scholarly journals Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals.

1988 ◽  
Vol 8 (11) ◽  
pp. 4829-4839 ◽  
Author(s):  
R M Denome ◽  
C N Cole
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Coding Region ◽  
S1 Nuclease ◽  
Cytoplasmic Rna ◽  
Sv40 Origin ◽  
Sv40 Infection ◽  
Simplex Virus ◽  
Polyadenylation Signals

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.

Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals

1988 ◽  
Vol 8 (11) ◽  
pp. 4829-4839
Author(s):  
R M Denome ◽  
C N Cole
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Coding Region ◽  
S1 Nuclease ◽  
Cytoplasmic Rna ◽  
Sv40 Origin ◽  
Sv40 Infection ◽  
Simplex Virus ◽  
Polyadenylation Signals

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.

scholarly journals Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells.

1985 ◽  
Vol 5 (9) ◽  
pp. 2443-2453 ◽  
Author(s):  
A Israel ◽  
S N Cohen
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Negative Regulation ◽  
Coding Region ◽  
Transient Expression Assay ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Pomc Gene ◽  
Simplex Virus ◽  
Nuclease Mapping

We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA.

Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells

1985 ◽  
Vol 5 (9) ◽  
pp. 2443-2453
Author(s):  
A Israel ◽  
S N Cohen
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Negative Regulation ◽  
Coding Region ◽  
Transient Expression Assay ◽  
S1 Nuclease ◽  
Protein Coding ◽  
Pomc Gene ◽  
Simplex Virus ◽  
Nuclease Mapping

We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA.

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

1989 ◽  
Vol 9 (10) ◽  
pp. 4248-4258 ◽  
Author(s):  
S Carswell ◽  
J C Alwine
Keyword(s):  
Steady State ◽  
Rna Stability ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Polyadenylation Site ◽  
Expression Vectors ◽  
Mrna Levels ◽  
The Difference ◽  
Polyadenylation Signals

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.

scholarly journals Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

1994 ◽  
Vol 14 (3) ◽  
pp. 2004-2010 ◽  
Author(s):  
A Graessmann ◽  
G Sandberg ◽  
E Guhl ◽  
M Graessmann
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Coding Region ◽  
Simplex Virus

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

scholarly journals Functionally Significant Secondary Structure of the Simian Virus 40 Late Polyadenylation Signal

2000 ◽  
Vol 20 (8) ◽  
pp. 2926-2932 ◽  
Author(s):  
Holly Hans ◽  
James C. Alwine
Keyword(s):  
Secondary Structure ◽  
Rna Processing ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Analysis Techniques ◽  
Downstream Region ◽  
Polyadenylation Signals ◽  
Structural Region

ABSTRACT The structure of the highly efficient simian virus 40 late polyadenylation signal (LPA signal) is more complex than those of most known mammalian polyadenylation signals. It contains efficiency elements both upstream and downstream of the AAUAAA region, and the downstream region contains three defined elements (two U-rich elements and one G-rich element) instead of the single U- or GU-rich element found in most polyadenylation signals. Since many reports have indicated that the secondary structure in RNA may play a significant role in RNA processing, we have used nuclease structure analysis techniques to determine the secondary structure of the LPA signal. We find that the LPA signal has a functionally significant secondary structure. Much of the region upstream of AAUAAA is sensitive to single-strand-specific nucleases. The region downstream of AAUAAA has both double- and single-stranded characteristics. Both U-rich elements are predominately sensitive to the double-strand-specific nuclease RNase V1, while the G-rich element is primarily single stranded. The U-rich element closest to AAUAAA contains four distinct RNase V1-sensitive regions, which we have designated structural region 1 (SR1), SR2, SR3, and SR4. Linker scanning mutants in the downstream region were analyzed both for structure and for function by in vitro cleavage analyses. These data show that the ability of the downstream region, particularly SR3, to form double-stranded structures correlates with efficient in vitro cleavage. We discuss the possibility that secondary structure downstream of the AAUAAA may be important for the functions of polyadenylation signals in general.

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

1989 ◽  
Vol 9 (10) ◽  
pp. 4248-4258
Author(s):  
S Carswell ◽  
J C Alwine
Keyword(s):  
Steady State ◽  
Rna Stability ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Polyadenylation Site ◽  
Expression Vectors ◽  
Mrna Levels ◽  
The Difference ◽  
Polyadenylation Signals

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.

scholarly journals Structure of Simian Virus 40 DNA Replicated by Herpes Simplex Virus Type 1

Virology ◽  
2000 ◽  
Vol 276 (2) ◽  
pp. 445-454 ◽  
Author(s):  
Johannes Blümel ◽  
Sascha Gräper ◽  
Bertfried Matz
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Simplex Virus

scholarly journals In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

1986 ◽  
Vol 6 (11) ◽  
pp. 4130-4132 ◽  
Author(s):  
S Hayashi ◽  
H Kondoh
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Fragments ◽  
Specific Expression ◽  
Mouse Cells ◽  
Gc Box ◽  
Simplex Virus

Expression of the chicken delta-crystallin gene 1 injected into the nuclei of mouse cells is lens specific. Coinjection of GC box-containing DNA fragments from delta-crystallin, simian virus 40 early, and herpes simplex virus type 1 tk promoters effectively suppressed delta-crystallin expression in the lens, but coinjection with DNA fragments not containing the GC box did not. This suppression was likely due to the competition of an Sp1-like transcription factor(s) and indicates involvement of the apparently ubiquitous factor(s) in the tissue-specific expression of the delta-crystallin gene.

The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells

1991 ◽  
Vol 11 (2) ◽  
pp. 1023-1029
Author(s):  
Y Li ◽  
D Li ◽  
K Osborn ◽  
L F Johnson
Keyword(s):  
Thymidylate Synthase ◽  
Cultured Cells ◽  
Transcriptional Start Site ◽  
Simian Virus 40 ◽  
Normal Growth ◽  
Housekeeping Gene ◽  
Simian Virus ◽  
Coding Region ◽  
Proliferating Cells ◽  
Early Promoter

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.

Sign in / Sign up

Export Citation Format

Share Document