Epigenetic interventions for brain rejuvenation：anchoring age-related transposons

Highlights：1.Iron and homocysteine accumulation in aging neurons alter genomic methylation.2.The altered methylome reactivates neuronal cell cycle,enabling transposable element mobilization.3.mi R29/p53 axis restores age-related methylation shifts,reactivating neuronal plasticity.4.Augmentation of mi R-29/p53 axis may preempt neurodegenerative disorders.

Effects of intrinsic and extrinsic noises on transposons kinetics

The absolute concentration robustness （ACR） steady state of a biochemical system can protect against changing a large concentration of the system＇s components. In this paper, a minimal model of autonomous-nonautonomous transposons driven by intrinsic and extrinsic noises is investigated. The effects of intrinsic and extrinsic noises on ACR steady state of the transposons kinetics are studied by numerical simulations. It is found that the predator-prey-like oscillations around the ACR steady state are induced by the intrinsic or extrinsic noises. Comparing with the case of intrinsic noises, the extrinsic noises can inhibit the amplitude of oscillations of transposon kinetics. To characterize the predator-prey-like oscillations, we calculate the probability distributions and the normalized correlation functions of a system in the stability domain. With the increasing of noise intensity, the peak of the probability distribution is shifted from the ACR steady state to the trivial steady state. The normalized autocorrelation and cross-correlation functions indicate that the state of the predator-prey oscillator is transmitted to 50 successive generations at least.

New Insights into Nested Long Terminal Repeat Retrotransposons in Brassica Species

Long terminal repeat （LTR） retrotransposons, one of the foremost types of transposons, continually change or modify gene function and reorganize the genome through bursts of dramatic proliferation. Many LTR-TEs preferen-tially insert within other LTR-TEs, but the cause and evolutionary significance of these nested LTR-TEs are not well under-stood. In this study, a total of 1.52 Gb of Brassica sequence containing 2020 bacterial artificial chromosomes （BACs） was scanned, and six bacterial artificial chromosome （BAC） clones with extremely nested LTR-TEs （LTR-TEs density： 7.24/kb） were selected for further analysis. The majority of the LTR-TEs in four of the six BACs were found to be derived from the rapid proliferation of retrotransposons originating within the BAC regions, with only a few LTR-TEs originating from the proliferation and insertion of retrotransposons from outside the BAC regions approximately 5-23 Mya. LTR-TEs also pref-erably inserted into TA-rich repeat regions. Gene prediction by Genescan identified 207 genes in the 0.84Mb of total BAC sequences. Only a few genes （3/207） could be matched to the Brassica expressed sequence tag （EST） database, indicating that most genes were inactive after retrotransposon insertion. Five of the six BACs were putatively centromeric. Hence, nested LTR-TEs in centromere regions are rapidly duplicated, repeatedly inserted, and act to suppress activity of genes and to reshuffle the structure of the centromeric sequences. Our results suggest that LTR-TEs burst and proliferate on a local scale to create nested LTR-TE regions, and that these nested LTR-TEs play a role in the formation of centromeres.

Potential of transposon-mediated cellular reprogramming towards cell-based therapies

Induced pluripotent stem(iPS)cells present a seminal discovery in cell biology and promise to support innovative treatments of so far incurable diseases.To translate iPS technology into clinical trials,the safety and stability of these reprogrammed cells needs to be shown.In recent years,different non-viral transposon systems have been developed for the induction of cellular pluripotency,and for the directed differentiation into desired cell types.In this review,we summarize the current state of the art of different transposon systems in iPS-based cell therapies.

<i>At</i>L1 a Non-LTR Retrotrasposon Fragment in the Genome of <i>Arabidopsis thaliana </i>with Homology to Plants and Animals

We report the isolation of AtL1, a 249 bp non-LTR retrotransposon fragment from Arabidopsis thaliana by fingerprinting mRNAs extracted from A. thaliana plants, ecotype Columbia, in different heat stress conditions. Southern blot and PCR analysis suggested that AtL1 occurs as a single- or low-copy insert in the genome of A. thaliana ecotype Columbia. The presence of AtL1 in the genome of different Arabidopsis ecotypes was confirmed by PCR amplification and sequencing thus excluding all possible contamination. A preliminary scan of the AtL1 nucleotide sequence against the EMBL and NCBI databases revealed a high degree of similarity to a group of LINE type L1 retrotransposons of mammals and with a cDNA sequence of Artemisia annua. A phylogenetic analysis of LINE elements from animals and plants placed AtL1 and A. annua sequences in close proximity to some mammalian sequences but distant from the other plants LINE elements including those from Arabidopsis.

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.

Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics. Isolation of genomic segments flanking the insertion Mutator （Mu） is a key step in insertion mutagenesis studies. Herein, we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize. The method consists of the following steps： 1） double-digestion of genomic DNA with Bgl II/Msp I and ligation of digested fragments to the Bgl II- and Msp I-adaptors; 2） enrichment of a subset of Bgl II/Msp I fragments followed by selective amplification of the Mu-flanking fragments; 3） simultaneous display of AFLP bands derived from the flanking regions for both insert and native Mu transposons; 4） identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5） confirmation of flanking fragments related to these Mu insertions. Using this approach, we have isolated flanking fragment（s） resulting from Mu insertion for every Mu-induced mutant, and one such fragment, M196-FF, is found to contain a partial sequence of the DNA topoisomerase I gene Topl. Moreover, the modified AFLP method including all restriction enzymes, adaptors and primers has been optimized in this study. The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.

TRANSPOSABLE GENETIC ELEMENTS IN SPIRULINA AND POTENTIAL APPLICATIONS FOR GENETIC ENGINEERING

Transposable elements in cyanobacteria are briefly reviewed. Evidence is presentedto show that transposable elements in Spirulina platensis is actually reflected on the phenotype change, i.e., helical to straight filaments. Transposition intermediates of DNA were isolated from the extrachromo-some and the transposition was related to helical variations in Spirulina. Uses of transposable elements formicroalgal recombination are discussed based on the transposition mechanism.

Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4’-Phosphatase, PGN_0524

Aim To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis （P. gingivalis）, exhibits towards the cationic antimicrobial peptide, polymyxin B. Methodology A genetic screen of P. gingivalis clones generated by a Tn4400-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B （PMB, 50μg·mL^-1）. Results P. gingivalis （ATCC 33277） is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations （200μg·mL^-1）. Approximately 2,700 independent Tn4400 ＇-derived mutants ofP. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose （50 μg·mL^-1）. A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400＇ transposon was integrated into the gene encoding the lipid A 4＇-phosphatase, PGN 0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400＇, was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural （MALDI-TOF MS） analyses revealed that lipid A isolates from 0524-Tn4400＂ and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P gingivalis lipid A spectrum. Finally, intact 0524- Tn4400＇ and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 （TLR4）-dependent E-selectin expression in human endothelial cells relative to intact wild-type P.. gingivalis or its corresponding LPS isolate. Conclusion The combined molecular evidence provided in this report suggests that PGN 0524, a lipid A 4＇-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P.. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

PiggyBac Transposon Mediated Efficient eGFP Expression in Porcine Somatic Cells and Cloned Embryos

PiggyBac transposon has demonstrated its long-term and stable transposition on genomes of various species but lacking of the evidence on farm animal genomes. In this study, we constructed a piggyBac transposon marked with enhanced green fluorescent protein （eGFP） and showed efficient transposition in porcine somatic cells and cloned embryos. Our results demonstrated that piggyBac transposase could efficiently catalyze transposition in porcine fetal fibroblast cells, as well as in embryos. PiggyBac transposition generated 18-fold more eGFP-positive cell colonies compared to pEGFP-C1 random insertion mutagenesis, but excessive transposase might affect the transfection rate. Also piggyBac mediated 4-fold more eGFP expression than random insertion in cells and 17-fold in cloned embryos at mRNA level. When the mutagenized cells were used for somatic cell nuclear transfer （SCNT）, the cleavage rate and blastocyst rate of constructed embryos harboring piggyBac transposition had no difference with random insertion group. This study provides key information on the piggyBac transposon system as a tool for creating transgenic pigs.

Disruption of chemotaxis-related genes affects multiple cellular processes and the virulence of pathogenic Vibrio harveyi

Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-TnlO/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and iden- tified. Sequence analysis revealed that the 465-bp fragment （GenBank accession number HM630274） fank- ing the transposon insertion site in mutant TS-CM1 had the highest identity （96.9%） with a hypothetical protein gene of V. harveyiATCC BAA-1116 and the second-highest identity （91.8%） with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence （GenBank accession number HM630275） also showed the highest identity （94.6%） with a hypothetical pro- tein gene of V. harveyi ATCC BAA-1116 and the second-highest identity （92.4%） with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhe- sion, and infectivity of the mutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and viru- lence of the pathogenic E harueyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi.

Waxy allele diversity in waxy maize landraces of Yunnan Province, China

Waxy maize is one of the main fresh-eating maize types,and a mutation of the waxy gene causes the waxy character of maize grains.China is rich in waxy maize landraces,and Yunnan and its surrounding areas,are the place of origin and genetic diversity center of Chinese waxy maize.The six known waxy alleles of Chinese waxy maize are wx-D7,wx-D10,wx-Cin4,wx-124,wx-Reina,and wx-Xuanwei.The mutation sites of these alleles all occur in the coding region of the waxy gene,however,the mechanism by which the waxy characteristic is caused by the mutation in the regulatory region has only been reported rarely in maize.In this study,405 waxy maize landraces from Yunnan were used as materials to identify the insertion and deletion of a large sequence fragment in the upstream~3.5 kb regulatory region of the waxy gene by molecular marker detection.Three different waxy alleles were identifed in this study:wx-PIF/Harbinger,wx-hAT and wxElote2.These three types of mutations all represented transposons inserted into the regulatory region of the waxy gene.Wx-PIF/Harbinger was a 304-bp MITE class transposon insertion belonging to the PIF/Harbinger family,while wx-hAT was a 560-bp MITE class transposon insertion belonging to the hAT family,and wx-Elote2 was a 6560-bp LTR-like transposon insertion.In this study,the alleles were identifed for more than 70%of the waxy maize landraces in Yunnan,which provids a basis for the utilization of these waxy maize landraces.

Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation

Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.

Sequence-based genetic mapping of Ds-tagged insertions to characterize malting-related traits in barley

Among various functional genomics tools used to characterize genes in plants, transposonbased mutagenesis approaches offer great potential, especially in barley and wheat, which possess large genomes and in which genetic transformation is not routine. Two Ds transposon flanking sequences(TNPs), TNP-29(27.4 c M(centi Morgan)) and TNP-79(70.3 c M), were mapped in the vicinity of a malting quality QTL located on chromosome 4H of barley. Reactivation of the Ds transposon sequence from these TNP lines led to the identification of genes in the malting QTL regions. Several Ds(dissociation) lines were generated by crossing TNP-29 and TNP-79 with an Ac TPase(activator) expressing line(25-B), and F2 progenies were subsequently screened for Ds insertions at new locations. To further characterize these Ds mutants, we mapped the new Ds flanking sequences on a barley genetic map and found that 29% of Ds were located in regions associated with the malting QTL located on chromosome 4H and in close proximity to other important malting-associated QTL across the barley chromosome. Using a sequence based approach, a linkage map was generated that confirmed the position of Ds loci in the barley genome map. Locating these Ds loci on the barley map opens avenues to dissect important malting QTL for facilitating identification of candidate malting genes.

Identification of genes involved in regulating MnSOD2 production and root colonization in Bacillus cereus 905

sodA2-encoding manganese-containing superoxide dismutase(MnSOD2)in Bacillus cereus 905 plays an essential role in antioxidative stress,nutrition utilization,rhizosphere and phyllosphere colonization.However,the genes involved in regulating the sod A2 expression have not been clearly elucidated in B.cereus.In this study,a genome-wide random insertion mutagenesis was constructed by using transposon TnYLB-1 to identify novel genes regulating the sodA2 expression.Seven mutants that changed the sodA2 expression at both m RNA and protein levels were finally obtained.Sequence analysis and BLAST data showed that the genes disrupted by TnYLB-1 in B.cereus 905 shared high conservations with those in the B.cereus type strain,ATCC 14579.These genes encode heat-inducible transcription repressor,chloride channel protein,recombinase A,ferrous iron transport protein,nucleoside diphosphate kinase,and histidine ammonia-lyase.Besides,we also provided the evidence that the genes regulating the sodA2 expression could influence colonization ability of B.cereus 905 on wheat roots.Specifically,those genes downregulating the sodA2 expression significantly reduced bacterial colonization on wheat roots,and mutants with increased MnSOD2 activities could enhance bacterial population densities on wheat roots to a certain degree.Our work provided information that multiple genes are involved in MnSOD2 production and wheat root colonization.The molecular regulatory pathways through which these genes modulate the sod A2 expression and root colonization need to be investigated extensively in the future.

Transformation of dissociation transposon in japonica rice Zhonghua 11 mediated by Agrobacterium

Dissociation (Ds) transposon is one of thetransposable elements in corn. The trans-posons can be transferred into other plantswhere the transposons were not found. Oncethe transposon was inserted into target gene ofthese plants, it could be used as a marker todistinguish and isolate the gene. The object ofthis study is to transfer Ds transposon to riceby Agrobacterium -mediated transformation.The calli of immature embryos, mature

Establishment of Real-Time Quantitative PCR Method for the Determination of Transposon Copy Number in Cronobacter sakazakii

[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.

高压静脉注射转染法建立脂源性炎症介导的肝癌小鼠模型

目的利用高压静脉注射转染法经小鼠尾静脉转染蛋白激酶B(AKT)、β联蛋白(β-catenin)及睡美人转座子质粒建立伴脂肪肝的炎癌转化小鼠肝癌模型并对该模型进行鉴定。方法选取6~8周龄雄性C57BL/6小鼠,通过高压静脉注射转染法在短时间内(5~9 s)经尾静脉注射含目的DNA的等渗生理溶液(20μg myrAKT、20μgΔN90-β-catenin和4μg Sleeping Beauty transposase溶于2 mL生理盐水),对照组采用相同方法注射对照质粒。于建模8、12、16周后,处死小鼠并收集血清及肝组织,提取RNA。HE染色检测肝脏组织结构及炎性细胞浸润程度;油红O染色检测肝细胞内脂质沉积情况;天狼星红和Masson染色检测肝组织纤维化程度;免疫组织化学染色检测肝组织KI-67抗原(ki67)表达显示细胞增殖活性、检测CD31表达显示微血管密度及分布情况;实时荧光定量PCR检测肝组织肿瘤标志物甲胎蛋白(AFP)的mRNA表达;流式细胞术检测肝组织内各免疫细胞亚群数量变化。结果小鼠经高压静脉注射转染法建模16周后,实验组小鼠成瘤率100%(20/20)。与对照组相比,实验组小鼠体质量增长缓慢,肝质量显著增加。HE染色结果显示,大量肝细胞出现脂肪变,炎性细胞浸润增多,肝癌组织内肿瘤细胞为类圆形或不规则形,核质比失调,异型性显著,呈团块状分布;ki67阳性细胞数增加。血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、γ谷氨酰转肽酶(γ-GT)、碱性磷酸酶(ALP)水平显著升高,AFP mRNA表达显著上升,CD31表达检测显示微血管密度增加且结构紊乱。流式细胞术分析肝脏中大量炎性细胞浸润,且以髓系及淋巴系为主。结论通过高压静脉注射转染法在肝细胞中激活AKT和β-catenin成功建立了伴发脂肪肝的炎癌转化小鼠肝癌模型,且方法简便,诱导时间短,成瘤率高,重复性强。

Characterization of Avirulent TnphoA Mutants in <i>Agrobacterium tumefaciens</i>to Enhance Transformation Efficiency

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane protein. Agrobacterium strain A6007 was mutagenized with E. coli strain mm294A plasmid pRK609 having TnphoA to obtain mutants defective in virulence. Because alkaline phosphatase activity is only detected when the PhoA gene product from the transposon is secreted out of the protoplasm, the virulence mutants are located in genes that code for transmembrane or periplasmic proteins. Attempts were made to obtain the sequences adjacent to the TnphoA inserts through several different approaches including Inverse PCR, Cloning, and Tail PCR. Transposon-adjacent sequence was obtained from one membrane anchor subunit in Bradyrhizobium japonicum i.e. succinate dehydrogenase which has enhanced transformation efficiency.

Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS)

Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.