Sensitive detection of pre-integration intermediates of LTR retrotransposons in crop plants

AbstractRetrotransposons have played an important role in the evolution of host genomes1,2. Their impact on host chromosomes is mainly deduced from the composition of DNA sequences, which have been fixed over evolutionary time. These studies provide important “snapshots” reflecting historical activities of transposons but do not predict current transposition potential. We previously reported Sequence-Independent Retrotransposon Trapping (SIRT) as a methodology that, by identification of extrachromosomal linear DNA (eclDNA), revealed the presence of active LTR retrotransposons in Arabidopsis9. Unfortunately, SIRT cannot be applied to large and transposon-rich genomes of crop plants. We have since developed an alternative approach named ALE-seq (amplification of LTR of eclDNAs followed by sequencing). ALE-seq reveals sequences of 5’ LTRs of eclDNAs after two-step amplification: in vitro transcription and subsequent reverse transcription. Using ALE-seq in rice, we detected eclDNAs for a novel Copia family LTR retrotransposon, Go-on, which is activated by heat stress. Sequencing of rice accessions revealed that Go-on has preferentially accumulated in indica rice grown at higher temperatures. Furthermore, ALE-seq applied to tomato fruits identified a developmentally regulated Gypsy family of retrotransposons. Importantly, a bioinformatic pipeline adapted for ALE-seq data analyses allows the direct and reference-free annotation of new active retroelements. This pipeline allows assessment of LTR retrotransposon activities in organisms for which genomic sequences and/or reference genomes are unavailable or are of low quality.

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A Nest of LTR Retrotransposons Adjacent the Disease Resistance-Priming Gene NPR1 in Beta vulgaris L. U.S. Hybrid H20

International Journal of Plant Genomics ◽

10.1155/2009/576742 ◽

2009 ◽

Vol 2009 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

David Kuykendall ◽

Jonathan Shao ◽

Kenneth Trimmer

Keyword(s):

Disease Resistance ◽

Nucleotide Sequence ◽

Dna Sequences ◽

Hypothetical Protein ◽

Ltr Retrotransposon ◽

Ltr Retrotransposons ◽

Nucleotide Sequence Alignment ◽

Genes Encoding ◽

Dna Elements ◽

Core Genes

A nest of long terminal repeat (LTR) retrotransposons (RTRs), discovered by LTR_STRUC analysis, is near core genes encoding the NPR1 disease resistance-activating factor and a heat-shock-factor-(HSF-) like protein in sugarbeet hybrid US H20. SCHULTE, a 10 833 bp LTR retrotransposon, with 1372 bp LTRs that are 0.7% divergent, has two ORFs with unexpected introns but encoding a reverse transcriptase with rve and Rvt2 domains similar to Ty1/copia-type retrotransposons and a hypothetical protein. SCHULTE produced significant nucleotide BLAST alignments with repeat DNA elements from all four families of plants represented in the TIGR plant repeat database (PRD); the best nucleotide sequence alignment was to ToRTL1 in Lycopersicon esculentum. A second sugarbeet LTR retrotransposon, SCHMIDT, 11 565 bp in length, has 2561 bp LTRs that share 100% identity with each other and share 98-99% nucleotide sequence identity over 10% of their length with DRVs, a family of highly repetitive, relatively small DNA sequences that are widely dispersed over the sugarbeet genome. SCHMIDT encodes a complete gypsy-like polyprotein in a single ORF. Analysis using LTR_STRUC of an in silico deletion of both of the above two LTR retrotransposons found that SCHULTE and SCHMIDT had inserted within an older LTR retrotransposon, resulting in a nest that is only about 10 Kb upstream of NPR1 in sugarbeet hybrid US H20.

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Chromosomal Locations of a Non-LTR Retrotransposon, Menolird18, in Cucumis melo and Cucumis sativus, and Its Implication on Genome Evolution of Cucumis Species

Cytogenetic and Genome Research ◽

10.1159/000511119 ◽

2020 ◽

Vol 160 (9) ◽

pp. 554-564

Author(s):

Agus B. Setiawan ◽

Chee H. Teo ◽

Shinji Kikuchi ◽

Hidenori Sassa ◽

Kenji Kato ◽

...

Keyword(s):

Genome Evolution ◽

Repetitive Dna ◽

Dna Sequences ◽

18S Rdna ◽

Cucumis Melo ◽

Repetitive Dna Sequences ◽

Ltr Retrotransposon ◽

Ltr Retrotransposons ◽

Mitotic Chromosomes ◽

Rna Genes

Mobile elements are major regulators of genome evolution through their effects on genome size and chromosome structure in higher organisms. Non-long terminal repeat (non-LTR) retrotransposons, one of the subclasses of transposons, are specifically inserted into repetitive DNA sequences. While studies on the insertion of non-LTR retrotransposons into ribosomal RNA genes and other repetitive DNA sequences have been reported in the animal kingdom, studies in the plant kingdom are limited. Here, using FISH, we confirmed that Menolird18, a member of LINE (long interspersed nuclear element) in non-LTR retrotransposons and found in Cucumis melo, was inserted into ITS and ETS (internal and external transcribed spacers) regions of 18S rDNA in melon and cucumber. Beside the 18S rDNA regions, Menolird18 was also detected in all centromeric regions of melon, while it was located at pericentromeric and sub-telomeric regions in cucumber. The fact that FISH signals of Menolird18 were found in centromeric and rDNA regions of mitotic chromosomes suggests that Menolird18 is a rDNA and centromere-specific non-LTR retrotransposon in melon. Our findings are the first report on a non-LTR retrotransposon that is highly conserved in 2 different plant species, melon and cucumber. The clear distinction of chromosomal localization of Menolird18 in melon and cucumber implies that it might have been involved in the evolutionary processes of the melon (C. melo) and cucumber (C. sativus) genomes.

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Use of a promoter-trap retrovirus to identify and isolate genes involved in differentiation of a myeloid progenitor cell line in vitro

Blood ◽

10.1182/blood.v87.5.1771.1771 ◽

1996 ◽

Vol 87 (5) ◽

pp. 1771-1779 ◽

Cited By ~ 13

Author(s):

JI Jonsson ◽

Q Wu ◽

K Nilsson ◽

RA Phillips

Keyword(s):

Dna Sequences ◽

Progenitor Cell ◽

High Efficiency ◽

Sequence Similarity ◽

Lacz Gene ◽

Developmentally Regulated ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cell ◽

Promoter Trap

Abstract Studies of gene regulation during early hematopoiesis and of the regulatory network that controls differentiation and lineage commitment are hampered by difficulties in isolating and growing stem cells and early progenitor cells. These difficulties preclude the application of standard molecular genetic approaches to these problems. As an alternative approach we have introduced a lacZ-containing promoter-trap retrovirus into hematopoietic cells. We used the interleukin-3- dependent mouse myeloid progenitor cell 32D as a model to identify transcriptionally active genes. The frequency of integrations that led to transcription of the lacZ gene was estimated to be 0.5% of all integrations, of which 14% were downregulated on differentiation of 32D cells towards neutrophils. Thus, one in every 1,000 to 2,000 integrations identified a developmentally regulated gene. Cellular DNA sequences upstream of proviral integrations were isolated by inverse polymerase chain reaction. Five were further characterized and we confirmed by RNA expression analysis that they were downregulated on differentiation. Sequence analysis revealed identification of novel genes with sequence similarity to known genes. Considering the high efficiency of retroviral infection, our study shows the feasibility of using promoter-trap vectors to identity and isolate developmentally regulated genes from early hematopoietic progenitors.

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Use of a promoter-trap retrovirus to identify and isolate genes involved in differentiation of a myeloid progenitor cell line in vitro

Blood ◽

10.1182/blood.v87.5.1771.bloodjournal8751771 ◽

1996 ◽

Vol 87 (5) ◽

pp. 1771-1779 ◽

Cited By ~ 1

Author(s):

JI Jonsson ◽

Q Wu ◽

K Nilsson ◽

RA Phillips

Keyword(s):

Dna Sequences ◽

Progenitor Cell ◽

High Efficiency ◽

Sequence Similarity ◽

Lacz Gene ◽

Developmentally Regulated ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cell ◽

Promoter Trap

Studies of gene regulation during early hematopoiesis and of the regulatory network that controls differentiation and lineage commitment are hampered by difficulties in isolating and growing stem cells and early progenitor cells. These difficulties preclude the application of standard molecular genetic approaches to these problems. As an alternative approach we have introduced a lacZ-containing promoter-trap retrovirus into hematopoietic cells. We used the interleukin-3- dependent mouse myeloid progenitor cell 32D as a model to identify transcriptionally active genes. The frequency of integrations that led to transcription of the lacZ gene was estimated to be 0.5% of all integrations, of which 14% were downregulated on differentiation of 32D cells towards neutrophils. Thus, one in every 1,000 to 2,000 integrations identified a developmentally regulated gene. Cellular DNA sequences upstream of proviral integrations were isolated by inverse polymerase chain reaction. Five were further characterized and we confirmed by RNA expression analysis that they were downregulated on differentiation. Sequence analysis revealed identification of novel genes with sequence similarity to known genes. Considering the high efficiency of retroviral infection, our study shows the feasibility of using promoter-trap vectors to identity and isolate developmentally regulated genes from early hematopoietic progenitors.

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Monitoring of r-Hirudin Anticoagulation during Cardiopulmonary Bypass – Assessment of the Whole Blood Ecarin Clotting Time

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656078 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0920-0925 ◽

Cited By ~ 139

Author(s):

Bernd Pötzsch ◽

Katharina Madlener ◽

Christoph Seelig ◽

Christian F Riess ◽

Andreas Greinacher ◽

...

Keyword(s):

Cardiopulmonary Bypass ◽

Whole Blood ◽

Heart Surgery ◽

Ex Vivo ◽

Open Heart Surgery ◽

Clotting Time ◽

Blood Samples ◽

Alternative Approach ◽

Ecarin Clotting Time

SummaryThe use of recombinant ® hirudin as an anticoagulant in performing extracorporeal circulation systems including cardiopulmonary bypass (CPB) devices requires a specific and easy to handle monitoring system. The usefulness of the celite-induced activated clotting time (ACT) and the activated partial thromboplastin time (APTT) for r-hirudin monitoring has been tested on ex vivo blood samples obtained from eight patients treated with r-hirudin during open heart surgery. The very poor relationship between the prolongation of the ACT and APTT values and the concentration of r-hirudin as measured using a chromogenic factor Ila assay indicates that both assays are not suitable to monitor r-hirudin anticoagulation. As an alternative approach a whole blood clotting assay based on the prothrombin-activating snake venom ecarin has been tested. In vitro experiments using r-hirudin- spiked whole blood samples showed a linear relationship between the concentration of hirudin added and the prolongation of the clotting times up to a concentration of r-hirudin of 4.0 µg/ml. Interassay coefficients (CV) of variation between 2.1% and 5.4% demonstrate the accuracy of the ecarin clotting time (ECT) assay. Differences in the interindividual responsiveness to r-hirudin were analyzed on r-hirudin- spiked blood samples obtained from 50 healthy blood donors. CV- values between 1.8% and 6% measured at r-hirudin concentrations between 0.5 and 4 µg/ml indicate remarkably slight differences in r-hirudin responsiveness. ECT assay results of the ex vivo blood samples linearily correlate (r = 0.79) to the concentration of r-hirudin. Moreover, assay results were not influenced by treatment with aprotinin or heparin. These findings together with the short measuring time with less than 120 seconds warrant the whole blood ECT to be a suitable assay for monitoring of r-hirudin anticoagulation in cardiac surgery.

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Identification of Differentially Expressed Genes Using cDNA-AFLP During Six Days In Vitro Tuberization of Potato

Zuriat ◽

10.24198/zuriat.v14i1.6751 ◽

2015 ◽

Vol 14 (1) ◽

Author(s):

Nono Carsono ◽

Christian Bachem

Keyword(s):

Gene Expression ◽

Developmental Process ◽

Expression Patterns ◽

Induction Medium ◽

In Vitro Tuberization ◽

Storage Organ ◽

Developmentally Regulated ◽

Study Gene Expression ◽

Differential Pattern

Tuberization in potato is a complex developmental process resulting in the differentiation of stolon into the storage organ, tuber. During tuberization, change in gene expression has been known to occur. To study gene expression during tuberization over the time, in vitro tuberization system provides a suitable tool, due to its synchronous in tuber formation. An early six days axillary bud growing on tuber induction medium is a crucial development since a large number of genes change in their expression patterns during this period. In order to identify, isolate and sequencing the genes which displaying differential pattern between tuberizing and non-tuberizing potato explants during six days in vitro tuberization, cDNA-AFLP fingerprint, method for the visualization of gene expression using cDNA as template which is amplified to generate an RNA-fingerprinting, was used in this experiment. Seventeen primer combinations were chosen based on their expression profile from cDNA-AFLP fingerprint. Forty five TDFs (transcript derived fragment), which displayed differential expressions, were obtained. Tuberizing explants had much more TDFs, which developmentally regulated, than those from non tuberizing explants. Seven TDFs were isolated, cloned and then sequenced. One TDF did not find similarity in the current databases. The nucleotide sequence of TDF F showed best similarity to invertase ezymes from the databases. The homology of six TDFs with known sequences is discussed in this paper.

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Anti-Mycobacterial Peroxides: A New Class of Agents for Development Against Tuberculosis

Medicinal Chemistry ◽

10.2174/1573406415666190430143535 ◽

2020 ◽

Vol 16 (3) ◽

pp. 392-402

Author(s):

Christiaan W. van der Westhuyzen ◽

Richard K. Haynes ◽

Jenny-Lee Panayides ◽

Ian Wiid ◽

Christopher J. Parkinson

Keyword(s):

Subsequent Development ◽

Antitubercular Activity ◽

Organic Peroxide ◽

New Drugs ◽

Skeletal Myoblast ◽

Artemisinin Derivatives ◽

Malaria Therapy ◽

New Class ◽

Alternative Approach

Background: With few exceptions, existing tuberculosis drugs were developed many years ago and resistance profiles have emerged. This has created a need for new drugs with discrete modes of action. There is evidence that tuberculosis (like other bacteria) is susceptible to oxidative pressure and this has yet to be properly utilised as a therapeutic approach in a manner similar to that which has proven highly successful in malaria therapy. Objective: To develop an alternative approach to the incorporation of bacterial siderophores that results in the creation of antitubercular peroxidic leads for subsequent development as novel agents against tuberculosis. Methods: Eight novel peroxides were prepared and the antitubercular activity (H37Rv) was compared to existing artemisinin derivatives in vitro. The potential for toxicity was evaluated against the L6 rat skeletal myoblast and HeLa cervical cancer lines in vitro. Results: The addition of a pyrimidinyl residue to an artemisinin or, preferably, a tetraoxane peroxidic structure results in antitubercular activity in vitro. The same effect is not observed in the absence of the pyrimidine or with other heteroaromatic substituents. Conclusion: The incorporation of a pyrimidinyl residue adjacent to the peroxidic function in an organic peroxide results in anti-tubercular activity in an otherwise inactive peroxidic compound. This will be a useful approach for creating oxidative drugs to target tuberculosis.

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Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

Alternatives to Laboratory Animals ◽

10.1177/026119299502300110 ◽

1995 ◽

Vol 23 (1) ◽

pp. 61-73

Author(s):

Coenraad Hendriksen ◽

Johan van der Gun

Keyword(s):

Quality Control ◽

Test Methods ◽

In Vitro Test ◽

Large Numbers ◽

Alternative Approach ◽

Inactivated Vaccines ◽

Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

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Genome-Wide Identification and Development of LTR Retrotransposon-Based Molecular Markers for the Melilotus Genus

Plants ◽

10.3390/plants10050890 ◽

2021 ◽

Vol 10 (5) ◽

pp. 890

Author(s):

Zifeng Ouyang ◽

Yimeng Wang ◽

Tiantian Ma ◽

Gisele Kanzana ◽

Fan Wu ◽

...

Keyword(s):

Group Method ◽

Ltr Retrotransposon ◽

Ltr Retrotransposons ◽

Important Data ◽

Breeding Programs ◽

Upgma Dendrogram ◽

Pair Group ◽

Medicinal Value ◽

Genome Wide ◽

Melilotus Albus

Melilotus is an important genus of legumes with industrial and medicinal value, partly due to the production of coumarin. To explore the genetic diversity and population structure of Melilotus, 40 accessions were analyzed using long terminal repeat (LTR) retrotransposon-based markers. A total of 585,894,349 bp of LTR retrotransposon sequences, accounting for 55.28% of the Melilotus genome, were identified using bioinformatics tools. A total of 181,040 LTR retrotransposons were identified and classified as Gypsy, Copia, or another type. A total of 350 pairs of primers were designed for assessing polymorphisms in 15 Melilotus albus accessions. Overall, 47 polymorphic primer pairs were screened for their availability and transferability in 18 Melilotus species. All the primer pairs were transferable, and 292 alleles were detected at 47 LTR retrotransposon loci. The average polymorphism information content (PIC) value was 0.66, which indicated that these markers were highly informative. Based on unweighted pair group method with arithmetic mean (UPGMA) dendrogram cluster analysis, the 18 Melilotus species were classified into three clusters. This study provides important data for future breeding programs and for implementing genetic improvements in the Melilotus genus.

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Unearthing LTR retrotransposon gag genes co-opted in the deep evolution of eukaryotes

Molecular Biology and Evolution ◽

10.1093/molbev/msab101 ◽

2021 ◽

Author(s):

Jianhua Wang ◽

Guan-Zhu Han

Keyword(s):

Gene Expression ◽

Selective Pressure ◽

Ltr Retrotransposon ◽

Ltr Retrotransposons ◽

Cellular Functions ◽

The Family ◽

Family Level ◽

Gene Expression Analyses ◽

Comprehensive Picture ◽

Gypsy Retrotransposons

Abstract LTR retrotransposons comprise a major component of the genomes of eukaryotes. On occasion, retrotransposon genes can be recruited by their hosts for diverse functions, a process formally referred to as co-option. However, a comprehensive picture of LTR retrotransposon gag gene co-option in eukaryotes is still lacking, with several documented cases exclusively involving Ty3/Gypsy retrotransposons in animals. Here we use a phylogenomic approach to systemically unearth co-option of retrotransposon gag genes above the family level of taxonomy in 2,011 eukaryotes, namely co-option occurring during the deep evolution of eukaryotes. We identify a total of 14 independent gag gene co-option events across more than 740 eukaryote families, eight of which have not been reported previously. Among these retrotransposon gag gene co-option events, nine, four, and one involve gag genes of Ty3/Gypsy, Ty1/Copia, and Bel-Pao retrotransposons, respectively. Seven, four, and three co-option events occurred in animals, plants, and fungi, respectively. Interestingly, two co-option events took place in the early evolution of angiosperms. Both selective pressure and gene expression analyses further support that these co-opted gag genes might perform diverse cellular functions in their hosts, and several co-opted gag genes might be subject to positive selection. Taken together, our results provide a comprehensive picture of LTR retrotransposon gag gene co-option events that occurred during the deep evolution of eukaryotes, and suggest paucity of LTR retrotransposon gag gene co-option during the deep evolution of eukaryotes.

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