Actin Contributes to the Hyperexpression of Baculovirus Polyhedrin (polh) and p10 as a Component of Transcription Initiation Complex (TIC)

Hyperexpression of polh and p10, two very late genes, is one of the remarkable characteristics in the baculovirus life cycle. However, the mechanisms underlying the hyperexpression of these two genes are still incompletely understood. In this study, actin was identified as a highly potential binding partner of polh and p10 promoters by conducting DNA pull-down and LC–MS/MS analyses. Inhibiting actin dynamics delayed and decreased the transcription of polh and p10. Actin interacted with viral RNA polymerase and transcription regulators, and the nuclear import of viral polymerase was inhibited with the disruption of actin dynamics. Simultaneously, the high enrichment of actin in polh and p10 promoters discovered via a chromatin immunoprecipitation (ChIP) assay indicated that actin was a component of the viral polymerase TIC. Moreover, overexpression of actin surprisingly upregulated the expression of luciferase (Luc) under the control of polh and p10 promoters. Taken together, actin participated in the hyperexpression of polh and p10 as a component of TIC. These results facilitate the promotion of the expression efficiency of foreign genes in the baculovirus expression vector system (BEVS).

Comparative Review of Plant Transformation Techniques

Plant transformation is now an important biotechnological tool in plant biology and a practical tool for transgenic plant development. There are many verified methods for stable introduction of novel genes into the nuclear genomes of diverse plant species. As a result, gene transfer and regeneration of transgenic plants are no longer the factors limiting the development and application of practical transformation systems for many plant species. However, the desire for higher transformation efficiency has stimulated work on not only improving various existing methods but also in inventing novel methods. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect methods of gene transfer have their own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy and eco-friendly method to transfer foreign genes. Many methods of genetic transformation have been proposed and tried in the laboratories, but most of them result to transient expressions. However, transformation work based on particle bombardment with DNA coated micro projectiles and Agrobacterium mediated transformation have proved to be promising in producing stable transgenic plants from a range of plant species.

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

Precise Characterization and Tracking of Stably Inherited Artificial Minichromosomes Made by Telomere-Mediated Chromosome Truncation in Brassica napus

Plant artificial minichromosomes are the next-generation technology for plant genetic engineering and represent an independent platform for expressing foreign genes and the tools for studying the structure and function of chromosomes. Minichromosomes have been successfully produced by telomere-mediated chromosome truncation in several plants. However, previous studies have primarily focused on the construction and rough characterization of minichromosomes, while the development of stably inherited minichromosomes and their precise characterization and tracking over different generations have rarely been demonstrated. In this study, a 0.35-kb direct repeat of the Arabidopsis telomeric sequence was transformed into Brassica napus to produce artificial minichromosomes, which were analyzed by multifluorescence in situ hybridization (multi-FISH), Southern hybridization, and primer extension telomere rapid amplification (PETRA). The stably inherited minichromosomes C2 and C4 were developed by crossing transgenic plants with wild-type plants and then selfing the hybrids. Notably, two truncation sites on chromosomes C2 and C4, respectively, were identified by resequencing; thus, the artificial minichromosomes were tracked over different generations with insertion site-specific PCR. This study provided two stably inherited minichromosomes in oilseed rape and describes approaches to precisely characterize the truncation position and track the minichromosomes in offspring through multi-FISH, genome resequencing, and insertion site-specific PCR.

The zinc-finger bearing xenogeneic silencer MucR in α-proteobacteria balances adaptation and regulatory integrity

Foreign AT-rich genes drive bacterial adaptation to new niches while challenging the existing regulation network. Here we report that MucR, a conserved regulator in α-proteobacteria, balances adaptation and regulatory integrity in Sinorhizobium fredii, a facultative microsymbiont of legumes. Chromatin immunoprecipitation sequencing coupled with transcriptomic data reveal that average transcription levels of both target and non-target genes, under free-living and symbiotic conditions, increase with their conservation levels. Targets involved in environmental adaptation and symbiosis belong to genus or species core and can be repressed or activated by MucR in a condition-dependent manner, implying regulatory integrations. However, most targets are enriched in strain-specific genes of lower expression levels and higher AT%. Within each conservation levels, targets have higher AT% and average transcription levels than non-target genes and can be further up-regulated in the mucR mutant. This is consistent with higher AT% of spacers between −35 and −10 elements of promoters for target genes, which enhances transcription. The MucR recruitment level linearly increases with AT% and the number of a flexible pattern (with periodic repeats of Ts) of target sequences. Collectively, MucR directly represses AT-rich foreign genes with predisposed high transcription potential while progressive erosions of its target sites facilitate regulatory integrations of foreign genes.

Effect of Gold Nanoparticles Size Capped with Surfactant on the Transformation of Plasmid into Escherichia coli Bacteria

Bacterial transformation has great importance in molecular biology, as it is used for introduction of foreign genes into bacterial cells either chemical or physical ways. Using calcium chloride to prepare competent cells and heat shock is the most widely used method for bacterial transformation. This method is an efficient and convenient technique but it has in some extent low transformation efficiency. Here we report the use of nanoparticles that significantly improve the transformation efficiency up to 10 times higher than the standard heat shock method by the assistance of (˜ 15, 25 nm) SDS capped gold nanoparticles in the transformation process that leads to the formation of temporary nano-channels across the bacterial cell wall to deliver plasmids into cells. Transformation of bacteria with plasmid was examined using Β-galactosidase assay.

Bdelloid rotifers use hundreds of horizontally acquired genes against fungal pathogens

Obligately asexual lineages are typically rare and short-lived. According to one hypothesis, they adapt too slowly to withstand relentlessly coevolving pathogens. Bdelloid rotifers seem to have avoided this fate, by enduring millions of years without males or sex. We investigated whether bdelloids' unusual capacity to acquire non-metazoan genes horizontally has enhanced their resistance to pathogens. We found that horizontally transferred genes are three times more likely than native genes to be upregulated in response to a natural fungal pathogen. This enrichment was twofold stronger than that elicited by a physical stressor (desiccation), and the genes showed little overlap. Among hundreds of upregulated non-metazoan genes were RNA ligases putatively involved in resisting fungal toxins and glucanases predicted to bind to fungal cell walls, acquired from bacteria. Our results provide evidence that bdelloids mitigate a predicted challenge of long-term asexuality in part through their ability to acquire and deploy so many foreign genes.

Evaluation of a new viral vaccine vector in mice and rhesus macaques: J paramyxovirus (JPV)-expressing HA of influenza A virus H5N1

H5N1, an avian influenza virus, is known to circulate in many Asian countries like Bangladesh, China, Cambodia, Indonesia, and Vietnam. The current FDA-approved H5N1 vaccine has a moderate level of efficacy. A safe and effective vaccine is needed to prevent the outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in humans. Non-segmented negative-sense single-stranded viruses (NNSVs) are widely used as a vector to develop vaccines for humans, animals, and poultry. NNSVs stably express foreign genes without integrating with the host genome. J Paramyxovirus (JPV) is a non-segmented negative-strand RNA virus and a member of the proposed genus Jeilongvirus in the family Paramyxoviridae . JPV-specific antibodies have been detected in rodents, bats, humans, and pigs, but the virus is not associated with disease in any species other than mice. JPV replicates in the respiratory tract of mice and efficiently expresses the virus-vectored foreign genes in tissue culture cells. In this work, we explored JPV as a vector for developing an H5N1 vaccine using intranasal delivery. We incorporated hemagglutinin (HA) of H5N1 into the JPV genome by replacing the small hydrophobic (SH) gene to generate a recombinant JPV expressing HA (rJPV-ΔSH-H5). A single intranasal administration of rJPV-ΔSH-H5 protected mice from a lethal HPAI H5N1 challenge. Intranasal vaccination of rJPV-ΔSH-H5 in rhesus macaques elicited antigen-specific humoral and cell-mediated immune responses. This work demonstrates that JPV is a promising vaccine vector. IMPORTANCE HPAI H5N1 outbreak in Southeast Asia destroyed millions of birds. Transmission of H5N1 into humans resulted in deaths in many countries. In this work, we developed a novel H5N1 vaccine candidate using JPV as a vector and demonstrated that JPV is an efficacious vaccine vector in animals. NNSVs stably express foreign genes without integrating into the host genome. JPV, an NNSV, replicates efficiently in the respiratory tract and induces robust immune responses.

Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA

In eukaryotes, 5-methylcytosine is the predominant DNA base modification, followed by N6-methyladenine. However, N4-methylcytosine (4mC) is confined to bacteria. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, freshwater invertebrates with transposon-poor genomes that are rich in foreign genes, lack C5-methyltransferases but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT introduces 4mC into DNA, and its chromodomain shapes the "histone-read-DNA-write" architecture together with a "DNA-read-histone-write" SETDB1/eggless H3K9me3 histone methyltransferase variant preferentially binding 4mC-DNA, to maintain 4mC and silent chromatin at transposons and tandem repeats. Our results bring the third base modification into the eukaryotic repertoire, demonstrate how non-native DNA methyl groups can reshape complex epigenetic systems to suppress transposon proliferation, and establish horizontal gene transfer as the source of regulatory innovation in eukaryotes.