Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs

Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMID-pcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Δpcna1 or Δpcna3) revealed that mutant cells could no longer be propagated, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.

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Marker recycling via 5-fluoroorotic acid and 5-fluorocytosine counter-selection in the white-rot agaricomycete Pleurotus ostreatus

Fungal Biology ◽

10.1016/j.funbio.2016.06.011 ◽

2016 ◽

Vol 120 (9) ◽

pp. 1146-1155 ◽

Cited By ~ 13

Author(s):

Takehito Nakazawa ◽

Masami Tsuzuki ◽

Toshikazu Irie ◽

Masahiro Sakamoto ◽

Yoichi Honda

Keyword(s):

Pleurotus Ostreatus ◽

White Rot ◽

Marker Recycling ◽

Counter Selection ◽

Fluoroorotic Acid

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Structural and functional organization of baculovirus genome

Biopolymers and Cell ◽

10.7124/bc.0005e3 ◽

2002 ◽

Vol 18 (1) ◽

pp. 3-12 ◽

Cited By ~ 2

Author(s):

L. I. Strokovskaya ◽

I. M. Kikhno ◽

R. A. Meleshko

Keyword(s):

Functional Organization ◽

Baculovirus Genome

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Revertants of an S49 cell mutant that expresses altered cyclic AMP-dependent protein kinase

Molecular and Cellular Biology ◽

10.1128/mcb.2.10.1229-1237.1982 ◽

1982 ◽

Vol 2 (10) ◽

pp. 1229-1237

Author(s):

T van Daalen Wetters ◽

P Coffino

Keyword(s):

Protein Kinase ◽

Regulatory Subunit ◽

Mutational Event ◽

Wild Type ◽

Cell Mutant ◽

Dependent Protein Kinase ◽

Cell Extracts ◽

Counter Selection ◽

Dependent Protein ◽

Gene Lesion

Dibutyryl adenosine 3',5'-phosphate (Bt2cAMP)-sensitive (Bt2cAMPS) revertants were isolated from a resistant S49 cell mutant carrying a structural gene lesion in the regulatory subunit of cAMP-dependent protein kinase (cA-PK). This was accomplished with a counter-selection in which, first, Bt2cAMP was used to reversibly arrest revertants, and then a sequence of treatments with bromodeoxyuridine, 33258 Hoechst dye, and white light was used to kill cycling mutant cells. Reversion rates in nonmutagenized cultures could not be accurately measured, but spontaneous revertants do occur and with frequencies of less than 10(-7) to 10(-5). The mutagens ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), and ICR191 increased the reversion frequency. In all cases, reversion to Bt2cAMP sensitivity was associated with restoration of wild-type levels and apparent activation constant for cAMP of cA-PK. MNNG induced revertants whose cell extracts contained cA-PK activity distinguishable from that of wild type by thermal liability. EMS did not. The counter-selection effectively isolates rare phenotypes and is therefore a useful tool in further somatic genetic experiments. The association of reversion with alterations in cA-PK function supports all previous data from this and other laboratories implicating cA-PK as the intracellular mediator of cAMP effects. Reversion is probably the result of a mutational event. Induction of reversion by ICR191 suggests the existence of a novel mechanism for generating revertants in somatic cells.

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Attitudes towards the Mentally Handicapped and Counter Selection in the Educational System

Systems of Education ◽

10.4324/9780203984222-16 ◽

2005 ◽

pp. 122-126

Keyword(s):

Educational System ◽

Mentally Handicapped ◽

Counter Selection

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Leveraging modern DNA assembly techniques for rapid, markerless genome modification

Biology Methods and Protocols ◽

10.1093/biomethods/bpw004 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 4

Author(s):

Ilya B Tikh ◽

James C Samuelson

Keyword(s):

High Efficiency ◽

Point Mutations ◽

Host Cells ◽

Dna Assembly ◽

E Coli ◽

Recombination Efficiency ◽

Genome Modification ◽

Counter Selection ◽

K 12 ◽

Polymerase Chain Reaction Primers

Abstract The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

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