[Analysis of cell specific transcription of the human cone transducin alpha subunit gene]

Abstract

Cone transducin alpha subunit (Tc alpha), together with the two other components; beta and gamma subunits, composes a heterotrimeric G protein and transmits visual signals in the optic system. Genomic Tc alpha gene was cloned from human placental library using a cDNA probe isolated from a human T-cell cell line, Jurkat. The 5' region of Tc alpha was sequenced and the mechanism of Tc alpha expression was analysed by primer extension and CAT assay. Furthermore DNase 1 hypersensitivity test and genomic sequencing were performed. Primer extension revealed two transcription initiation sites. A TATA box and an inverted CCAAT box were identified just upstream of the transcription initiation sites, which constituted a typical promoter structure. In further upstream region, an octamer sequence (ATGCAAAT) and an inverted GGCCCC were found. The latter sequence is frequently present upstream of the genes expressed exclusively in retinal cells. No specific DNA element enhancing the expression of Tc alpha in Y79 cells (a Tc alpha expressing cell line) was found by CAT assay. However, DNaseI hypersensitivity test revealed the region to be in an active chromatin structure in Y79 as opposed to HeLa (a Tc alpha non-expressing cell line). By further study utilizing genomic sequencing, HeLa DNA was methylated in contrast to Y79, suggesting methylation as one of the transcription-regulating factors of this region.