Copies Of Genetic Material Research Articles

Electron Microscopy for Structural Determination and Analysis of Viruses

Viruses are known to be associated with large-scale, dynamic conformational changes that take place to facilitate cell entry and genome delivery. It is also known that a replication machinery is involved in the advanced stage of the infectious cycle that enables to read and synthesize nucleic acid strands. This process results in the generation of new copies of genetic material. In this process, the function of structural proteins helps to assemble and package the appropriate contents to produce new infectious particles. Lately, there has been a great deal of research interest on structural elucidation of these events. This interest is primarily driven by the significance of virus structural identification, which helps understand these processes and also their inhibition by antiviral agents such as neutralizing antibodies and drugs. To this end, the development of electron microscopy (EM) techniques for studies in virology has played a major role for structural determination and analysis of viruses. In this mini-review, we have highlighted some of the latest developments in this field. We have briefly described important role of EM in virology. We have also discussed notable application examples of EM in elucidating various virus structures to gain insights into identification of deadly pathogens and other infectious agents and outbreaks along with anti-viral developmental strategies.

Rational Selection of CRISPR-Cas9 Guide RNAs for Homology-Directed Genome Editing

Homology-directed repair (HDR) of a DNA break allows copying of genetic material from an exogenous DNA template and is frequently exploited in CRISPR-Cas9 genome editing. However, HDR is in competition with other DNA repair pathways, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), and the efficiency of HDR outcomes is not predictable. Consequently, to optimize HDR editing, panels of CRISPR-Cas9 guide RNAs (gRNAs) and matched homology templates must be evaluated. We report here that CRISPR-Cas9 indel signatures can instead be used to identify gRNAs that maximize HDR outcomes. Specifically, we show that the frequency of deletions resulting from MMEJ repair, characterized as deletions greater than or equal to 3bp, better predicts HDR frequency than consideration of total indel frequency. We further demonstrate that tools that predict gRNA indel signatures can be repurposed to identify gRNAs to promote HDR. Finally, by comparing indels generated by S.aureus and S.pyogenes Cas9 targeted to the same site, we add to the growing body of data that the targeted DNA sequence is a major factor governing genome editing outcomes.

Template-Directed Catalysis of a Multistep Reaction Pathway for Nonenzymatic RNA Primer Extension.

Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5′-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed primer extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and primer extension. In the presence of the catalytic metal ion Mg2+, >90% of the template-bound intermediate reacts with the adjacent primer to generate the primer extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction.

Dissecting Virus Infectious Cycles by Cryo-Electron Microscopy.

In response to events such as receptor binding and endocytic triggers, viruses undergo large-scale, dynamic conformational changes necessary for cell entry and genome delivery. In later stages of the infectious cycle, replication machinery must read and synthesize nucleic acid strands to generate new copies of genetic material, and structural proteins must assemble and package the appropriate contents in order to produce new infectious particles. Structural elucidation of these events is key to understanding them and their inhibition by antiviral agents such as neutralizing antibodies and drugs. Electron microscopy is a versatile technique that offers the ability to resolve three-dimensional structures of individual viral proteins and whole virions in multiple functional states, even in cells at different stages of infection (Fig 1). Here we focus on the use of transmission electron microscopy of frozen-hydrated specimens, i.e., cryo-electron microscopy (cryo-EM). A major advantage of cryo-EM over other structural approaches is that samples of a broad range of sizes can be imaged under near-physiological conditions with native hydration intact. This approach does not require the specimen to be fixed, stained, or coaxed into a crystalline lattice. This versatility has enabled cryo-EM to expand the envelope of structural virology and opened new avenues for understanding the molecular and cellular processes of virus infection and pathogenesis. Fig 1 Cryo-electron microscopy (cryo-EM) enables one to gain insight into viruses over many levels of structural hierarchy, ranging from determination of near-atomic resolution structures of subunits and symmetrical virus assemblies to ultrastructural analysis ... In this micro-review, we examine general approaches and recent applications of cryo-EM to virology. The reader is referred to a number of excellent reviews for more in-depth discussion of current methodology (e.g., [1,2]). Electron microscopy and structural virology have a long, shared history dating to the first images of bacteriophages gathered using some of the earliest electron microscopes [3]. As the technique of cryogenically preserving unfixed samples was being developed, again, viruses were among the first test subjects that helped to demonstrate the utility of cryo-EM for characterizing structure of large biological assemblies [4–6].

The evolutionary enhancement of genotype-phenotype linkages in the presence of multiple copies of genetic material.

Genetic evolutionary mechanisms employed by protolife developed without accompanying regulatory mechanisms for the amounts of genetic material in protocells. When many copies of genetic material are present, inactive copies generated by mutations are not effectively excluded through phenotypic selection. We demonstrate a model of gene evolution initiated with different amounts of DNA inside artificial protocells. We adopted transcription- and translation-coupled RNA replication and liposome-based in vitro compartmentalization. Despite the fact that the average number of DNA copies in each liposome was 6.4, DNA encoding active genes was maintained until the 16th selection round. Our experimental and theoretical results indicated that gene evolution can occur in the presence of multiple DNA copies. Most genetic material became junk code through gene mutations, and consequently the linkage between genotype and phenotype was enhanced through the associated decreases in active genetic material.

High genotype diversity and lack of isolation by distance in theAlternaria solanipopulations fromChina

A genomic library was used to develop sevenSSRmarkers for studying the population genetics ofAlternaria solani, a pathogenic fungus causing early blight disease of potato and tomato worldwide. Population genetic analysis of 268 isolates ofA. solanisampled from four locations, each representing one of four potato production systems inChina, indicates that theseSSRmarkers are moderately diverse, selectively neutral and possibly unlinked. Population genetic analysis also indicated that genetic variation ofA. solaniinChina is high. About 2/3 of 123 genotypes were detected only once and genotype diversity measured by the standardizedShannon index ranged between 0·82 and 0·92 in the populations. Although clones were detected in multiple populations separated by thousands of kilometres, random association amongSSRloci was found in half of the populations assayed. On average, nearly six copies of genetic material were exchanged among these populations each generation and no isolation by distance was detected. It is hypothesized that the joint effects of cryptic sexual reproduction and human‐mediated gene flow may account for the observed population genetic structure ofA. solaniinChina.

Protein expression from exogenous mRNA: Uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains of Saccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in the G1 stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.