Fine-structure analysis of the processing and polyadenylation region of the herpes simplex virus type 1 thymidine kinase gene by using linker scanning, internal deletion, and insertion mutations.

Zhang, Fang; Denome, Roger; Cole, Charles N.

doi:10.1128/mcb.6.12.4611

Cited by 41 publications

(45 citation statements)

References 38 publications

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“…It is likely that the apparent contradictions are attributable to differences in plasmid constructions and experimental designs. Significantly, no small linker scanning mutation within the more 5'-proximal element could prevent in vitro cleavage (57), a result similar to that found with downstream elements from other polyadenylation signals (30,58). This result suggests that the downstream regions within polyadenylation signals may contain functionally redundant elements (57).…”

Section: Discussionsupporting

confidence: 66%

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Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

Carswell¹,

Alwine²

1989

Mol. Cell. Biol.

169

141

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The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.Most eucaryotic mRNAs are cleaved postranscriptionally at a specific site in the 3' untranslated region between 10 and 30 nucleotides downstream of a consensus AAUAAA sequence (42). Subsequent to cleavage, a polyadenosine chain with an average length of about 200 to 300 nucleotides is covalently attached to the RNA at the cleavage site (reviewed in reference 8). The purpose of 3'-end cleavage and polyadenylation is not clearly understood. However, studies with mutants have implicated polyadenylation as an essential process for the production of stable mRNA (reviewed in reference 5). In the present study, we compared the relative efficiencies of polyadenylation at the simian virus 40 (SV40) early and late polyadenylation sites. We found that the late polyadenylation signal is substantially more efficient than the early one in vivo. The findings in this work support the existence of a gene expression mechanism by which the relative efficiency of polyadenylation site utilization affects the steady-state levels of mRNA. We also found that the SV40 late polyadenylation signal contains an unusual element upstream of its AAUAAA hexanucleotide that is required for efficient usage of the late polyadenylation site.SV40 is a temporally regulated DNA virus, with early and late phases of infection. During a lytic infection of permissive monkey cells, early RNA is transcribed and the major early protein, T antigen, is made. T antigen is a multifunctional protein that enables replication of viral DNA as well as down-regulation of its own transcription (reviewed in reference 1) and activa...

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Section: Discussionsupporting

confidence: 66%

“…7). Previous work has shown that readthrough RNAs accumulate as products of primary transcripts when polyadenylation is inefficient (9,27,30,48,58). An absence of increased levels of these large RNAs in mutant transfections might, thus, indicate that mutant primary transcripts are less stable than wild-type primary transcripts.…”

Section: Discussionmentioning

confidence: 99%

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

Carswell¹,

Alwine²

1989

Mol. Cell. Biol.

169

141

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“…2B) was generally conserved across eukaryotes, even though in budding yeast greater variation in these cis elements appears to be tolerated (Graber et al 1999). One notable feature of PAS is that, depending on exactly how the AAUAAA and DSE signals are positioned and defined, the actual site of poly(A) addition can vary by several to tens of nucleotides (Zhang et al 1986;Sheets et al 1990;Tian et al 2005). This is especially evident in yeast PAS, which, perhaps due to lower sequence conservation, do not accurately define the position of polyadenylation (Zhao et al 1999).…”

Section: Polyadenylation Signals and 39 Noncoding Rna (Ncrna) Sequencesmentioning

confidence: 99%

Ending the message: poly(A) signals then and now

Proudfoot¹

2011

Genes Dev.

520

486

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Polyadenylation [poly(A)] signals (PAS) are a defining feature of eukaryotic protein-coding genes. The central sequence motif AAUAAA was identified in the mid1970s and subsequently shown to require flanking, auxiliary elements for both 39-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination. More recent genomic analysis has established the generality of the PAS for eukaryotic mRNA. Evidence for the mechanism of mRNA 39-end formation is outlined, as is the way this RNA processing reaction communicates with RNA polymerase II to terminate transcription. The widespread phenomenon of alternative poly(A) site usage and how this interrelates with pre-mRNA splicing is then reviewed. This shows that gene expression can be drastically affected by how the message is ended. A central theme of this review is that while genomic analysis provides generality for the importance of PAS selection, detailed mechanistic understanding still requires the direct analysis of specific genes by genetic and biochemical approaches.

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“…The common 3' terminus of the US10/ll/12 transcripts lies approximately 160 bp from the XbaI site in 1802 DNA. This reduction is unlikely to be a direct effect of the introduced sequences on mRNA processing since the signals required for efficient 3' end formation are contained within 40 to 50 bp beyond the poly(A) site of HSV mRNAs Zhang et al, 1986). One possibility is that transcriptional readthrough from the inserted sequences in vFJ7 and vFJl0 could lower the amount of USll mRNA available for translation either by production of anti-sense mRNA or transcriptional interference.…”

Section: Discussionmentioning

confidence: 99%

Insertion of DNA Sequences at a Unique Restriction Enzyme Site Engineered for Vector Purposes into the Genome of Herpes Simplex Virus Type 1

Rixon¹,

McLauchlan²

1990

Journal of General Virology

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We describe the construction of a novel herpes simplex virus (HSV) vector containing a unique XbaI restriction enzyme cloning site in an intergenic position in the short unique genome region. Sequences can be inserted at this site with high efficiency by ligation with Xbaldigested vector DNA. A series of plasmids has been designed for use with this vector. These allow protein coding sequences to be placed under the control of various transcriptional regulation signals and then isolated as XbaI fragments ready for insertion into the vector. The XbaI fragments also contain the /~-galactosidase gene thereby facilitating selection of recombinant virus by screening for blue plaques. A variant of the vector has been made based on the temperature-sensitive (ts) mutant tsK, which expresses only immediate early (IE) genes at non-permissive temperatures. Chloramphenicol acetyltransferase was used as a reporter gene to assess the fidelity of expression of sequences cloned into this position. Under these circumstances IE and early HSV promoters were shown to behave as expected in both wild-type and ts vectors.

show abstract

Fine-structure analysis of the processing and polyadenylation region of the herpes simplex virus type 1 thymidine kinase gene by using linker scanning, internal deletion, and insertion mutations.

Cited by 41 publications

References 38 publications

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

Ending the message: poly(A) signals then and now

Insertion of DNA Sequences at a Unique Restriction Enzyme Site Engineered for Vector Purposes into the Genome of Herpes Simplex Virus Type 1

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