Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei

1985 ◽  
Vol 5 (1) ◽  
pp. 70-74
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Kanamycin Resistance ◽  
Linear Plasmid ◽  
Wild Type ◽  
Cell Nuclei ◽  
Heteroduplex Dna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
A Cell

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.

scholarly journals Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

1985 ◽  
Vol 5 (1) ◽  
pp. 70-74 ◽  
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Kanamycin Resistance ◽  
Linear Plasmid ◽  
Wild Type ◽  
Cell Nuclei ◽  
Heteroduplex Dna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
A Cell

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 400-406
Author(s):  
W P Deng ◽  
J A Nickoloff
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Wild Type ◽  
Heteroduplex Dna ◽  
Frameshift Mutations ◽  
Single Strand Annealing ◽  
Extrachromosomal Recombination ◽  
Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

scholarly journals The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs

2019 ◽  
Vol 366 (8) ◽  
Author(s):  
Thomas Jacobsen ◽  
Chunyu Liao ◽  
Chase L Beisel
Keyword(s):  
Dna Cleavage ◽  
Mammalian Cells ◽  
Consensus Sequence ◽  
Wild Type ◽  
E Coli ◽  
Cleavage Efficiency ◽  
Protospacer Adjacent Motif ◽  
Short Palindromic Repeat ◽  
A Cell ◽  
Validation Experiments

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.

scholarly journals Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei.

1985 ◽  
Vol 5 (1) ◽  
pp. 59-69 ◽  
Author(s):  
K R Folger ◽  
K Thomas ◽  
M R Capecchi
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
Recombinant Dna ◽  
Dna Duplexes ◽  
Dna Molecules ◽  
Cell Nuclei ◽  
High Concentration ◽  
Length Polymorphisms

We have examined the mechanism of homologous recombination between plasmid molecules coinjected into cultured mammalian cells. Cell lines containing recombinant DNA molecules were obtained by selecting for the reconstruction of a functional Neor gene from two plasmids that bear different amber mutations in the Neor gene. In addition, these plasmids contain restriction-length polymorphisms within and near the Neor gene. These polymorphisms did not confer a selectable phenotype but were used to identify and categorize selected and nonselected recombinant DNA molecules. The striking conclusion from this analysis is that the predominant mechanism for the exchange of information between coinjected plasmid molecules over short distances (i.e., less than 1 kilobase) proceeds via nonreciprocal homologous recombination. The frequency of homologous recombination between coinjected plasmid molecules in cultured mammalian cells is extremely high, approaching unity. We demonstrate that this high frequency requires neither a high input of plasmid molecules per cell nor a localized high concentration of plasmid DNA within the nucleus. Thus, it appears that plasmid molecules, once introduced into the nucleus, have no difficulty seeking each other out and participating in homologous recombination even in the presence of a vast excess of host DNA sequences. Finally, we show that most of the homologous recombination events occur within a 1-h interval after the introduction of plasmid DNA into the cell nucleus.

scholarly journals Proteolysis by calpains: a possible contribution to degradation of p53.

1997 ◽  
Vol 17 (5) ◽  
pp. 2806-2815 ◽  
Author(s):  
M Pariat ◽  
S Carillo ◽  
M Molinari ◽  
C Salvat ◽  
L Debüssche ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Tertiary Structure ◽  
P53 Protein ◽  
Calcium Ionophore ◽  
Expression Vectors ◽  
Wild Type ◽  
Experimental Conditions ◽  
Null Cell ◽  
Wide Range ◽  
Wild Type P53

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

p53 functions as a cell cycle control protein in osteosarcomas

1990 ◽  
Vol 10 (11) ◽  
pp. 5772-5781
Author(s):  
L Diller ◽  
J Kassel ◽  
C E Nelson ◽  
M A Gryka ◽  
G Litwak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Control ◽  
P53 Gene ◽  
Tumor Formation ◽  
Wild Type ◽  
Gene Encoding ◽  
Wide Range ◽  
A Cell ◽  
Wild Type P53

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

scholarly journals Effect of the Molecular Nature of Mutation on the Efficiency of Intrachromosomal Gene Conversion in Mouse Cells

Genetics ◽  
1987 ◽  
Vol 117 (4) ◽  
pp. 759-769
Author(s):  
Anthea Letsou ◽  
R Michael Liskay
Keyword(s):  
Gene Conversion ◽  
Mammalian Cells ◽  
Directed Path ◽  
Wild Type ◽  
Heteroduplex Dna ◽  
Single Base ◽  
Conversion Rates ◽  
Mouse Cells ◽  
Single Base Pair ◽  
Molecular Nature

ABSTRACT With the intent of further exploring the nature of gene conversion in mammalian cells, we systematically addressed the effects of the molecular nature of mutation on the efficiency of intrachromosomal gene conversion in cultured mouse cells. Comparison of conversion rates revealed that all mutations studied were suitable substrates for gene conversion; however, we observed that the rates at which different mutations converted to wild-type could differ by two orders of magnitude. Differences in conversion rates were correlated with the molecular nature of the mutations. In general, rates of conversion decreased with increasing size of the molecular lesions. In comparisons of conversion rates for single base pair insertions and deletions we detected a genotype-directed path for conversion, by which an insertion was converted to wild-type three to four times more efficiently than was a deletion which maps to the same site. The data are discussed in relation to current theories of gene conversion, and are consistent with the idea that gene conversion in mammalian cells can result from repair of heteroduplex DNA (hDNA) intermediates.

scholarly journals Complementation of mutant and wild-type human mitochondrial DNAs coexisting since the mutation event and lack of complementation of DNAs introduced separately into a cell within distinct organelles

1994 ◽  
Vol 14 (4) ◽  
pp. 2699-2712
Author(s):  
M Yoneda ◽  
T Miyatake ◽  
G Attardi
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Wild Type ◽  
Mitochondrial Dna Mutation ◽  
Experimental Conditions ◽  
Chloramphenicol Resistance ◽  
Dna Mutation ◽  
Mitochondrial Dnas ◽  
A Cell ◽  
Type Gene

The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

scholarly journals The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

2020 ◽  
Author(s):  
William A. Michaud ◽  
Genevieve M. Boland ◽  
S. Alireza Rabi
Keyword(s):  
Viral Entry ◽  
Vaccine Development ◽  
Viral Evolution ◽  
Wild Type ◽  
Single Cycle ◽  
Competition Assay ◽  
Functional Studies ◽  
Wide Range ◽  
Direct Competition ◽  
A Cell

AbstractEarly in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.

scholarly journals Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 400-406 ◽  
Author(s):  
W P Deng ◽  
J A Nickoloff
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Wild Type ◽  
Heteroduplex Dna ◽  
Frameshift Mutations ◽  
Single Strand Annealing ◽  
Extrachromosomal Recombination ◽  
Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

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