Voices of biotech leaders

Nature Biotechnology asks a selection of leaders from across biotech to look at the future of the sector and make some predictions for the coming years

Voices of biotech leaders

Nature Biotechnology asks a selection of leaders from across biotech to look at the future of the sector and make some predictions for the coming years.</p

Publisher Correction:Voices of biotech leaders (Nature Biotechnology, (2021), 39, 6, (654-660), 10.1038/s41587-021-00941-4)

In the version of this article initially published, an author name was given as Abasi Ene Abong. The correct name is Abasi Ene-Obong. Also, the affiliation for Sebastian Giwa was given as Elevian, Pagliuca Harvard Life Lab, Allston, MA, USA. The correct affiliations are Biostasis Research Institute, Berkeley, CA, USA; Sylvatica Biotech, North Charleston, SC, USA; and Humanity Bio, Kensington, CA, USA. An affiliation for Jeantine Lunshof was given as Department of Genetics, Harvard Medical School, Boston, MA, USA. The correct affiliation is Wyss Institute for Biological Engineering, Harvard University, Boston, MA, USA. The errors have been corrected in the PDF and HTML versions of the article

Differential Requirements for MCM Proteins in DNA Replication in Drosophila S2 Cells

BackgroundThe MCM2-7 proteins are crucial components of the pre replication complex (preRC) in eukaryotes. Since they are significantly more abundant than other preRC components, we were interested in determining whether the entire cellular content was necessary for DNA replication in vivo.Methodology/Principle FindingsWe performed a systematic depletion of the MCM proteins in Drosophila S2 cells using dsRNA-interference. Reducing MCM2-6 levels by >95–99% had no significant effect on cell cycle distribution or viability. Depletion of MCM7 however caused an S-phase arrest. MCM2-7 depletion produced no change in the number of replication forks as measured by PCNA loading. We also depleted MCM8. This caused a 30% reduction in fork number, but no significant effect on cell cycle distribution or viability. No additive effects were observed by co-depleting MCM8 and MCM5.Conclusions/SignificanceThese studies suggest that, in agreement with what has previously been observed for Xenopus in vitro, not all of the cellular content of MCM2-6 proteins is needed for normal cell cycling. They also reveal an unexpected unique role for MCM7. Finally they suggest that MCM8 has a role in DNA replication in S2 cells

Quantitation of cell cycle parameters of Drosophila MCM mutants.

<p>Quantitation of cell cycle parameters of Drosophila MCM mutants.</p

FACS analysis profiles of cells targeted with dsRNA against MCM2-7 proteins as compared to control targeted cells.

<p>The data presented is one complete representative data set from multiple repetitions.</p

Immunoblot to show the effect of MCM depletions on the levels of MCM2, MCM5 ORC5 and CDC45 bound to chromatin.

<p>Coomassie stained histones are included as a loading control. The amount of each protein was compared to the histones using Alpha Innotech imager software. Horizontal numbers show the MCM protein targeted by RNAi. Several repetitions of this experiment were performed and this represents 1 complete data set.</p

Immunoblot to show the effect of MCM depletions on the levels of PCNA bound to chromatin.

<p>Coomassie stained histones are included as a loading control. The amount of PCNA was compared to the histones using Alpha Innotech imager software. Horizontal numbers show the MCM protein targeted by RNAi.</p

Dpa¹ mutants show abnormal chromosome condensation and unorganised mitotic spindles.

<p>Neuroblasts of heterozygous or homozygous third instar larvae (prepared as in 4) were stained for: centrosomes (red) using CP190 antibodies (a kind gift from Will Whitfield) and Goat anti-rabbit TxRed (Jackson labs); spindles (green) using anti-tubulin (Clone DM1A-Sigma) and goat anti-mouse Alexa 488 (Jackson Labs); and DNA (blue) using DAPI (Sigma). Heterozygous neuroblasts showed normal spindles with correct localization of CP190 at the poles in metaphase (A/B) and anaphase (B), and normally condensed chromosomes. Homozygous neuroblasts (C/D) showed fragmented, hyper-condensed and aneuploid chromosomes, and disorganised mitotic spindles usually lacking CP190.</p