Requirement for the 3' flanking region of the bovine growth hormone gene for accurate polyadenylylation.

Abstract

We examined whether the sequence extending 3' to the polyadenylylation site of the bovine growth hormone gene contains any signal that affects the polyadenylylation of the growth hormone mRNA. For this purpose, cloned copies of this gene, each containing a different length of growth hormone-specific sequence 3' to the wild-type polyadenylylation site, were used to transfect COS-1 cells. The polyadenylylation site on the mRNAs produced from the exogenously added growth hormone genes were analyzed with an S1 nuclease mapping procedure. We found that a gene containing 84 base pairs of its own 3' flanking sequence is capable of producing an accurately polyadenylylated mRNA. On the other hand, genes containing only 1, 10, or 13 base pairs of 3' flanking sequence were principally polyadenylylated at discrete sites either upstream or downstream from the wild-type position. Using a computer program, we examined whether secondary structures on the primary growth hormone transcript correlated with the site where the mRNA is polyadenylylated.

Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.

“…Even when the AATAAA signal was retained in the ADA cDNA, use of the polyadenylation signal contributed by the SV40 vector predominated. This may be due, in part, to the nature of the sequences located 3' to the cDNA insert, as it is now apparent that downstream sequences affect the efficiency with which putative poly(A) addition sites are utilized (23,41). …”

Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites.

“…In addition, it is possible that there are even more elements since in one series of constructions, sequences distal to +43 could restore activity. (29).…”

Different 3' End Regions Strongly Influence the Level of Gene Expression in Plant Cells

“…In animal cells, formation of most mRNA 3' ends involves endonucleolytic cleavage of larger precursors followed by the addition of up to 250 adenylate residues [the poly(A) tract] (for review, see Birnstiel, Busslinger, and Strub, 1985;Proudfoot and Whitelaw, 1987). Extensive deletion analysis and site-specific mutagenesis have defined two cis-acting elements that are both essential for accurate and efficient 3' end formation: the highly conserved AAUAAA, located 10 nucleotides to 30 nucleotides upstream from the poly(A) addition site (Proudfoot and Brownlee, 1976;Fitzgerald and Shenk, 1981;Montell et al, 1983;Wickens and Stephenson, 1984) and the less conserved (G)T-rich sequences immediately 3' to the cleavage site (Gil and Proudfoot, 1984;Woychik et al, 1984;Hart, McDevitt, and Nevins, 1985;McLauchlan et al, 1985;McDevitt et al, 1986;Gil and Proudfoot, 1987). In addition to these sequences, the 3'-untranslated region can also contain regulatory sequences that affect the mRNA stability.…”