Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside

Norgard, Michael V.; Emigholz, K; Monahan, John J.

doi:10.1128/jb.138.1.270-272.1979

Cited by 135 publications

(31 citation statements)

References 14 publications

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“…For the preparation of plasmid DNA, bacteria containing the desired plasmid were grown in enriched M9 medium to an ODwo of 0.15. Cytidine was then added to a final concentration of 1 mg/ml (Norgard et al, 1979) and…”

Section: Extraction Of Nucleic Acidsmentioning

confidence: 99%

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Oren¹,

Bienz²,

Rechavi

et al. 1983

The EMBO Journal

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Three cDNA clones, corresponding to two non‐overlapping regions of the mRNA coding for the mouse p53 cellular tumor antigen, were isolated and characterized. In hybridization‐selection assays, these clones were capable of selectively binding p53 mRNA, as demonstrated by in vitro translation and immunoprecipitation with anti‐p53 monoclonal antibodies. The p53 mRNA appeared to be the only messenger species specifically selected by these clones. The size of the p53 mRNA was found to be approximately 2 kb, and its levels to vary substantially among different types of transformed cells. Evidence was found for the existence of two distinct p53‐specific genes in mouse genomic DNA. Two partially overlapping recombinant phage clones were obtained, both derived from the same p53‐specific genomic DNA region. The orientation of the various cDNA clones relative to that of the p53 mRNA was established by S1 analysis and the relationship between the cDNA clones and the genomic ones was determined by comparative restriction enzyme mapping and nucleic acid hybridization.

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Section: Extraction Of Nucleic Acidsmentioning

confidence: 99%

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Oren¹,

Bienz²,

Rechavi

et al. 1983

The EMBO Journal

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“…DNA fragments sized on low-gel-temperature agarose (Bio-Rad) were used for ligation without extraction (Frischauf et al, 1980). Plasmid DNA was isolated as described by Wilkie et al (1979) after induction with uridine (Norgard et al, 1979). 5' End labelling of restriction fragments and DNA sequencing were carried out by the method of Maxam and Gilbert (1980).…”

Section: Enzymesmentioning

confidence: 99%

DNA sequences required for transcription in vivo of the human corticotropin-beta-lipotropin precursor gene.

Mishina¹,

Kurosaki²,

Yamamoto³

et al. 1982

The EMBO Journal

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The cloned human corticotropin‐beta‐lipotropin precursor gene, when joined with an SV40 vector and introduced into COS monkey cells, is transcribed from its own promoter. The DNA sequences required for promoter function have been identified by using 5′ deletion mutants of the fusion gene AT which contains the 5′‐flanking sequence and capping site of the human corticotropin‐beta‐lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The deletion of the sequence located between 53 and 59 bp upstream of the capping site enhances the transcription approximately 3‐fold, while the deletion of the TATA box region abolishes the transcription.

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“…For Cm amplification, cultures were grown to an absorbance (A) of about 0.8 at 500 nm [8], after this Cm (VEB Berlin-Chemie) at a final concentration of 25 or 50 /~g/ml was added. In some experiments the cells were cultivated in the presence of 1 mg uridine/ml to increase the plasmid yield per cell [6]. The plasmid DNA was determined in cells harvested during different growth phases (as indicated in the legends) and about 15 h after Cm treatment according to [10].…”

Section: Methodsmentioning

confidence: 99%

“…For cloning experiments new methods are constantly being sought to improve the yields of plasmid DNA from cultures of bacterial cells [6,7]. To isolate pBR322 DNA in large amounts, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…Studying this problem we have shown that in E. coli relA strains which cannot produce ppGpp upon amino acid limitation pBR322 DNA is amplified after isoleucine starvation [1]. These resuits support our assumption of a negative control of pBR322 DNA replication by ppGpp.For cloning experiments new methods are constantly being sought to improve the yields of plasmid DNA from cultures of bacterial cells [6,7]. To isolate pBR322 DNA in large amounts, e.g.…”

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confidence: 99%

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Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Hecker¹,

Schroeter²,

Mach³

1985

FEMS Microbiology Letters

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We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli reiA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yidd per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli. INTRODUCTIONE. coil [2][3][4]. However, experiments, involving amino acid starvation to inhibit protein synthesis only resulted in a low enrichment of plasmid DNA. In both cases protein synthesis is inhibited but only in amino acid-starved cells ppGpp is synthesized whereas Cm treatment prevents the formation of ppGpp [1,5]. Possibly ppGpp can inhibit plasmid amplification in amino acid-starved cells. Studying this problem we have shown that in E. coli relA strains which cannot produce ppGpp upon amino acid limitation pBR322 DNA is amplified after isoleucine starvation [1]. These resuits support our assumption of a negative control of pBR322 DNA replication by ppGpp.For cloning experiments new methods are constantly being sought to improve the yields of plasmid DNA from cultures of bacterial cells [6,7]. To isolate pBR322 DNA in large amounts, e.g. for recombinant DNA research, the method of plasmid amplification by Cm is widely used [8]. In this study the yields of plasmid DNA amplified either by Cm or by amino acid starvation of the E. coli relA strain CP79 will be compared. Our results indicate that E. coli relA strains might be suitable hosts for amplification of pBR322 plasmid DNA.Inhibition of protein synthesis by Cm causes a rapid amplification of ColEl-related plasmids in 0378-1097/85/$03.30

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Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside

Cited by 135 publications

References 14 publications

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen.

DNA sequences required for transcription in vivo of the human corticotropin-beta-lipotropin precursor gene.

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

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