear extract of the CD8(+) VL3 T lymph oma cell line revealed proteins of similar to 110-130 kDa (called L2a- P1) and > 200 kDa (called L2a-P2) which bind within a 100 nucleotide r egion of this fragment (called L2a) to yield band 1 and 2 respectively , and which may play a role in regulation of Lyt-2 gene transcription.