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.

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 Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

2020 ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
Chemical Production ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Shock Proteins

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter were powerful tools to mediate the protein expression. Moreover, Escherichia coli W3110 strain possesses more advantages than the B strain due to more heat shock proteins and higher tolerance to chemicals. Therefore, implementation of T7-based system in W3110 strain is a conceivable strategy to develop the cell factory. Results Three novel W3110 strains with chromosome-equipped T7RNAP (i.e W3110:IL5, W3110::L5 and W3110::pI) were engineered to demonstrate the feasibility on protein expression and chemical production. At first, the LacZ and T7RNAP with IPTG induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), Cas9, carbonic anhydrase (CA) and lysine decarboxylase (CadA). All the proteins were expressed higher and enzymatic functions were better in W3110::L5 and W3110::pI. Moreover, the highest cadaverine production, lysine consumption and the yield were obtained in W3110::L5(+) strain with pET28a(+)-CadA which reached 32.2 g/L, 45 g/L and 91.7% at 24 h, while the W3110::pI(-) strain with pSU-T7-CadA achieved 36.9 g/L, 43.8 g/L and 103.4% at 12 h which is unnecessary of inducer. Conclusion Inducer and lacI/lacO regulators on chromosome and plasmid have been investigated in W3110 strains with T7RNAP. The newly engineered W3110::L5 and W3110:pI both possessed similar protein expression compared to commercial BL21(DE3). Furthermore, among all strains, W3110::pI displayed the greatest potential as cell factory in the future.

scholarly journals Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Wan-Wen Ting ◽  
Shih-I Tan ◽  
I-Son Ng
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Rna Polymerase ◽  
Green Fluorescence Protein ◽  
T7 Rna Polymerase ◽  
Cell Factory ◽  
T7 Promoter ◽  
Iptg Induction ◽  
E Coli ◽  
A Cell

Abstract Background Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy. Results Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively. Conclusion Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.

scholarly journals Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

1982 ◽  
Vol 2 (12) ◽  
pp. 1595-1607 ◽  
Author(s):  
Timothy J. Miller ◽  
Janet E. Mertz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Structural Requirements ◽  
Infected Monkey ◽  
Full Complement ◽  
Sv40 Dna

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei ofXenopus laevisoocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 minichromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

scholarly journals Transcription of hepatitis B virus by RNA polymerase II.

1983 ◽  
Vol 3 (10) ◽  
pp. 1766-1773 ◽  
Author(s):  
L B Rall ◽  
D N Standring ◽  
O Laub ◽  
W J Rutter
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Simian Virus 40 ◽  
Virus Genome ◽  
Simian Virus ◽  
Free System ◽  
B Virus

We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.

Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site

1990 ◽  
Vol 10 (2) ◽  
pp. 653-661
Author(s):  
A L Means ◽  
P J Farnham
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Simian Virus ◽  
Sequence Element ◽  
Initiator Element

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

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