Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene.

Cole, Charles N.; Santangelo, George M.

doi:10.1128/mcb.3.2.267

Cited by 45 publications

(39 citation statements)

References 74 publications

(61 reference statements)

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“…Whether these internal AAUAAA's are not recognized as signals for polyadenylation because they are not in the proper nucleotide sequence context (i.e., additional nucleotides are required or nucleotides actually present nearby are inhibitory) or, alternatively, because the secondary structure of the RNAs is such that nonutilized AAUAAA's are not accessible to the poly(A) polymerase complex, is not yet clear. Support for the later notion is provided by the observation that when a segment of SV40 DNA encoding the internal, unused AAUAAA sequence sequence mentioned above was transferred to the 3' end of a gene from which the normal polyadenylation signal had been deleted, the cryptic AAUAAA signal was now apparently used (7).…”

Section: Discussionmentioning

confidence: 96%

RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

Manley¹,

Yu²,

Ryner³

1985

Mol. Cell. Biol.

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To determine whether a specific nucleotide sequence is required to direct polyadenylation of a simian virus 40 early pre-mRNA in a soluble HeLa whole-cell lysate, we constructed a series of rearranged and deleted DNA templates, transcribed them in vitro, and determined whether the resultant RNAs could be polyadenylated when incubated in whole-cell lysate. When a 237-base-pair DNA fragment encoding the 3' end of the simian virus 40 early pre-mRNA was transferred to recombinant plasmids encoding RNAs that were not substrates for polyadenylation, the resultant RNAs could now be polyadenylated efficiently. In one case, the chimeric RNA was polyadenylated even more efficiently than was the original simian virus 40 early transcript. Analysis of the RNAs produced from the deletion mutant templates revealed that only RNAs containing at least one copy of the AAUAAA sequence situated near the 3' end and implicated in 3'-end formation and polyadenylation in vivo could be polyadenylated in vitro. Surprisingly, this sequence directed polyadenylation of pre-mRNAs not only when near the RNA 3' end, i.e., 50 nucleotides or less away, but also when the 3' end was situated over 400 nucleotides downstream. Thus, our results show that a polyadenylic acid polymerase activity in HeLa lysates can recognize a specific nucleotide sequence in pre-mRNA and then, in the absence of the nucleolytic cleavage that presumably occurs in vivo, locate the RNA 3' end and use it as a primer for polyadenylic acid synthesis.

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Section: Discussionmentioning

confidence: 96%

RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

Manley¹,

Yu²,

Ryner³

1985

Mol. Cell. Biol.

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“…This proposal gains some support from the demonstration that an 88-bp fragment from the middle of the coding region of SV40 large T antigen, containing the sequence AAUAAA which is not normally involved in transcriptional processing, can specify such processing when positioned behind the thymidine kinase gene (10), although it must be stressed that processing promoted in this manner occurs inefficiently. However, it is clear that the AATAAA hexanucleotide can lie unrecognized within the coding region of an mRNA, as in the middle of the early region of SV40 (18,40) and within the adenovirus type 12 Ela transcriptional unit (37).…”

Section: Discussionmentioning

confidence: 98%

“…Removal of the sequence specifying polyadenylation from the herpes simplex virus thymidine kinase gene has been shown to result in the abolition of thymidine kinase expression (10). Although the sequence 5'-TATAAA located at position 104 resembled the consensus eucaryotic polyadenylation signal, we sought confirmation that this hexanucleotide represented the sequence specifying polyadenyation of the surface antigen transcripts.…”

Section: Construction Of Vectorsmentioning

confidence: 99%

Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids.

Simonsen¹,

Levinson²

1983

Mol. Cell. Biol.

159

104

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We examined the transcription of the hepatitis B virus surface antigen (HBsAg) gene in COS cells transfected with simian virus 40-based recombinant plasmids. When positioned behind the simian virus 40 late promoter, three transcripts were identified which hybridized to the HBsAg gene: a 2,000-nucleotide transcript colinear with a gene, a 1,100-nucleotide transcript representing a spliced molecule in which a major portion of the sequences encoding HBsAg were deleted, and an 800-nucleotide transcript derived primarily from sequences 3' to the HBsAg gene. The splice acceptor site utilized by the 1,100-nucleotide transcript is located immediately upstream of an open reading frame of unknown function contained within the 3' nontranslated region of the HBsAg gene. The HBsAg-specific mRNA species terminate 12 to 19 base pairs 3' of the sequence UAUAAA, similar to the concensus hexanucleotide which is thought to promote polyadenylation (AAUAAA). We constructed a series of plasmids with progressive deletions from the region surrounding where these transcripts terminate. Analysis of mRNA produced by cells transfected with these plasmids indicated that the signal hexanucleotide is in itself unable to promote the efficient processing of mRNA in the absence of downstream hepatitis B virus sequences. Processing proceeds properly, however, from plasmids containing an additional 30 nucleotides 3' of this signal.

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“…4B; this structure is substantially less stable than the structure that is shown for the delta+84 transcript. 3. S1 nuclease mapping of the 3' termini of growth hormone mRNAs transcribed from the expression plasmids delta-37, delta+1, delta+10, delta+13, delta+84, and pSVB3/Ba.…”

Section: Discussionmentioning

confidence: 99%

“…This evolutionarily conserved hexanucleotide has been determined to be essential for the production of polyadenylylated mRNAs (2,3), and is an important signal for efficient processing of the 3' terminus of the primary RNA transcript (4,5). However, this sequence cannot be the only signal that regulates the polyadenylylation reaction because some genes contain additional A-A-T-A-A-A hexanucleotides that do not appear to function as polyadenylylation signals (6)(7)(8).…”

mentioning

confidence: 99%

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

Woychik¹,

Lyons²,

Post³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

110

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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.

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Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene.

Cited by 45 publications

References 74 publications

RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

RNA sequence containing hexanucleotide AAUAAA directs efficient mRNA polyadenylation in vitro.

Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids.

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

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