Common RNA polymerase I, II, and III upstream elements in mouse 7SK gene locus revealed by the inverse polymerase chain reaction

Abstract

7SK RNA in mammalian cells is derived from a gene or genes belonging to a middle-repetitive family in the genome. Standard library search techniques applied to isolating such genes are complicated by the finding of multiple truncated or otherwise modified versions of the sequence, whereas the true gene loci can sometimes be eliminated from amplified libraries. After an unsuccessful search for the 7SK RNA gene in four mouse genomic libraries, we used the inverse polymerase chain reaction (IPCR) on fractionated genomic DNA to characterize sequences containing complete copies of 7SK plus flanking regions for analysis of putative transcription regulatory sequences. Direct sequence of IPCR-amplified material allowed for selection of upstream and downstream primers which could then be used for direct PCR, sequencing, and characterization of the mouse 7SK gene locus. So far, we found only one complete copy of the canonical 7SK gene that differed from the human sequence in only 4 bases. The gene is flanked by a very well-conserved upstream control region that includes a TATA motif, two direct repeats, and a proximal sequence element common to mammalian genes transcribed by all three RNA polymerases. The 3' region contains multiple stretches of T residues, typical of class III terminators.