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-nonautono...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.展开更多
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 pref...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.展开更多
[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.Fals...[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.False positive was identified by PCR and then removed.Mice were infected to detect the virulence of mutants.The bionomics of attenuated strains were detected,too.[Result] The attenuated mutants showed similar reproductive activity to that of wild strain.The virulence of mutants was still stable after 30 passages.[Conclusion] This study provided foundation for exploring the virulence factors and pathogenic mechanism of HPS.展开更多
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 ...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.展开更多
[Objective]The research aimed to construct deficient strain generated by Selenomonas ruminantium mutant with the acetic acid and analyze its fermentation characteristics.[Method]Based on the transposon tagging method,...[Objective]The research aimed to construct deficient strain generated by Selenomonas ruminantium mutant with the acetic acid and analyze its fermentation characteristics.[Method]Based on the transposon tagging method,Selenomonas ruminantium（recipient strain）was carried out the transposon mutagenesis via the transposon donor strain E.coli S17-1/pZJ25∷Tn5.The zygote was screened by using the selective medium which included kanamycin and sodium fluoroacetate.[Result]Seven transposon engineered strains which had the stable resistance to kanamycin and fluoroethanoic acid were screened.Selenomonas ruminantium mutant was carried out 16S rRNA and Tn5 PCR identification.Moreover,the specific activities of AK and PTA were analyzed.The mutant belonged to fluoroethanoic acid resistance strain with pta gene deficiency.[Conclusion]The research laid the foundation for further studying the cellular metabolic network and regulation of acetic acid in rumen microorganism of ruminant animal.展开更多
[Objective]The research aimed at investigating mechanisms of the changes of pharbitis.nil flower colors and leaf colors.[Method]The recombinant plasmid pMD18-T/Tpn was constructed by amplifying the target fragments fr...[Objective]The research aimed at investigating mechanisms of the changes of pharbitis.nil flower colors and leaf colors.[Method]The recombinant plasmid pMD18-T/Tpn was constructed by amplifying the target fragments from young leaf tissues of the blue and white mutant of Pharbitis nil with PCR technology and investigating in the fields.[Result]The results of investigations in the fields suggested that the mutant was segregated in F3 generation,and the mutations of leaf colors did not affect the changes of flower colors.The results of PCR amplification showed that there was a band about 330 bp in genomic DNA of Lanbai,Chunlan,Dahua,respectively,however,no bands were found in genomic DNA of Chunguang,Baixue,Ping＇anhong and Chuiman.[Conclusion]The sequence of recombinant plasmid of the mutant was indeed a fragment of the transposon of Pharbitis nil.展开更多
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. So...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.展开更多
The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PC...The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PCR. Results showed that 65.4% of the T-DNA was integrated in different locations of rice genome, and some T-DNA flanking sequences were located on certain chromosomes. A number of T-DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac x Ds and Ds x Ac were constructed. The excision frequency of Ds element trans-activated by Ac transposase was 22.7% in the F-2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to 'cut-paste' mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.展开更多
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 ...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.展开更多
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 studi...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.展开更多
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 fila...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.展开更多
Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens.The R gene mediates resistance,in most cases,dependent on the co-existence of its cognate pathogen-derived avirul...Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens.The R gene mediates resistance,in most cases,dependent on the co-existence of its cognate pathogen-derived avirulence (Avr) gene.The rice blast R gene Piz-t corresponds in gene-for-gene fashion to the Magnaporthe oryzae Avr gene AvrPiz-t.In this study,we determined and compared the genomic sequences surrounding the AvrPiz-t gene in both avirulent and virulent isolates,designating as AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively.The sequence of the AvrPiz-t-ZB15 region is 120966 bp whereas avrPiz-t-70-15 is 146292 bp in length.The extreme sequence similarity and good synteny in gene order and content along with the absence of two predicted genes in the avrPiz-t-70-15 region were observed in the predicted protein-coding regions in the AvrPiz-t locus.Nevertheless,frequent presence/absence and highly dynamic organization of transposable elements (TEs) were identified,representing the major variation of the AvrPiz-t locus between different isolates.Moreover,TEs constitute 27.3% and 43.2% of the genomic contents of the AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively,indicating that TEs contribute largely to the organization and evolution of AvrPiz-t locus.The findings of this study suggest that M.oryzae could benefit in an evolutionary sense from the presence of active TEs in genes conferring avirulence and provide an ability to rapidly change and thus to overcome host R genes.展开更多
African cultivated rice,Oryza glaberrima,is characterized by its glabrous glumes.During domestication,the pubescent glumes of its wild ancestor,Oryza barthii,lost their trichomes,and in this study,we show that glabrou...African cultivated rice,Oryza glaberrima,is characterized by its glabrous glumes.During domestication,the pubescent glumes of its wild ancestor,Oryza barthii,lost their trichomes,and in this study,we show that glabrous glume 5(GLAG5),a WUSCHEL-like homeobox transcription factor gene on chromosome 5,is required for trichome development.DNA methylation associated with an hATtransposable element inserted in the promoter region of GLAG5 is found to reduce its expression,leading to the formation of glabrous glumes and leaves in African cultivated rice.Among 82 African cultivated rice varieties investigated in this study,59(approximately 71%)lines exhibit glabrous glumes and harbor the hAT transposon;however,the other 23 varieties(approximately 29%),which exhibit pubescent glumes,lack the hAT transposon,indicating that glag5 had undergone strong artificial selection.Theπ;/π;ratios also show the hAT transposon insertions influence the genetic diversity of an approximately 150-kb interval encompassing the GLAG5 locus.The identification of the GLAG5 gene provides new insights into the domestication of cultivated rice in Africa.We speculate that the selection of varieties with mutations in their promoter regions is an important aspect of crop domestication.展开更多
In plants,transposable element(TE)-triggered mutants are important resources for functional genomic studies.However,conventional approaches for genome-wide identification of TE insertion sites are costly and laborious...In plants,transposable element(TE)-triggered mutants are important resources for functional genomic studies.However,conventional approaches for genome-wide identification of TE insertion sites are costly and laborious.This study developed a novel,rapid,and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing(TEAseq).Using TEAseq,we systemically profiled the Dissociation(Ds)insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background.The Ac-containing individuals were excluded for getting rid of the potential somatic insertions.We characterized 35,696 germinal Ds insertions tagging 10,323 genes,representing approximately 23.3%of the total genes in the maize genome.The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci,and insertions occurred preferentially in gene body regions.Furthermore,we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development.We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations.Overall,our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.展开更多
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 gene...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.展开更多
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 ...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.展开更多
Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generat...Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZIO by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxj1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxj1b. Although normal expression offoxj1b is dramatically reduced, T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxjlb expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.展开更多
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-in...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.展开更多
Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism...Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism.Nevertheless,the comprehensive analysis of endogenous piggyBac-like elements(PLEs)is important before using piggyBac,because they may influence the genetic stability of transgenic lines.Herein,we conducted a genome-wide analysis of PLEs in the brown planthopper(BPH)Nilaparvata lugens(St?l)(Hemiptera:Delphacidae),and identified a total of 28 PLE sequences.All N.lugens piggy Bac-like elements(NlPLEs)were present as multiple copies in the genome of BPH.Among the identified NlPLEs,NlPLE25 had the highest copy number and it was distributed on five chromosomes.The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals,as well as a single open reading frame transposase encoding546 amino acids.Furthermore,NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision.A cross-recognition between the NlPLE25 transposon and the piggy Bac transposon was also revealed in this study.These findings provide useful information for the construction of transgenic insect lines.展开更多
文摘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.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11775091 and 11474117)
文摘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.
文摘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.
基金Supported by National High Technology Research and Development Program of China(2006AA10A206)National Natural Science Foundation of China(31001072)the Fund of Beijing Academy of Agriculture and Forestry Sciences for Young Scholars(QNJJ201012)~~
文摘[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.False positive was identified by PCR and then removed.Mice were infected to detect the virulence of mutants.The bionomics of attenuated strains were detected,too.[Result] The attenuated mutants showed similar reproductive activity to that of wild strain.The virulence of mutants was still stable after 30 passages.[Conclusion] This study provided foundation for exploring the virulence factors and pathogenic mechanism of HPS.
文摘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.
基金Supported by National Natural Science Fund Item(30230260,30600441)~~
文摘[Objective]The research aimed to construct deficient strain generated by Selenomonas ruminantium mutant with the acetic acid and analyze its fermentation characteristics.[Method]Based on the transposon tagging method,Selenomonas ruminantium（recipient strain）was carried out the transposon mutagenesis via the transposon donor strain E.coli S17-1/pZJ25∷Tn5.The zygote was screened by using the selective medium which included kanamycin and sodium fluoroacetate.[Result]Seven transposon engineered strains which had the stable resistance to kanamycin and fluoroethanoic acid were screened.Selenomonas ruminantium mutant was carried out 16S rRNA and Tn5 PCR identification.Moreover,the specific activities of AK and PTA were analyzed.The mutant belonged to fluoroethanoic acid resistance strain with pta gene deficiency.[Conclusion]The research laid the foundation for further studying the cellular metabolic network and regulation of acetic acid in rumen microorganism of ruminant animal.
文摘[Objective]The research aimed at investigating mechanisms of the changes of pharbitis.nil flower colors and leaf colors.[Method]The recombinant plasmid pMD18-T/Tpn was constructed by amplifying the target fragments from young leaf tissues of the blue and white mutant of Pharbitis nil with PCR technology and investigating in the fields.[Result]The results of investigations in the fields suggested that the mutant was segregated in F3 generation,and the mutations of leaf colors did not affect the changes of flower colors.The results of PCR amplification showed that there was a band about 330 bp in genomic DNA of Lanbai,Chunlan,Dahua,respectively,however,no bands were found in genomic DNA of Chunguang,Baixue,Ping＇anhong and Chuiman.[Conclusion]The sequence of recombinant plasmid of the mutant was indeed a fragment of the transposon of Pharbitis nil.
文摘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.
文摘The Agrobacterium mediated transgenic rice ( Oryza saliva L.) population with inserts of maize transposon Activator/Dissociation (Ac/Ds) was investigated. DNA sequences flanking the T-DNA were analyzed with inverse PCR. Results showed that 65.4% of the T-DNA was integrated in different locations of rice genome, and some T-DNA flanking sequences were located on certain chromosomes. A number of T-DNA was found to have inserted into protein coding regions. In order to induce transposition of the inserted Ds elements, 354 crosses of Ac x Ds and Ds x Ac were constructed. The excision frequency of Ds element trans-activated by Ac transposase was 22.7% in the F-2 populations, and the transposition was confirmed with analyses of DNA sequences flanking the Ds elements. In addition to the transposition due to 'cut-paste' mechanism, Ds can replicate itself and integrate into a new locus, and inaccurate excisions were also found. A proportion of DNA segments flanking the Ds elements showed no homologies to sequences published in GenBank, of which two were registered under the accession numbers AF355153 and AF355770. The strategy of using transposon tagging for rice genomics study was discussed.
基金National Research Foundation of Korea(NRF-2017R1A2B3004972)IPET(No.109023–05-5-CG000)The BK21 PLUS Program for Creative Veterinary Science Research
文摘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.
文摘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.
文摘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.
基金Project supported by the National Natural Science Foundation of China (Nos.30700450 and 30971878)the Fundamental Research Funds for the Central Universities (No.2009QNA6024),China
文摘Plants utilize multiple layers of defense mechanisms to fight against the invasion of diverse pathogens.The R gene mediates resistance,in most cases,dependent on the co-existence of its cognate pathogen-derived avirulence (Avr) gene.The rice blast R gene Piz-t corresponds in gene-for-gene fashion to the Magnaporthe oryzae Avr gene AvrPiz-t.In this study,we determined and compared the genomic sequences surrounding the AvrPiz-t gene in both avirulent and virulent isolates,designating as AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively.The sequence of the AvrPiz-t-ZB15 region is 120966 bp whereas avrPiz-t-70-15 is 146292 bp in length.The extreme sequence similarity and good synteny in gene order and content along with the absence of two predicted genes in the avrPiz-t-70-15 region were observed in the predicted protein-coding regions in the AvrPiz-t locus.Nevertheless,frequent presence/absence and highly dynamic organization of transposable elements (TEs) were identified,representing the major variation of the AvrPiz-t locus between different isolates.Moreover,TEs constitute 27.3% and 43.2% of the genomic contents of the AvrPiz-t-ZB15 and avrPiz-t-70-15 regions,respectively,indicating that TEs contribute largely to the organization and evolution of AvrPiz-t locus.The findings of this study suggest that M.oryzae could benefit in an evolutionary sense from the presence of active TEs in genes conferring avirulence and provide an ability to rapidly change and thus to overcome host R genes.
基金supported by the National Natural Science Foundation of China(31925029)。
文摘African cultivated rice,Oryza glaberrima,is characterized by its glabrous glumes.During domestication,the pubescent glumes of its wild ancestor,Oryza barthii,lost their trichomes,and in this study,we show that glabrous glume 5(GLAG5),a WUSCHEL-like homeobox transcription factor gene on chromosome 5,is required for trichome development.DNA methylation associated with an hATtransposable element inserted in the promoter region of GLAG5 is found to reduce its expression,leading to the formation of glabrous glumes and leaves in African cultivated rice.Among 82 African cultivated rice varieties investigated in this study,59(approximately 71%)lines exhibit glabrous glumes and harbor the hAT transposon;however,the other 23 varieties(approximately 29%),which exhibit pubescent glumes,lack the hAT transposon,indicating that glag5 had undergone strong artificial selection.Theπ;/π;ratios also show the hAT transposon insertions influence the genetic diversity of an approximately 150-kb interval encompassing the GLAG5 locus.The identification of the GLAG5 gene provides new insights into the domestication of cultivated rice in Africa.We speculate that the selection of varieties with mutations in their promoter regions is an important aspect of crop domestication.
基金the Ministry of Science and Technology of China(2016YFD0101000 and 2016YFD0101001)the Natural Science Foundation of China(31901595).
文摘In plants,transposable element(TE)-triggered mutants are important resources for functional genomic studies.However,conventional approaches for genome-wide identification of TE insertion sites are costly and laborious.This study developed a novel,rapid,and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing(TEAseq).Using TEAseq,we systemically profiled the Dissociation(Ds)insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background.The Ac-containing individuals were excluded for getting rid of the potential somatic insertions.We characterized 35,696 germinal Ds insertions tagging 10,323 genes,representing approximately 23.3%of the total genes in the maize genome.The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci,and insertions occurred preferentially in gene body regions.Furthermore,we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development.We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations.Overall,our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.
文摘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.
基金Supported by the National Projects of Genetic Modified Organism Breeding Technology (2008ZX08006-002)the State Transgenic Research Programme of China (2008ZX08006-002)
文摘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.
基金supported by the National High-tech R&D Program (863 Program) (No. 2006AA02Z167)the Major Science Programs of China (No. 2006CB943401)the National Basic Research Program of China (No. 2005CB522502)
文摘Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZIO by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxj1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxj1b. Although normal expression offoxj1b is dramatically reduced, T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxjlb expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.
基金The National Natural Science Foundation of China under contract Nos 31272699 and 41176115National Department Public Benefit Research Foundation of China under contract No. 200903029+1 种基金the Natural Science Foundation of Fujian Province under contract No.2011J06014the National Hi-Tech Research and Development Program of China (863 Program) under contract No. 2007AA09Z115
文摘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.
基金the Foundation of Guangzhou Science and Technology Key Project(No.201904020041),China。
文摘Piggy Bac is a transposable DNA element originally discovered in the cabbage looper moth(Trichoplusia ni).The T.ni piggyBac transposon can introduce exogenous fragments into a genome,constructing a transgenic organism.Nevertheless,the comprehensive analysis of endogenous piggyBac-like elements(PLEs)is important before using piggyBac,because they may influence the genetic stability of transgenic lines.Herein,we conducted a genome-wide analysis of PLEs in the brown planthopper(BPH)Nilaparvata lugens(St?l)(Hemiptera:Delphacidae),and identified a total of 28 PLE sequences.All N.lugens piggy Bac-like elements(NlPLEs)were present as multiple copies in the genome of BPH.Among the identified NlPLEs,NlPLE25 had the highest copy number and it was distributed on five chromosomes.The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals,as well as a single open reading frame transposase encoding546 amino acids.Furthermore,NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision.A cross-recognition between the NlPLE25 transposon and the piggy Bac transposon was also revealed in this study.These findings provide useful information for the construction of transgenic insect lines.
