Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes

1990 ◽  
Vol 10 (1) ◽  
pp. 353-360
Author(s):  
B M Benton ◽  
W K Eng ◽  
J J Dunn ◽  
F W Studier ◽  
R Sternglanz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Target Genes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Yeast Cells ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
T7 Promoter

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

scholarly journals Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes.

1990 ◽  
Vol 10 (1) ◽  
pp. 353-360 ◽  
Author(s):  
B M Benton ◽  
W K Eng ◽  
J J Dunn ◽  
F W Studier ◽  
R Sternglanz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Target Genes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Yeast Cells ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
T7 Promoter

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

scholarly journals Feline Calicivirus Capsid Protein Expression and Capsid Assembly in Cultured Feline Cells

1999 ◽  
Vol 73 (1) ◽  
pp. 834-838 ◽  
Author(s):  
Klaus Geissler ◽  
Karla Schneider ◽  
Andrea Fleuchaus ◽  
Colin R. Parrish ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Capsid Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T7 Rna Polymerase ◽  
Feline Calicivirus ◽  
T7 Promoter ◽  
Mature Size

ABSTRACT The capsid protein of feline calicivirus (FCV) was expressed by using plasmids containing cytomegalovirus, simian virus 40, or T7 promoters. The strongest expression was achieved with the T7 promoter and coinfection with vaccinia virus expressing the T7 RNA polymerase (MVA/T7pol). The FCV precursor capsid protein was processed to the mature-size protein, and these proteins were assembled in to virus-like particles.

scholarly journals Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (3) ◽  
pp. 947-957 ◽  
Author(s):  
N J Axelrod ◽  
G G Carmichael ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Evolutionary Relationship ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Yeast Cells ◽  
Upstream Activation Sequence ◽  
Beta Galactosidase ◽  
Activation Sequence

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.

scholarly journals Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

1985 ◽  
Vol 5 (3) ◽  
pp. 563-568 ◽  
Author(s):  
H Ariga ◽  
Z Tsuchihashi ◽  
M Naruto ◽  
M Yamada
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Yeast Cells ◽  
Dna Fragments ◽  
Replication System ◽  
Sv40 Dna

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.

Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (3) ◽  
pp. 947-957
Author(s):  
N J Axelrod ◽  
G G Carmichael ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Evolutionary Relationship ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Simian Virus ◽  
Yeast Cells ◽  
Upstream Activation Sequence ◽  
Beta Galactosidase ◽  
Activation Sequence

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synthesis, and three fragments from the polyomavirus enhancer region stimulated moderate levels. Three of the four controls of random DNA sequences failed to stimulate significant levels, and the fourth stimulated moderate levels. The stimulation in all cases was independent of the orientation of the inserted fragment. Two series of clones were examined in which between one and six tandemly arranged copies of a fragment were inserted into the XhoI site of the vector. Very interestingly, we detected an apparent exponential relationship between the number of copies of a fragment and the amount of beta-galactosidase produced. Southern analysis showed that increases in enzyme activity were not a result of increased plasmid copy number. Rather, quantitative S1 nuclease analysis demonstrated that the increases were correlated with steady-state levels of lacZ-specific mRNA. We suggest that there may be an evolutionary relationship between some transcriptional activation sequences in yeast cells and the higher eucaryotic regulatory elements that we tested.

Development of an inducible T7 expression system in Bacillus subtilis ATCC 6051a for production of α-L-arabinofuranosidase

2020 ◽  
Author(s):  
Minghua Ji ◽  
Sijie Li ◽  
Yunhui Liu ◽  
Haiying Wu ◽  
Qiao Chen ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Fermentation Broth ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
T7 Promoter ◽  
Subtilis Atcc ◽  
Bacillus Subtilis Atcc

Abstract Background: Xylan is the second most abundant polysaccharide biomass on the earth, the polymers have a backbone of β-1,4-linked xylose residues with various side-chain substitutions, such as arabinose, acetic acid, glucuronic acid, and other esterified groups, thus, the removal of arabinose side groups by α-L-arabinofuranosidases is helpful in various industrial processes involving xylan treatment. Bacillus subtilis ATCC 6051a is known for its excellent capacity of secretory production of recombinant peptides, however, poor experience in genetic manipulation and lack of universal expression elements impede this strain for wider application.Results: Xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, and the transformation efficiency of the engineered strain B. subtilis 164S was increased by more than 1000 folds. B. subtilis 164S was further modified to generate B. subtilis 164T7P which incorporates a D-xylose inducible T7 RNA polymerase. The recombinant GFP expressed by 164T7P is more than thirteen times that achieved by P43 promoter, representing the most efficient expression system that had been ever reported in B. subtilis . Subsequently, abfA1 , encoding a glycoside hydrolase (GH) family 51 enzyme was cloned and overexpressed in 164T7P. The activity of recombinant α-L-arabinofuranosidase (AbfA1) reached 90.6±2.0 U mL -1 in the fermentation broth. Using p NPA as a substrate, kinetic parameters of the crude enzyme were Km of 1.4±0.1 mM and kcat of 139.4 s −1 . The optimum temperature and pH of the recombinant AbfA1 towards p NPA were observed to be 45 °C and pH 6.5, respectively.Conclusion: With enhanced cellular competence and introduction of T7 RNA polymerase, B. subtilis ATCC 6051a was engineered as a versatile cell tool for recombinant production of heterologous peptides employing T7 promoter. The novel expression kit demonstrated a very low level of leaky expression of target genes. The efficiency and applicability of the system were demonstrated by high-level production of a bacterial type α-L-arabinofuranosidase.

scholarly journals RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.

1997 ◽  
Vol 17 (9) ◽  
pp. 5077-5086 ◽  
Author(s):  
R A Ach ◽  
T Durfee ◽  
A B Miller ◽  
P Taranto ◽  
L Hanley-Bowdoin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cysteine Residue ◽  
T Antigen ◽  
Yeast Cells ◽  
Binding Potential ◽  
Adenovirus E1a ◽  
Retinoblastoma Tumor ◽  
Related Proteins

Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.

scholarly journals Simian Virus 40 T-Antigen-Mediated Gene Regulation in Enterocytes Is Controlled Primarily by the Rb-E2F Pathway

2009 ◽  
Vol 83 (18) ◽  
pp. 9521-9531 ◽  
Author(s):  
Abhilasha V. Rathi ◽  
M. Teresa Sáenz Robles ◽  
Paul G. Cantalupo ◽  
Robert H. Whitehead ◽  
James M. Pipas
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Cell Transformation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rb Pathway ◽  
Mouse Small Intestine ◽  
Mouse Intestine ◽  
Intestinal Hyperplasia

ABSTRACT Simian virus 40 large T antigen (TAg) contributes to cell transformation, in part, by targeting two well-characterized tumor suppressors, pRb and p53. TAg expression affects the transcriptional circuits controlled by Rb and by p53. We have performed a microarray analysis to examine the global change in gene expression induced by wild-type TAg (TAgwt) and TAg mutants, in an effort to link changes in gene expression to specific transforming functions. For this analysis we have used enterocytes from the mouse small intestine expressing TAg. Expression of TAgwt in the mouse intestine results in hyperplasia and dysplasia. Our analysis indicates that practically all gene expression regulated by TAg in enterocytes is dependent upon its binding and inactivation of the Rb family proteins. To further dissect the role of the Rb family in the induction of intestinal hyperplasia, we have screened several lines of transgenic mice expressing a truncated TAg (TAgN136), which is able to interfere with the Rb pathway but lacks the functions associated with the carboxy terminus of the protein. This analysis confirmed the pivotal association between the Rb pathway and the induction of intestinal hyperplasia and revealed that upregulation of p53 target genes is not associated with the tumorigenic phenotype. Furthermore, we found that TAgN136 was sufficient to induce intestinal hyperplasia, although the appearance of dysplasia was significantly delayed.

scholarly journals Activation of RNA Polymerase III Transcription in Cells Transformed by Simian Virus 40

1999 ◽  
Vol 19 (7) ◽  
pp. 4927-4934 ◽  
Author(s):  
Christopher G. C. Larminie ◽  
Josephine E. Sutcliffe ◽  
Kerrie Tosh ◽  
Andrew G. Winter ◽  
Zoe A. Felton-Edkins ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Mrna Levels ◽  
Large T Antigen ◽  
Tumor Viruses ◽  
Sv40 Transformation ◽  
Pol Iii

ABSTRACT RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses.

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