In PNAS this week, University of Oxford researchers present their replication and segregation of an Escherichia coli chromosome with two replication origins. The team constructed E. coli cells with two wild-type origins separated by one megabase. "Productive bidirectional replication initiated synchronously at both spatially separate origins. Newly replicated DNA from both origins was segregated sequentially as replication progressed, with two temporally and spatially separate replication termination events," the authors write. They found that replication initiation occurs at a cell volume identical to that of cells with a single origin, "showing that initiation control is independent of cellular and chromosomal oriC concentration."
Also in PNAS this week, researchers in South Korea say that microRNA-mediated posttranscriptional regulation is required for maintaining undifferentiated properties of blastoderm and primordial germ cells in chickens. The team conducted miRNA expression profiling to test specific miRNA signatures in undifferentiated blastoderm and primordial germ cells, and found that seven miRNAs are highly expressed in blastoderms and 10 are expressed in primordial germ cells. When they further analyzed miR-302a and miR-456 for blastoderm and miR-181a* for primordial germ cells, the researchers found that "both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation." They add, "Moreover, miR-181a* showed a bifunctional role in primordial germ cells by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of primordial germ cells by silencing homeobox A1 expression. Additionally, miR-181a* prevented primordial germ cells from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript."
Researchers in France explore direct visualization of siRNA electrotransfer into cancer cells at the single cell level in PNAS this week. The team investigates the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing EGFP. "The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode," the authors write. They found that siRNA was inefficient for gene silencing because it didn't penetrate the cells when added after electropulsation. Therefore, the team suggests, an electric field can act on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules.
And finally in PNAS this week, researchers at Columbia University in New York report that p53 binding to nucleosomes within the p21 promoter in vivo leads to nucleosome loss and transcriptional activation. In their study, the authors show that both distal and proximal p53 response elements within the promoter of the p21 gene in unstressed HCT116 colon carcinoma cells are "localized within a region of relatively high nucleosome occupancy." When the cells are unstressed, p53 is prebound to both p21 response elements within the nucleosomal DNA of these cells, but treatment of the cells with doxorubicin or Nutlin-3 is accompanied by p53-dependent subsequent loss of nucleosomes associated with such p53 response elements, the team writes. "We show that in vitro p53 can bind to mononucleosomal DNA containing the distal p21 response element, provided the binding site is not close to the diad center of the nucleosome. In line with this, our data indicate that the p53 distal response element within the p21 gene is located close to the end of the nucleosome," it adds.