DNA, Volume 2, Issue 1 (March 2022) – 7 articles

Endogenous retroviruses (ERVs) represent genomic components of retroviral origin that are found integrated in the genomes of various species of vertebrates. These genomic elements have been widely characterized in model organisms and humans. However, composition and abundances of ERVs have not been categorized fully in all domestic animals. The advent of next generation sequencing technologies, development of bioinformatics tools, availability of genomic databases, and molecular cytogenetic techniques have revolutionized the exploration of the genome structure. Here, we investigated the nature, abundance, organization and assembly of ERVs and complete genomes of Jaagsiekte sheep retrovirus (JSRV) from high-throughput sequencing (HTS) data from two Iraqi domestic sheep breeds. We used graph-based read clustering (RepeatExplorer), frequency analysis of short motifs (k-mers), alignment to reference genome assemblies and fluorescent in situ hybridization (FISH). Three classes of ERVs were identified with the total genomic proportions of 0.55% from all analyzed whole genome sequencing raw reads, while FISH to ovine metaphase chromosomes exhibited abundant centromeric to dispersed distribution of these ERVs. Furthermore, the complete genomes of JSRV of two Iraqi sheep breeds were assembled and phylogenetically clustered with the known enJSRV proviruses in sheep worldwide. Characterization of partial and complete sequences of mammalian ERVs is valuable in providing insights into the genome landscape, to help with future genome assemblies, and to identify potential sources of disease when ERVs become active. Full article

DNA replication stress is a constant threat that cells must manage to proliferate and maintain genome integrity. DNA replication stress responses, a subset of the broader DNA damage response (DDR), operate when the DNA replication machinery (replisome) is blocked or replication forks collapse during S phase. There are many sources of replication stress, such as DNA lesions caused by endogenous and exogenous agents including commonly used cancer therapeutics, and difficult-to-replicate DNA sequences comprising fragile sites, G-quadraplex DNA, hairpins at trinucleotide repeats, and telomeres. Replication stress is also a consequence of conflicts between opposing transcription and replication, and oncogenic stress which dysregulates replication origin firing and fork progression. Cells initially respond to replication stress by protecting blocked replisomes, but if the offending problem (e.g., DNA damage) is not bypassed or resolved in a timely manner, forks may be cleaved by nucleases, inducing a DNA double-strand break (DSB) and providing a means to accurately restart stalled forks via homologous recombination. However, DSBs pose their own risks to genome stability if left unrepaired or misrepaired. Here we focus on replication stress response systems, comprising DDR signaling, fork protection, and fork processing by nucleases that promote fork repair and restart. Replication stress nucleases include MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, and FEN1. Replication stress factors are important in cancer etiology as suppressors of genome instability associated with oncogenic mutations, and as potential cancer therapy targets to enhance the efficacy of chemo- and radiotherapeutics. Full article

DNA nanoengineering, in particular, DNA origami has potential applications in a variety of areas including, for example, nanoelectronics, biomedical diagnostics, and therapeutics. To fully realize the potential of DNA self-assembly in these and other areas, methods must be available for economical, scalable, and reliable production of single-stranded DNA (ssDNA) scaffolds from virtually any source. In this review, we will describe the virtues and liabilities of four strategies for generating ssDNA, including Rolling Circle Amplification (RCA), strand-specific exonuclease digestion, chemical denaturation, and asymmetric PCR (aPCR), with suggestions for approaches to optimize the use of each method. Full article

Cell lines are a widely used pre-clinical models for biomedical research. The accessibility and the relative simplicity of facilities necessary for the use of cell lines, along with the large number of potential applications, encourage many researchers to choose this model. However, the access to cell lines from a non-confident source or through the interlaboratory exchange results in uncontrollable cell lines of uncertain quality. Furthermore, the possibility of using cell lines as an endless resource through multiple passages can contribute to this uncontrolled scenario, the main consequence of which is the lack of reproducibility between the research results. Different initiatives have emerged to promote the best practices regarding the use of cell lines and minimize the effect on the scientific results reported, including comprehensive quality control in the frame of Good Cell Culture Practice (GCCP). Cell Banks, research infrastructures for the professional distribution of biological material of high and known quality and origin, are committed with these initiatives. Many of the quality controls used to test different attributes of cell lines are based on DNA. This review describes quality control protocols of cell lines whose target molecule is DNA, and details the scope or purpose and their corresponding functionality. Full article

To date, our understanding of how DNA is packaged in the cell nucleus, condensed from chromatin into chromosomes, and organized throughout the cell cycle remains sparse. Three dimensional (3D) ultrastructural imaging is an important tool for unravelling the organizational structure of chromosomes. For large volume 3D imaging of biological samples, serial block-face scanning electron microscopy (SBFSEM) has been applied, whereby ultrastructural information is achieved by analyzing 3D reconstructions acquired from measured data sets. In this review, we summarize the contribution of SBFSEM for obtaining 3D images of chromosomes to investigate their ultrastructure and organization in the cell and its nucleus. Furthermore, this review highlights the potential of SBFSEM for advancing 3D chromosome research. Full article

Avian cell culture is widely applied for cytogenetic studies, the improvement of which increasingly allows for the production of high-quality chromosomes, essential to perform both classical and molecular cytogenetic studies. Among these approaches, there are two main types: fibroblast and bone marrow culture. Despite its high cost and complexity, fibroblast culture is considered the superior approach due to the quality of the metaphases produced. Short-term bone marrow cultivation provides more condensed chromosomes but nonetheless is quicker and easier. In the search for a quicker, cheaper way to prepare metaphases without losing quality, the present work developed a novel, widely applicable protocol for avian chromosome preparation. Twenty-one bird embryos from distinct families were sampled: Icteridae, Columbidae, Furnariidae, Estrildidae, Thraupidae, Troglodytidae and Ardeidae. The protocol was based on a combination of modified fibroblast culture and bone marrow cultivation, taking the advantages of both. The results show that all species consistently presented good mitotic indexes and high-quality chromosomes. Overall, the application of this protocol for bird cytogenetics can optimize the time, considering that most fibroblast cultures take at least 3 days and often much longer. However, our protocol can be performed in 3 h with a much-reduced cost of reagents and equipment. Full article

Zinc fingers consist of one of the most abundant motifs in transcription factors and DNA-binding proteins. Recent studies provide evidence on the pathological implication of zinc finger proteins in various neurodevelopmental disorders and malignancies but their role in pediatric brain tumors is largely unexplored. To this end, we investigated the differential expression of zinc finger-containing genes along with relevant biological processes and pathways among four main brain tumor categories (pilocytic astrocytomas, ependymomas, medulloblastomas and glioblastomas). By employing an extended bioinformatic toolset, we performed a preliminary in silico study in order to identify the expression of zinc finger-containing genes and associated functions in pediatric brain tumors. Our data analysis reveals the prominent role of C₂H₂-type zinc finger-containing genes in the molecular mechanisms underlying pediatric brain tumors followed by the Ring and PHD finger types. Significant dysregulation of ABLIM2 and UHFR1 genes was detected in all tumor types drawing attention to the dysregulation of cell polarization process and Ubiquitin-Proteasome System (UPS) in the pathogenesis of pediatric brain tumors. Moreover, significant gene clustering was observed in multiple locations with two highly visible clusters revealing a contrast in gene regulation between medulloblastomas and the other three brain tumor types, indicating a promising area of future research. Full article