Characterized bGH 3'-flanking sequence requirements showing that bGH polyA signal contains a diffuse efficiency element rather than discrete GU- or U-rich motifs found in other signals. Deletion and block mutation analysis revealed that bGH polyadenylation efficiency depends on distributed sequence elements, establishing molecular basis for its unique properties and widespread use in expression vectors.
The 3'-flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation
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Key findings
Abstract
In addition to the conserved AAUAAA hexanucleotide, GU- and U-rich sequences in the 3'-flanking region are thought to be critical for efficient polyadenylation. The 3'-flanking sequence requirements for efficient and accurate polyadenylation of the bovine growth hormone (bGH) gene were determined by quantitative S1 nuclease analysis of transcripts derived from various bGH 3' deletions and block mutations transiently transfected into COS-1 cells. Though the bGH 3'-flanking sequence contains a portion of the putative GU efficiency element, we find that mutation of this element leads to a marginal decrease in efficiency similar to that from mutation of other sequences that do not contain recognizable GU- or U-rich motifs. The data are consistent with a diffuse efficiency element in the bGH polyadenylation signal rather than a discrete element as is thought to exist in other mammalian signals. We have also determined that a region from 18 to 27 nucleotides downstream of the cleavage site contains sequences required for correctly positioning the cleavage site.