scholarly journals A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells

1999 ◽  
Vol 27 (2) ◽  
pp. 426-428 ◽  
Author(s):  
C. Piechaczek ◽  
C. Fetzer ◽  
A. Baiker ◽  
J. Bode ◽  
H. J. Lipps
Keyword(s):  
Cho Cells ◽  
Origin Of Replication ◽  
Sv40 Origin

scholarly journals Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content

1992 ◽  
Vol 288 (2) ◽  
pp. 511-518 ◽  
Author(s):  
L M Shantz ◽  
I Holm ◽  
O A Jänne ◽  
A E Pegg
Keyword(s):  
Cho Cells ◽  
Translational Regulation ◽  
Decarboxylase Activity ◽  
Origin Of Replication ◽  
Sv40 Promoter ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Mammalian Cell Lines ◽  
Sv40 Origin ◽  
Polyamine Content

The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMh1, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMh1. The pSAMh1 cDNA is missing 72 nucleotides from the 5′ end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMh1 in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.

Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence

1988 ◽  
Vol 8 (4) ◽  
pp. 1509-1517
Author(s):  
R Kumar ◽  
K P Yoon ◽  
K N Subramanian
Keyword(s):  
Transcriptional Activation ◽  
Base Pair ◽  
Copy Number ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Replication Efficiency ◽  
Sv40 Origin ◽  
Multiple Copies ◽  
In Cis

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.

scholarly journals Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence.

1988 ◽  
Vol 8 (4) ◽  
pp. 1509-1517 ◽  
Author(s):  
R Kumar ◽  
K P Yoon ◽  
K N Subramanian
Keyword(s):  
Transcriptional Activation ◽  
Base Pair ◽  
Copy Number ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Origin Of Replication ◽  
Replication Efficiency ◽  
Sv40 Origin ◽  
Multiple Copies ◽  
In Cis

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.

Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication

Cell ◽  
1991 ◽  
Vol 65 (6) ◽  
pp. 1083-1091 ◽  
Author(s):  
Jill Bargonetti ◽  
Paula N. Friedman ◽  
Scott E. Kern ◽  
Bert Vogelstein ◽  
Carol Prives
Keyword(s):  
Mutant P53 ◽  
Origin Of Replication ◽  
Wild Type ◽  
Sv40 Origin
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