Thus, our present protocol requires an additional fosmid DNA isolation step and transformation into a TrfA-producing strain to enable transient copy-number induction.
It is transient, and eternal.
However, with the help of the ST-VSG, the DGS delivers the least transient power to copy with the load mutation as can be seen in Fig. 7c.
In this case, non-replicating mtDNAs are removed and replicating mtDNAs are capable of fast enough replication to survive the transient drop in copy number.
One of the critical factors for transient transfection is plasmid copy number (PCN), for which we here provide an optimized qPCR based protocol.
However, this regime yields a transient period of mtDNA copy number depletion, while the non-replicating mtDNAs are degrading but the (small) population of replicating agents remains low.
However, with multiple, nonoverlapping replication forks on archaeal chromosomes the transient variation in gene copy number during the cell cycle should be significantly reduced.
Collectively, this defective process, caused by the lack of RNase H1, ceased the progress of DNA synthesis in the strand-coupled DNA replication and prevented restoration of mtDNA copy number after transient mtDNA depletion.
That knockdown of DNA2 or both DNA2 and FEN1 in the present investigation did not induce even a modest delay in the recovery of mtDNA copy number after transient mtDNA depletion or any increase of susceptibility of the replication intermediates to S1 nuclease fits well with the above hypothesis.
Although the reason for the presence of the two replication modes for mtDNA is unclear, a substantial fraction of the mtDNA replication intermediates are products of the strand-coupled DNA synthesis mode, and this mode appears to be predominant when cells amplify mtDNA to restore the copy number after transient mtDNA depletion [3,5].
To avoid potential saturation effects resulting from multi-copy expression in transient transfections, we generated cell lines expressing a stably integrated version of the wildtype or sequence-optimized mip-1α gene.
