Rat gene 33: analysis of its structure, messenger RNA and basal promoter activity

Several overlapping cDNA and genomic DNA clones corresponding to mRNA g33 and gene 33, respectively, were isolated and characterized. The mRNA g33 molecule is 2971 nt in length, exclusive of a poly(A+) tail, and encodes a putative 459 amino acid protein (49,946 daltons). The 13.2 kbp gene consists of four exons, three introns, and has two initiation sites located 27 and 30 bp downstream from a TATA box. Transfection of H4IIE cells with a fusion gene 33 (-1900 to +32)/luciferase construct (pSL330A) gave rise to readily detectable luciferase activity. In addition, primer extension analysis of the gene 33/luciferase mRNA transcribed in these experiments showed that transcription initiates in the gene 33 DNA at two sites consistent with those found in vivo. Analysis of the effect of 5' deletions on basal promoter activity showed that, in relation to the promoter activity of pSL330A, approximately 40% of the activity is lost between -1643 and -1050, another approximately 40% between -550 and -475, and deletion to -55 causes a total loss of detectable luciferase activity.