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.

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.

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells

1983 ◽  
Vol 3 (12) ◽  
pp. 2203-2210
Author(s):  
J W Innis ◽  
W A Scott
Keyword(s):  
Chromatin Structure ◽  
Plasmid Dna ◽  
Base Pair ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
Dnase I ◽  
Viral Origin ◽  
African Green Monkey Kidney

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.

In Vitro Assessment of the Cytotoxicity of Nisin, Pediocin, and Selected Colicins on Simian Virus 40–Transfected Human Colon and Vero Monkey Kidney Cells with Trypan Blue Staining Viability Assays

2003 ◽  
Vol 66 (5) ◽  
pp. 847-853 ◽  
Author(s):  
S. E. MURINDA ◽  
K. A. RASHID ◽  
R. F. ROBERTS
Keyword(s):  
Mammalian Cells ◽  
Trypan Blue ◽  
Calf Serum ◽  
Simian Virus 40 ◽  
Human Colon ◽  
Vero Cells ◽  
Simian Virus ◽  
Monkey Kidney ◽  
Blue Staining ◽  
Trypan Blue Staining

Gram-positive bacterial bacteriocins (nisin and pediocin) and gram-negative bacterial bacteriocins (colicins [Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured simian virus 40–transfected human colon (SV40-HC) and Vero monkey kidney (Vero) cells. Bacteriocin-treated cells were assessed for viability by trypan blue staining. Monolayers of SV40-HC and Vero cells were cultured in tissue culture plates (35°C, 10% CO2 in humidified air) with the use of Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) calf serum. Actively growing cells in the log phase (ca. 104 cells per ml) were treated with individual partially purified bacteriocin preparations at 170, 350, and 700 activity units per ml. Duplicate culture plates for each bacteriocin treatment and untreated controls were withdrawn after 16, 32, and 48 h of incubation. Cells were dissociated with trypsin and treated with trypan blue and were then counted in a hemocytometer with the use of a phase-contrast microscope. Viability assays indicated dose-dependent toxicity for some bacteriocins. Nisin, pediocin, and Col E6 were the most cytotoxic bacteriocins; SV40-HC cells demonstrated greater sensitivity than Vero cells did. Some bacteriocins can be toxic to mammalian cells; therefore, bacteriocins intended for use as biopreservatives must be evaluated for toxicity to mammalian cells and for other toxicities. Col E1, Col E3, Col E7, and Col K demonstrated little toxicity at the activities tested, indicating that they are safe and thus have potential for use as food biopreservatives.

scholarly journals Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

1983 ◽  
Vol 3 (12) ◽  
pp. 2203-2210 ◽  
Author(s):  
J W Innis ◽  
W A Scott
Keyword(s):  
Chromatin Structure ◽  
Plasmid Dna ◽  
Base Pair ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
Dnase I ◽  
Viral Origin ◽  
African Green Monkey Kidney

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.

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