scholarly journals Whole-Genome Resequencing using Next-Generation and Nanopore Sequencing for Molecular Characterization of T-DNA Integration in Transgenic Poplar 741

2020 ◽  
Author(s):  
Xinghao Chen ◽  
Yan Dong ◽  
Yali Huang ◽  
Jianmin Fan ◽  
Minsheng Yang ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Insertion Site ◽  
Nanopore Sequencing ◽  
Transgenic Poplar ◽  
Next Generation ◽  
Genome Resequencing ◽  
Dna Integration ◽  
Dna Insertion ◽  
Insertion Sites ◽  
Safety Assessments

Abstract BackgroundWith the rapid development of transgenic technology, transgenic plants have been planted all over the world, and transgenic forest trees have also been commercialized. At the same time, the potential threat of transgenic plants to human health and the natural environment has aroused widespread concern. Therefore, safety assessments before field release and commercial planting of transgenic plants are necessary. By determining the copy number and integration sites of T-DNA in transgenic plants, the safety of transgenic plants at the genomic level can be assessed.ResultsIn this study, we performed genome resequencing of Pb29, a transgenic high-resistance poplar 741 line that has been commercialized, using next-generation and Nanopore sequencing. The results revealed that there are two T-DNA insertion sites, located at 9,283,905–9,283,937 bp on chromosome 3 (Chr03) and 10,868,777–10,868,803 bp on Chr10. The accuracy of the T-DNA insertion locations and directions was verified using polymerase chain reaction amplification. Through sequence alignment, different degrees of base deletions were detected on the T-DNA left and right border sequences, and in the flanking sequences of the insertion sites. An unknown fragment was inserted between the Chr03 insertion site and the right flanking sequence, but the Pb29 genome did not undergo chromosomal rearrangement. It is worth noting that we did not detect the API gene in the Pb29 genome, indicating that Pb29 is a transgenic line containing only the BtCry1AC gene. On Chr03, the insertion of T-DNA disrupted a gene encoding TAF12 protein, but the transcriptional abundance of this gene did not change significantly in the leaves of Pb29. Additionally, except for the gene located closest to the T-DNA integration site, the expression levels of four other neighboring genes did not change significantly in the leaves of Pb29. ConclusionsThis study provides important molecular information for safety assessments and management of transgenic poplar 741. Our findings also provide a theoretical basis for safety assessments of other transgenic poplar.

scholarly journals Gibberellin 20-Oxidase Gene OsGA20ox3 Regulates Plant Stature and Disease Development in Rice

2013 ◽  
Vol 26 (2) ◽  
pp. 227-239 ◽  
Author(s):  
Xue Qin ◽  
Jun Hua Liu ◽  
Wen Sheng Zhao ◽  
Xu Jun Chen ◽  
Ze Jian Guo ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Critical Role ◽  
Insertion Site ◽  
Insertion Mutant ◽  
Wild Type Plant ◽  
Wild Type ◽  
Oxidase Gene ◽  
Dna Insertion ◽  
Flanking Sequences ◽  
Plant Stature

Gibberellin (GA) 20-oxidase (GA20ox) catalyses consecutive steps of oxidation in the late part of the GA biosynthetic pathway. A T-DNA insertion mutant (17S-14) in rice, with an elongated phenotype, was isolated. Analysis of the flanking sequences of the T-DNA insertion site revealed that an incomplete T-DNA integration resulted in enhanced constitutively expression of downstream OsGA20ox3 in the mutant. The accumulation of bioactive GA1 and GA4 were increased in the mutant in comparison with the wild-type plant. Transgenic plants overexpressing OsGA20ox3 showed phenotypes similar to those of the 17S-14 mutant, and the RNA interference (RNAi) lines that had decreased OsGA20ox3 expression exhibited a semidwarf phenotype. Expression of OsGA20ox3 was detected in the leaves and roots of young seedlings, immature panicles, anthers, and pollens, based on β-glucuronidase (GUS) activity staining in transgenic plants expressing the OsGA20ox3 promoter fused to the GUS gene. The OsGA20ox3 RNAi lines showed enhanced resistance against rice pathogens Magnaporthe oryzae (causing rice blast) and Xanthomonas oryzae pv. oryzae (causing bacterial blight) and increased expression of defense-related genes. Conversely, OsGA20ox3-overexpressing plants were more susceptible to these pathogens comparing with the wild-type plants. The susceptibility of wild-type plants to X. oryzae pv. oryzae was increased by exogenous application of GA3 and decreased by S-3307 treatment. Together, the results provide direct evidence for a critical role of OsGA20ox3 in regulating not only plant stature but also disease resistance in rice.

scholarly journals Major Chromosomal Rearrangements Induced by T-DNA Transformation in Arabidopsis

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 641-650 ◽  
Author(s):  
Philippe Nacry ◽  
Christine Camilleri ◽  
Béatrice Courtial ◽  
Michel Caboche ◽  
David Bouchez
Keyword(s):  
Chromosomal Rearrangements ◽  
The Other ◽  
Chromosome 3 ◽  
Head Orientation ◽  
Dna Transformation ◽  
Chromosome 2 ◽  
Dna Integration ◽  
Dna Insertion ◽  
Insertion Sites ◽  
Flanking Regions

Abstract We show that major chromosomal rearrangements can occur upon T-DNA transformation of Arabidopsis thaliana. In the ACL4 line, two T-DNA insertion loci were found; one is a tandem T-DNA insert in a head-to-head orientation, and the other is a truncated insert with only the left part of the T-region. The four flanking DNA regions were isolated and located on the Arabidopsis chromosomes; for both inserts, one side of the T-DNA maps to chromosome 2, whereas the other side maps to chromosome 3. Both chromosome 3 flanking regions map to the same location, despite a 1.4-kb deletion at this point, whereas chromosome 2 flanking regions are located 40 cM apart on the bottom arm of chromosome 2. These results strongly suggest a reciprocal translocation between chromosomes 2 and 3, with the breakpoints located at the T-DNA insertion sites. The interchanged fragments roughly correspond to the 20-cM distal ends of both chromosomes. Moreover, a large inversion, spanning 40 cM on the genetic map, occurs on the bottom arm of chromosome 2. This was confirmed by genetic analyses that demonstrated a strong reduction of recombination in the inverted region. Models for T-DNA integration and the consequences for T-DNA tagging are discussed in light of these results.

T-DNA integration patterns in transgenic plants mediated by Agrobacterium tumefaciens

2011 ◽  
Vol 33 (12) ◽  
pp. 1327-1334 ◽  
Author(s):  
Lin YANG ◽  
Feng-Ling FU ◽  
Wan-Chen LI
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Transgenic Plants ◽  
Dna Integration

scholarly journals Tn5406, a New Staphylococcal Transposon Conferring Resistance to Streptogramin A and Related Compounds Including Dalfopristin

2002 ◽  
Vol 46 (8) ◽  
pp. 2337-2343 ◽  
Author(s):  
Julien Haroche ◽  
Jeanine Allignet ◽  
Névine El Solh
Keyword(s):  
Staphylococcus Aureus ◽  
Insertion Site ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Plasmid Copy ◽  
Acid Identity ◽  
Abc Protein ◽  
Single Plasmid ◽  
Insertion Sites ◽  
Related Compounds

ABSTRACT We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.

Characterization of T-DNA Insertion Sites in Arabidopsis thaliana and the Implications for Saturation Mutagenesis

2002 ◽  
Vol 6 (2) ◽  
pp. 163-174 ◽  
Author(s):  
Patrick J. Krysan ◽  
Jeffery C. Young ◽  
Peter J. Jester ◽  
Sean Monson ◽  
Greg Copenhaver ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Saturation Mutagenesis ◽  
Dna Insertion ◽  
Insertion Sites

A comparison of transparent polyurethane and dry gauze dressings for peripheral i.v. catheter sites: rates of phlebitis, infiltration, and dislodgment by patients

1997 ◽  
Vol 6 (5) ◽  
pp. 377-381 ◽  
Author(s):  
KA Tripepi-Bova ◽  
KD Woods ◽  
MC Loach
Keyword(s):  
Direct Observation ◽  
Meta Analysis ◽  
Insertion Site ◽  
Direct Visualization ◽  
Gauze Dressing ◽  
Insertion Sites ◽  
To Receive ◽  
Lower Frequencies

BACKGROUND: Before a meta-analysis by Hoffman et al was published, polyurethane dressings were used at insertion sites for peripheral i.v. catheters at our institution. On the basis of the results of the meta-analysis, we began to use gauze dressings. The change from polyurethane dressings to gauze dressings limited direct observation of the i.v. insertion site, and i.v. catheters were anecdotally reported not to be anchored as securely as before. OBJECTIVES: The purpose of this study was to compare the effects of the use of transparent polyurethane dressings and gauze dressings at insertion sites for peripheral i.v. catheters on the frequency of phlebitis, infiltration, and catheter dislodgment by patients. METHODS: Two hundred twenty-nine patients were randomized to receive either gauze (n = 121) or transparent polyurethane (n = 108) dressings, and observations were recorded. RESULTS: The frequency of catheter dislodgment by the patient was significantly higher (P < .05) in patients with the gauze dressing (15%) than in patients with the transparent polyurethane dressing (6%). A trend toward lower frequencies of phlebitis (1.8% vs 3.3%) and infiltration (17.6% vs 20.7%) was noted in the patients with the transparent polyurethane dressings. DISCUSSION: The clinical advantages of the transparent polyurethane dressings lie in the ease of direct visualization of the i.v. insertion site and the securement of the i.v. catheter. CONCLUSION: At our institution, given the decreased disruption of the i.v. therapy with the transparent polyurethane dressings and the lack of differences in the rates of phlebitis or infiltration with the two types of dressings, we prefer to use transparent polyurethane rather than gauze dressings at insertion sites for peripheral i.v. catheters.

Patency of radial arterial catheters

2001 ◽  
Vol 10 (2) ◽  
pp. 104-111 ◽  
Author(s):  
J Kaye ◽  
GR Heald ◽  
J Morton ◽  
T Weaver
Keyword(s):  
Sampling Methods ◽  
Insertion Site ◽  
Blood Sampling ◽  
Monitoring Systems ◽  
Site Location ◽  
Pressure Monitoring ◽  
Radiocarpal Joint ◽  
Background Data ◽  
Isotonic Sodium Chloride ◽  
Insertion Sites

BACKGROUND: Data on the influence of flush methods, blood-sampling methods, and site location on the patency of radial arterial catheters used for pressure monitoring are sparse. OBJECTIVES: To determine the effects of flush and blood-sampling methods, insertion site, and sex of patients on catheter patency. METHODS: In a randomized trial, 174 patients requiring radial arterial pressure monitoring were assigned to 4 groups: fast flush as needed and nonwaste blood sampling; fast flush as needed and waste blood sampling; fast flush every 4 hours and waste blood sampling; and fast flush every 4 hours and nonwaste blood sampling. All site locations were evaluated for patency, and all monitoring systems were maintained with isotonic sodium chloride solution. RESULTS: Nonpatent catheters were 4.23 times more likely in patients with insertion sites 3 cm or higher above the bend of the wrist than in patients with lower sites (P = .01). Duration of patency did not differ between catheters maintained with fast flush every 4 hours and those flushed as needed or between catheters according to the method of blood sampling. Women were 3.05 times more likely than men to have nonpatent catheters (P = .02). With insertion sites 3 cm or higher above the radiocarpal joint, nonpatency was 7.3 times more likely in women than in men (P < .001). CONCLUSIONS: Insertion sites closest to the bend of the wrist increase chances of maintaining patency. Catheters can be maintained with as-needed flushes, and either waste or nonwaste blood sampling can be used.

scholarly journals Insertion of Foreign DNA into an Established Mammalian Genome Can Alter the Methylation of Cellular DNA Sequences

1999 ◽  
Vol 73 (2) ◽  
pp. 1010-1022 ◽  
Author(s):  
Ralph Remus ◽  
Christina Kämmer ◽  
Hilde Heller ◽  
Birgit Schmitz ◽  
Gudrun Schell ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Mammalian Genome ◽  
Dna Integration ◽  
Bhk21 Cell ◽  
Cellular Genes ◽  
Cell Clones ◽  
Dna Insertion ◽  
Foreign Dna ◽  
Methylation Patterns

ABSTRACT The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage λ and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated λ DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage λ DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-λ-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after λ DNA integration might have existed prior to λ DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5′-CG-3′ methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated λ DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

scholarly journals Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

2015 ◽  
Vol 84 (3) ◽  
pp. 701-710 ◽  
Author(s):  
Madeleine G. Moule ◽  
Natasha Spink ◽  
Sam Willcocks ◽  
Jiali Lim ◽  
José Afonso Guerra-Assunção ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Insertion Site ◽  
Wild Type ◽  
Content Type ◽  
Growth And Survival ◽  
New Genes ◽  
Insertion Sites

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

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