Semiconservative transmission of DNA N 6-adenine methylation in a unicellular eukaryote.

Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

A Simple and Rapid Cryopreservation Technique for Ciliates: A Long-Term Storage Procedure Used for Marine Scuticociliates.

Pseudocohnilembus persalinus is a free-living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, long-term laboratory maintenance for this species is mainly achieved by subculture that requires rigorous culture environments and, too often, cultures of the organism die out for a variety of reasons. Successful transport of viable cultures also poses problems for researchers. This study describes a simple and rapid protocol for long-term cryopreservation of P. persalinus. The effects of physiological states of individuals before freezing, the type and concentration of cryoprotectant, and optimal temperatures for freezing and thawing were assessed. A cryopreservation protocol, using a mixture of 30% glycerol and 70% concentrated P. persalinus cell culture, incorporating rate-controlled freezing at -80 °C before liquid nitrogen storage, maintained a high recovery efficiency after 8 wk of storage. These results suggest that broader application of this protocol to build a cryopreserved marine protozoa culture bank for biological studies may be possible.

Genome-wide identification of ATP-binding cassette transporter B subfamily, focusing on its structure, evolution and rearrangement in ciliates.

ATP-binding cassette subfamily B (ABCB) has been implicated in various essential functions such as multidrug resistance, auxin transport and heavy metal tolerance in animals and plants. However, the functions, the genomic distribution and the evolutionary history have not been characterized systematically in lower eukaryotes. As a lineage of highly specialized unicellular eukaryotes, ciliates have extremely diverse genomic features including nuclear dimorphism. To further understand the genomic structure and evolutionary history of this gene family, we investigated the ABCB gene subfamily in 11 ciliates. The results demonstrate that there is evidence of substantial gene duplication, which has occurred by different mechanisms in different species. These gene duplicates show consistent purifying selection, suggesting functional constraint, in all but one species, where positive selection may be acting to generate novel function. We also compare the gene structures in the micronuclear and macronuclear genomes and find no gene scrambling during genome rearrangement, despite the abundance of such scrambling in two of our focal species. These results lay the foundation for future analyses of the function of these genes and the mechanisms responsible for their evolution across diverse eukaryotic lineages.

Convolutional Neural Networks Based on Sequential Spike Predict the High Human Adaptation of SARS-CoV-2 Omicron Variants.

The COVID-19 pandemic has frequently produced more highly transmissible SARS-CoV-2 variants, such as Omicron, which has produced sublineages. It is a challenge to tell apart high-risk Omicron sublineages and other lineages of SARS-CoV-2 variants. We aimed to build a fine-grained deep learning (DL) model to assess SARS-CoV-2 transmissibility, updating our former coarse-grained model, with the training/validating data of early-stage SARS-CoV-2 variants and based on sequential Spike samples. Sequential amino acid (AA) frequency was decomposed into serially and slidingly windowed fragments in Spike. Unsupervised machine learning approaches were performed to observe the distribution in sequential AA frequency and then a supervised Convolutional Neural Network (CNN) was built with three adaptation labels to predict the human adaptation of Omicron variants in sublineages. Results indicated clear inter-lineage separation and intra-lineage clustering for SARS-CoV-2 variants in the decomposed sequential AAs. Accurate classification by the predictor was validated for the variants with different adaptations. Higher adaptation for the BA.2 sublineage and middle-level adaptation for the BA.1/BA.1.1 sublineages were predicted for Omicron variants. Summarily, the Omicron BA.2 sublineage is more adaptive than BA.1/BA.1.1 and has spread more rapidly, particularly in Europe. The fine-grained adaptation DL model works well for the timely assessment of the transmissibility of SARS-CoV-2 variants, facilitating the control of emerging SARS-CoV-2 variants.

An Optimized and Versatile Counter-Flow Centrifugal Elutriation Workflow to Obtain Synchronized Eukaryotic Cells.

Cell synchronization is a powerful tool to understand cell cycle events and its regulatory mechanisms. Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior and/or cell cycle progression, however, adjusting specific parameters in a cell type/equipment-dependent manner can be challenging. In this paper, we used the unicellular eukaryotic model organism, Tetrahymena thermophila as a testing system for optimizing CCE workflow. Firstly, flow cytometry conditions were identified that reduced nuclei adhesion and improved the assessment of cell cycle stage. We then systematically examined how to achieve the optimal conditions for three critical factors affecting the outcome of CCE, including loading flow rate, collection flow rate and collection volume. Using our optimized workflow, we obtained a large population of highly synchronous G1-phase Tetrahymena as measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into nascent DNA strands, bulk DNA content changes by flow cytometry, and cell cycle progression by light microscopy. This detailed protocol can be easily adapted to synchronize other eukaryotic cells.

FIS activates glnAp2 in Escherichia coli: role of a DNA bend centered at -55, upstream of the transcription start site.

A binding site for the Escherichia coli nucleoid binding protein FIS (factor for inversion stimulation) was identified upstream of a sigma54-dependent promoter, glnAp2. The binding and bending center of FIS is positioned at -55 with respect to the transcription start site (+1). Binding of FIS at this site activates the transcription of glnAp2 both in vivo and in vitro. Furthermore, we substituted the FIS-mediated DNA bending with other protein (cAMP receptor protein or integration host factor)-mediated DNA bending, without changing the position of the bending center. In vitro transcription assays indicated that all DNA bends centered at -55 activate transcriptional initiation of glnAp2, especially when linear templates were used.