In Vitro Replication Assay with Mammalian Cell Extracts

2009 ◽  
pp. 203-216 ◽  
Author(s):  
Wasia Rizwani ◽  
Srikumar P. Chellappan
Keyword(s):  
Mammalian Cell ◽  
Replication Assay ◽  
Cell Extracts ◽  
In Vitro Replication

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

scholarly journals Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon

2005 ◽  
Vol 86 (11) ◽  
pp. 3075-3080 ◽  
Author(s):  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Replication Assay ◽  
Cell Extracts ◽  
Genotype 1B

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts

1999 ◽  
pp. 301-315 ◽  
Author(s):  
Guido Frosina ◽  
Enrico Cappelli ◽  
Paola Fortini ◽  
Eugenia Dogliotti
Keyword(s):  
Mammalian Cell ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Cell Extracts ◽  
Base Excision

Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

Biochimie ◽  
2013 ◽  
Vol 95 (2) ◽  
pp. 320-328
Author(s):  
Seiki Nakao ◽  
Sufang Zhang ◽  
Markku Vaara ◽  
Juhani E. Syväoja ◽  
Marietta Y. Lee ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mammalian Cell ◽  
Gap Filling ◽  
Free System ◽  
Cell Free System ◽  
Replication Assay ◽  
System A

scholarly journals Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

1992 ◽  
Vol 12 (4) ◽  
pp. 1605-1612 ◽  
Author(s):  
G Dianov ◽  
A Price ◽  
T Lindahl
Keyword(s):  
Mammalian Cell ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repair Synthesis ◽  
Single Nucleotide ◽  
Cell Extracts ◽  
Dna Repair Synthesis

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.

In Vitro Replication Assay for Merkel Cell Polyomavirus (MCPyV)

2015 ◽  
Vol 38 (1) ◽  
Author(s):  
Friederike Neumann ◽  
Manja Czech‐Sioli ◽  
Adam Grundhoff ◽  
Nicole Fischer
Keyword(s):  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
Replication Assay ◽  
In Vitro Replication

In Vitro RNA Splicing in Mammalian Cell Extracts

2001 ◽  
Vol 18 (1) ◽  
Author(s):  
Nanette Rooke ◽  
Jason Underwood
Keyword(s):  
Mammalian Cell ◽  
Rna Splicing ◽  
Cell Extracts

In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts

2003 ◽  
pp. 301-315 ◽  
Author(s):  
Guido Frosina ◽  
Enrico Cappelli ◽  
Paola Fortini ◽  
Eugenia Dogliotti
Keyword(s):  
Mammalian Cell ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Cell Extracts ◽  
Base Excision

Generation of single-nucleotide repair patches following excision of uracil residues from DNA

1992 ◽  
Vol 12 (4) ◽  
pp. 1605-1612
Author(s):  
G Dianov ◽  
A Price ◽  
T Lindahl
Keyword(s):  
Mammalian Cell ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repair Synthesis ◽  
Single Nucleotide ◽  
Cell Extracts ◽  
Dna Repair Synthesis

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.

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