Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Conserved and Widespread Expression of piRNA-Like Molecules and PIWI-Like Genes Reveal Dual Functions of Transposon Silencing and Gene Regulation in Pinctada fucata (Mollusca)

PIWI proteins and PIWI-interacting RNAs (piRNAs) suppress transposon activity in animals, thus safeguarding the genome from detrimental insertion mutagenesis. Recent studies revealed additional targets and functions of piRNAs in various animals. piRNAs are ubiquitously expressed in somatic tissues of the pearl oyster Pinctada fucata, however, the role of somatic piRNAs has not well characterized. This study reports the PIWI/piRNA pathway, including piRNA biogenesis and piRNA-mediated transposon silencing, and gene regulation in P. fucata. The biogenesis factors of PIWI, Zucchini, and HEN1, which are ubiquitous in somatic and gonadal tissues, were first identified in P. fucata using transcriptome analysis. Bioinformatics analyses suggested that different populations of piRNAs participate in the ping-pong amplification loop in a tissue-specific manner. In addition, a total of 69 piRNA clusters were identified in the genome of P. fucata based on the expression of piRNAs, which contained 26% transposons and enhanced for DNA/Crypton, LINE/CR1, SINE/Deu, and DNA/Academ. The expression patterns of the piRNAs and piRNA clusters in somatic tissues were not substantially different, but varied significantly between the somatic and gonadal tissues. Furthermore, locked-nucleic-acid modified oligonucleotide (LNA-antagonist) was used to silence single piRNA (piRNA0001) expression in P. fucata. Hundreds of endogenous genes were differentially expressed after piRNA silencing in P. fucata. Target prediction showed that some endogenous genes were targeted by piRNA0001, including twelve upregulated and nine downregulated genes after piRNA0001 silencing. The results indicated that piRNAs from somatic tissues may be related to gene regulation, whereas piRNAs from gonadal tissues are more closely associated to transposon silencing. This study will enhance our understanding of the role of piRNAs in mollusks, transposon silencing, and the regulatory function of the PIWI/piRNA pathway on protein-coding genes outside of germ line cells in P. fucata.

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

Background Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. Results We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. Conclusions Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

Hydroxyurea induces a stress response that alters DNA replication and nucleotide metabolism in Bacillus subtilis

Hydroxyurea (HU) is classified as a ribonucleotide reductase (RNR) inhibitor and has been widely used to stall DNA replication by depleting deoxyribonucleoside triphosphate (dNTP) pools. Recent evidence in E. coli shows that HU readily forms breakdown products that damage DNA directly, indicating that toxicity is a result of secondary effects. Because HU is so widely used in the laboratory and as a clinical therapeutic, it is important to understand the biological effects of HU. To determine how Bacillus subtilis responds to HU-induced stress, we performed saturating transposon insertion mutagenesis followed by deep-sequencing (Tn-seq), transcriptomic analysis (RNA-seq), and measured replication fork progression. Our data show that B. subtilis cells elongate and replication fork progression is slowed following HU challenge. The transcriptomic data show that B. subtilis cells initially mount a metabolic response likely caused by dNTP pool depletion before inducing the DNA damage response (SOS) after prolonged exposure. To compensate for reduced nucleotide pools, B. subtilis upregulates the purine and pyrimidine biosynthetic machinery and downregulates the enzymes producing ribose 5-phosphate. We show that overexpression of RNR genes nrdEF suppresses the growth interference caused by HU, suggesting that RNR is an important target of HU in B. subtilis. Although genes involved in nucleotide and carbon metabolism showed considerable differential expression, we also find that genes of unknown function (y-genes) represent the largest class of differentially expressed genes. Deletion of individual y-genes caused moderate growth interference in the presence of HU, suggesting that cells have several ways of coping with HU-induced metabolic stress. IMPORTANCE Hydroxyurea (HU) has been widely used as a clinical therapeutic and an inhibitor of DNA replication. Some evidence suggests that HU inhibits ribonucleotide reductase depleting dNTP pools while other evidence shows that toxic HU breakdown products are responsible for growth inhibition and genotoxic stress. Here, we use multiple, complementary approaches to characterize the response of Bacillus subtilis to HU. B. subtilis responds by upregulating expression of purine and pyrimidine biosynthesis. We show HU challenge reduced DNA replication, and that overexpression of the ribonucleotide reductase operon suppressed growth interference on HU. Our results demonstrate that HU targets RNR and several other metabolic enzymes contributing to toxicity in bacteria.

Large scale genomic rearrangements in selectedArabidopsis thalianaT-DNA lines are caused by T-DNA insertion mutagenesis

BackgroundExperimental proof of gene function assignments in plants is heavily based on mutant analyses. T- DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions ofArabidopsis thalianagenes during the last decades.ResultsWe sequenced the genomes of 14A. thalianaGABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, suggesting that the number of T-DNA insertions per line was underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10% of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days.ConclusionLong read sequencing was demonstrated to be a very effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype- to-phenotype relations.

BipA exerts temperature-dependent translational control of biofilm-associated colony morphology in Vibrio cholerae

Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22°C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae’s complex biofilm regulatory network.

Whole Genome Sequencing and Tn5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Potential silencing of gene expression by PIWI-interacting RNAs (piRNAs) in somatic tissues in mollusk

PIWI/piRNA suppress transposon activity in animals, thereby safeguarding the genome from detrimental insertion mutagenesis. Recently, evidence revealed additional piRNA targets and functions in various animals. Although piRNAs are ubiquitously expressed in somatic tissues of the pearl oyster Pinctada fucata, their role is not well-characterized. Here, we report a PIWI/piRNA pathway, including piRNA biogenesis and piRNA-mediated gene regulation in P. fucata. A locked-nucleic-acid modified oligonucleotide (LNA-antagonist) was used to silence a single piRNA (piRNA0001) expression in P. fucata, which resulted in the differential expression of hundreds of endogenous genes. Target prediction analysis revealed that, following silencing, tens of endogenous genes were targeted by piRNA0001, including twelve up-regulated and nine down-regulated genes. Bioinformatic analyses suggested that different piRNA populations participate in the ping-pong amplification loop in a tissue-specific manner. These findings have improved our knowledge of the role of piRNA in mollusks, and provided evidence to understand the regulatory function of the PIWI/piRNA pathway on protein-coding genes outside of germline cells.

Loss of ZIP facilitates JAK2-STAT3 activation in tamoxifen-resistant breast cancer

Tamoxifen, a widely used modulator of the estrogen receptor (ER), targets ER-positive breast cancer preferentially. We used a powerful validation-based insertion mutagenesis method to find that expression of a dominant-negative, truncated form of the histone deacetylase ZIP led to resistance to tamoxifen. Consistently, increased expression of full-length ZIP gives the opposite phenotype, inhibiting the expression of genes whose products mediate resistance. An important example isJAK2. By binding to two specific sequences in the promoter, ZIP suppressesJAK2expression. Increased expression and activation of JAK2 when ZIP is inhibited lead to increased STAT3 phosphorylation and increased resistance to tamoxifen, both in cell culture experiments and in a mouse xenograft model. Furthermore, data from human tumors are consistent with the conclusion that decreased expression of ZIP leads to resistance to tamoxifen in ER-positive breast cancer.

The NHEJ Repair of DNA Double Strand Breaks in Physcomitrella patens Depends on the Kleisin NSE4 of the SMC5/6 Complex

Structural maintenance of chromosomes (SMC) complexes are involve in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play imminent role in DNA stabilization during repair by encircling DNA at the site of lesion by bridging the heteroduplex of SMC5 and SMC6 by non SMC kleisin components NSE1, 3 and 4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to genotoxic stress, kinetics of DSB repair and insertion mutagenesis. In the moss Physcomitrella patens SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight attenuation of transcription by dCas9 binding was enough to obtain stable lines with DSB repair defect and specific bleomycin sensitivity. Whereas survival after bleomycin or MMS treatment fully depends on active SMC6, NSE4 has little or negligible effect. We conclude that whereas circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the immediate NHEJ DSB repair response, other functions associated with SMC5/6 complex are critical to survive DNA damage.