Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells

1990 ◽  
Vol 10 (7) ◽  
pp. 3343-3356
Author(s):  
M A Labow ◽  
S B Baim ◽  
T Shenk ◽  
A J Levine
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Transcriptional Activator ◽  
Transcription Unit ◽  
Lac Repressor ◽  
Coding Region ◽  
Virion Protein ◽  
Sv40 Promoter ◽  
Large Tumor ◽  
Lac Operator

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.

scholarly journals Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

1990 ◽  
Vol 10 (7) ◽  
pp. 3343-3356 ◽  
Author(s):  
M A Labow ◽  
S B Baim ◽  
T Shenk ◽  
A J Levine
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Transcriptional Activator ◽  
Transcription Unit ◽  
Lac Repressor ◽  
Coding Region ◽  
Virion Protein ◽  
Sv40 Promoter ◽  
Large Tumor ◽  
Lac Operator

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.

scholarly journals Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

1992 ◽  
Vol 12 (10) ◽  
pp. 4271-4278 ◽  
Author(s):  
D E Syroid ◽  
R I Tapping ◽  
J P Capone
Keyword(s):  
Mammalian Cells ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Lac Repressor ◽  
Trna Genes ◽  
Coding Region ◽  
Lac Operator ◽  
Cell Extracts

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

scholarly journals Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants.

1984 ◽  
Vol 4 (11) ◽  
pp. 2406-2412 ◽  
Author(s):  
W J Muller ◽  
M A Naujokas ◽  
J A Hassell
Keyword(s):  
Cell Lines ◽  
Tumor Antigens ◽  
Simian Virus 40 ◽  
Mouse Cell ◽  
Simian Virus ◽  
Transcription Unit ◽  
T Antigen ◽  
Coding Region ◽  
Large Tumor ◽  
Early Promoter

Construction of polyomavirus vectors, analysis of mutant viruses, and rescue of integrated polyomavirus genomes would be considerably aided by the availability of transformed, permissive mouse cell lines capable of producing the viral tumor antigens. To isolate such cell lines, we constructed a hybrid transcription unit composed of the simian virus 40 early promoter fused to the coding region for the polyomavirus tumor antigens. This hybrid transcription unit was used to transform NIH 3T3 cells. Independent foci of transformed cells were isolated, recloned, and characterized. Among 10 lines initially analyzed, 7 supported the replication of origin-bearing plasmid DNAs. Three cell lines were characterized in greater detail. Each line contained one or two independent insertions of polyomavirus DNA and synthesized all three viral tumor antigens. Moreover, the large tumor antigen in two of three lines bound with specificity to sequences about the polyomavirus origin and early promoter. These cell lines should prove useful for studying not only the replication of polyomavirus but also the expression of foreign genes in a mouse cell environment.

Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system

1992 ◽  
Vol 12 (10) ◽  
pp. 4271-4278
Author(s):  
D E Syroid ◽  
R I Tapping ◽  
J P Capone
Keyword(s):  
Mammalian Cells ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Lac Repressor ◽  
Trna Genes ◽  
Coding Region ◽  
Lac Operator ◽  
Cell Extracts

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants

1984 ◽  
Vol 4 (11) ◽  
pp. 2406-2412
Author(s):  
W J Muller ◽  
M A Naujokas ◽  
J A Hassell
Keyword(s):  
Cell Lines ◽  
Tumor Antigens ◽  
Simian Virus 40 ◽  
Mouse Cell ◽  
Simian Virus ◽  
Transcription Unit ◽  
T Antigen ◽  
Coding Region ◽  
Large Tumor ◽  
Early Promoter

Construction of polyomavirus vectors, analysis of mutant viruses, and rescue of integrated polyomavirus genomes would be considerably aided by the availability of transformed, permissive mouse cell lines capable of producing the viral tumor antigens. To isolate such cell lines, we constructed a hybrid transcription unit composed of the simian virus 40 early promoter fused to the coding region for the polyomavirus tumor antigens. This hybrid transcription unit was used to transform NIH 3T3 cells. Independent foci of transformed cells were isolated, recloned, and characterized. Among 10 lines initially analyzed, 7 supported the replication of origin-bearing plasmid DNAs. Three cell lines were characterized in greater detail. Each line contained one or two independent insertions of polyomavirus DNA and synthesized all three viral tumor antigens. Moreover, the large tumor antigen in two of three lines bound with specificity to sequences about the polyomavirus origin and early promoter. These cell lines should prove useful for studying not only the replication of polyomavirus but also the expression of foreign genes in a mouse cell environment.

scholarly journals Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

1982 ◽  
Vol 2 (9) ◽  
pp. 1044-1051 ◽  
Author(s):  
C M Gorman ◽  
L F Moffat ◽  
B H Howard
Keyword(s):  
Promoter Region ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chloramphenicol Acetyltransferase ◽  
Repeat Sequence ◽  
Monkey Kidney ◽  
Sv40 Promoter ◽  
Hindiii Site ◽  
African Green Monkey Kidney

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

scholarly journals In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation

2005 ◽  
Vol 25 (11) ◽  
pp. 4552-4564 ◽  
Author(s):  
Pernette J. Verschure ◽  
Ineke van der Kraan ◽  
Wim de Leeuw ◽  
Johan van der Vlag ◽  
Anne E. Carpenter ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Large Scale ◽  
Chromosome Region ◽  
Chromatin Condensation ◽  
Regulation Of Gene Expression ◽  
Lac Repressor ◽  
Homologous Proteins ◽  
Lac Operator

ABSTRACT Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) α (HP1α) and HP1β on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1α and HP1β in vivo targeting. HP1α targeting causes the recruitment of endogenous HP1β to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1α and HP1β targeting is sufficient to induce heterochromatin formation.

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

1982 ◽  
Vol 2 (9) ◽  
pp. 1044-1051
Author(s):  
C M Gorman ◽  
L F Moffat ◽  
B H Howard
Keyword(s):  
Promoter Region ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chloramphenicol Acetyltransferase ◽  
Repeat Sequence ◽  
Monkey Kidney ◽  
Sv40 Promoter ◽  
Hindiii Site ◽  
African Green Monkey Kidney

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

scholarly journals 5-Azacytidine Induces Transgene Silencing by DNA Methylation in Chinese Hamster Cells

1999 ◽  
Vol 19 (4) ◽  
pp. 3198-3204 ◽  
Author(s):  
Limor Broday ◽  
Yong-Woo Lee ◽  
Max Costa
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Mammalian Cells ◽  
De Novo ◽  
Chromatin Condensation ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Transgene Silencing ◽  
Sv40 Promoter ◽  
Mutagenic Potential

ABSTRACT The cytosine analog 5-azacytidine (5-AzaC) is a demethylating agent that is also known to induce mutagenesis in mammalian cells. In this study, the mutagenic potential of this drug was tested in the G10 and G12 transgenic Chinese hamster cell lines, which have a single bacterial gpt gene integrated into the genome at different sites, with its expression driven by a simian virus 40 (SV40) promoter. We show that the mutation frequencies following a 48-h exposure to different concentrations of 5-AzaC were 10 to 20 times higher than those of any of the other numerous mutagens that have been tested in the G10-G12 system. Moreover, the mutation frequencies were much higher in the G10 cell line than in the G12 cells. Detailed molecular analysis of the 6-thioguanine (6-TG)-resistant variants demonstrated that transgene silencing by de novo DNA methylation and increased chromatin condensation in the SV40 promoter was the major factor responsible for this high level of 6-TG resistance. As would be expected, exposure to 5-AzaC lowered the overall genomic DNA methylation levels, but it unexpectedly caused hypermethylation and increased chromatin condensation of the transgene in both the G10 and G12 cell lines. These results provide the first evidence that 5-AzaC may also induce transgene-specific DNA methylation, a phenomenon that can further be used for the elucidation of the mechanism that controls silencing of foreign DNA.

scholarly journals Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen

1989 ◽  
Vol 86 (17) ◽  
pp. 6479-6483 ◽  
Author(s):  
I J Mohr ◽  
Y Gluzman ◽  
M P Fairman ◽  
M Strauss ◽  
D McVey ◽  
...  
Keyword(s):  
Tumor Antigen ◽  
Mammalian Cells ◽  
Expression System ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Wild Type ◽  
Large Tumor ◽  
Large Tumor Antigen

A bacterial expression system was used to produce simian virus 40 large tumor antigen (T antigen) in the absence of the extensive posttranslational modifications that occur in mammalian cells. Wild-type T antigen produced in bacteria retained a specific subset of the biochemical activities displayed by its mammalian counterpart. Escherichia coli T antigen functioned as a helicase and bound to DNA fragments containing either site I or the wild-type origin of replication in a manner identical to mammalian T antigen. However, T antigen purified from E. coli did not efficiently bind to site II, an essential cis element within the simian virus 40 origin of replication. It therefore could not unwind origin-containing plasmids or efficiently replicate simian virus 40 DNA in vitro. The ability of protein phosphorylation to modulate the intrinsic preference of full-length T antigen for either site I or site II is discussed.

Sign in / Sign up

Export Citation Format

Share Document