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In JoVE (1)
Other Publications (11)
- Journal of Molecular Biology
- Molecular and Cellular Biology
- Nucleic Acids Research
- Proceedings of the National Academy of Sciences of the United States of America
- Nucleic Acids Research
- Journal of Molecular Biology
- Genetics
- Fungal Genetics and Biology : FG & B
- Mobile Genetic Elements
- Mobile Genetic Elements
- Plant Science : an International Journal of Experimental Plant Biology
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Articles by Shawn Christensen in JoVE
JoVE Immunology and Infection
Kwantificering van schimmelkolonisatie, Sporogenesis, en de productie van mycotoxinen Met behulp van Kernel Bioassays
Plant Pathology and Microbiology, Texas A&M University
De verwoesting van de graangewassen door zaad-infecterende schimmels heeft ertoe geleid dat tal van onderzoeksinspanningen om beter te begrijpen plant-pathogeen interacties. Om zaad-schimmel interacties in een laboratorium te bestuderen, ontwikkelden we een robuuste methode voor de kwantificering van de schimmel reproductie, biomassa en verontreiniging met mycotoxinen met behulp van kernel bioassays.
Other articles by Shawn Christensen on PubMed
Footprint of the Retrotransposon R2Bm Protein on Its Target Site Before and After Cleavage
R2 elements are non-long terminal repeat (non-LTR) retrotransposons that specifically integrate into the 28 S rRNA genes of their host. These elements encode a single open reading frame with a genome-specific endonuclease and a reverse transcriptase that uses the cleaved chromosomal target site to prime reverse transcription. Cleavage of the DNA strand that is used to prime reverse transcription is an efficient process that occurs in the presence or absence of RNA. Cleavage of the second DNA strand is much less efficient and requires RNA. Reverse transcription occurs before second strand cleavage and only if the RNA bound to the protein contains the 3' untranslated region of the R2 element. Thus a complex series of protein interactions with the DNA and conformational changes in the protein are likely to occur during this retrotransposition reaction. Here, we conduct electrophoretic mobility-shift assays and DNase I footprint studies on the binding of the R2 protein to the DNA target in the presence and absence of RNA both before and after first strand cleavage. While the total expanse of the protein footprint on the DNA eventually covers five helical turns, before cleavage the footprint only extends from 17 bp to 40 bp upstream of the cleavage site. This footprint is the same in the presence and absence of RNA. We hypothesize that the active site of the endonuclease domain is analogous to type IIS restriction enzymes in that it is located on a flexible domain that is not tightly bound to the cleavage site. After first strand cleavage the protein footprint extends beyond the cleavage site. We suggest that this increased protection after cleavage is the RT domain that is positioned over the free DNA end to begin reverse transcription on the nicked DNA substrate.
R2 Target-primed Reverse Transcription: Ordered Cleavage and Polymerization Steps by Protein Subunits Asymmetrically Bound to the Target DNA
R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.
Role of the Bombyx Mori R2 Element N-terminal Domain in the Target-primed Reverse Transcription (TPRT) Reaction
R2 is a site-specific non-long terminal repeat (non-LTR) retrotransposon encoding a single polypeptide with reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The current model of R2 retrotransposition involves an ordered series of cleavage and polymerization steps carried out by at least two R2 protein subunits, one bound upstream and one bound downstream of the integration site. The role in the retrotransposition reaction of two conserved DNA-binding motifs, a C2H2 zinc finger (ZF) and a Myb motif, located within the N-terminal domain of the protein are explored in this report. These motifs do not appear to play a role in RT or the ability of the protein to bind the R2 RNA transcript. Methylation and missing nucleoside interference-based DNA footprints using polypeptides to the N-terminal domain suggest the ZF and Myb motifs bind to regions -3 to -1 and +10 to +15 with reference to the insertion site. Mutations in these DNA sites or of the N-terminal protein domain blocked binding and the activity of the downstream subunit. Mutations of the protein domain also affected binding of the upstream subunit but not its function, suggesting the primary path to DNA target recognition by R2 involves both upstream and downstream subunits.
RNA from the 5' End of the R2 Retrotransposon Controls R2 Protein Binding to and Cleavage of Its DNA Target Site
Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.
Isoenergetic Penta- and Hexanucleotide Microarray Probing and Chemical Mapping Provide a Secondary Structure Model for an RNA Element Orchestrating R2 Retrotransposon Protein Function
LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2' oxygen and 4' carbon of ribose) and 2,6-diaminopurine were incorporated into 2'-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5' coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5' RNA). This R2Bm 5' RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5' RNA.
Secondary Structures for 5' Regions of R2 Retrotransposon RNAs Reveal a Novel Conserved Pseudoknot and Regions That Evolve Under Different Constraints
Sequences from the 5' region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for a segment of this RNA is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five conserved secondary structures are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5' segment to a protein coding function near the 3' end. This suggests that local regions evolved under separate functional constraints (structural, coding, or both).
Convergently Recruited Nuclear Transport Retrogenes Are Male Biased in Expression and Evolving Under Positive Selection in Drosophila
The analyses of gene duplications by retroposition have revealed an excess of male-biased duplicates generated from X chromosome to autosomes in flies and mammals. Investigating these genes is of primary importance in understanding sexual dimorphism and genome evolution. In a particular instance in Drosophila, X-linked nuclear transport genes (Ntf-2 and ran) have given rise to autosomal retroposed copies three independent times (along the lineages leading to Drosophila melanogaster, D. ananassae, and D. grimshawi). Here we explore in further detail the expression and the mode of evolution of these Drosophila Ntf-2- and ran-derived retrogenes. Five of the six retrogenes show male-biased expression. The ran-like gene of D. melanogaster and D. simulans has undergone recurrent positive selection. Similarly, in D. ananassae and D. atripex, the Ntf-2 and ran retrogenes show evidence of past positive selection. The data suggest that strong selection is acting on the origin and evolution of these retrogenes. Avoiding male meiotic X inactivation, increasing level of expression of X-linked genes in male testes, and/or sexual antagonism might explain the recurrent duplication of retrogenes from X to autosomes. Interestingly, the ran-like in D. yakuba has mostly pseudogenized alleles. Disablement of the ran-like gene in D. yakuba indicates turnover of these duplicates. We discuss the possibility that Dntf-2r and ran-like might be involved in genomic conflicts during spermatogenesis.
The Lipid Language of Plant-fungal Interactions
Lipid mediated cross-kingdom communication between hosts and pathogens is a rapidly emerging field in molecular plant-fungal interactions. Amidst our growing understanding of fungal and plant chemical cross-talk lies the distinct, yet little studied, role for a group of oxygenated lipids derived from polyunsaturated fatty acids, termed oxylipins. Endogenous fungal oxylipins are known for their roles in carrying out pathogenic strategies to successfully colonize their host, reproduce, and synthesize toxins. While plant oxylipins also have functions in reproduction and development, they are largely recognized as agents that facilitate resistance to pathogen attack. Here we review the composition and endogenous functions of oxylipins produced by both plants and fungi and introduce evidence which suggests that fungal pathogens exploit host oxylipins to facilitate their own virulence and pathogenic development. Specifically, we describe how fungi induce plant lipid metabolism to utilize plant oxylipins in order to promote G-protein-mediated regulation of sporulation and mycotoxin production in the fungus. The use of host-ligand mimicry (i.e. coronatine) to manipulate plant defense responses that benefit the fungus are also implicated.
Independently Derived Targeting of 28S RDNA by A- and D-clade R2 Retrotransposons: Plasticity of Integration Mechanism
Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through a target primed reverse transcription mechanism (TPRT). R2 elements have been used as a model system for investigating non-LTR retrotransposon integration. We previously demonstrated that R2 retrotransposons require two subunits of the element-encoded multifunctional protein to integrate-one subunit bound upstream of the insertion site and one bound downstream. R2 elements have been phylogenetically categorized into four clades: R2-A, B, C and D, that diverged from a common ancestor more than 850 million years ago. All R2 elements target the same sequence within 28S rDNA. The amino-terminal domain of R2Bm, an R2-D clade element, contains a single zinc finger and a Myb motif that are responsible for binding R2 protein downstream of the insertion site. Target site recognition is of interest as it is the first step in the integration reaction and may help elucidate evolutionary history and integration mechanism. The amino-terminal domain of R2-A clade members contains three zinc fingers and a Myb motif. We show here that R2Lp, an R2-A clade member, uses its amino-terminal DNA binding motifs to bind upstream of the insertion site. Because the R2-A and R2-D clade elements recognize 28S rDNA differently, we conclude the A- and D-clades represent independent targeting events to the 28S site. Our results also indicate a certain plasticity of insertional mechanics exists between the two clades.
Targeting Novel Sites: The N-terminal DNA Binding Domain of Non-LTR Retrotransposons is an Adaptable Module That is Implicated in Changing Site Specificities
Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through target primed reverse transcription (TPRT). RLE-bearing elements have been used as a model system for investigating non-LTR retrotransposon integration. R2 elements target a specific site in the 28S rDNA gene. We previously demonstrated that the two major sub-classes of R2 (R2-A and R2-D) target the R2 insertion site in an opposing manner with regard to the pairing of known DNA binding domains and bound sequences-indicating that the A- and D-clades represent independently derived modes of targeting that site. Elements have been discovered that group phylogenetically with R2 but do not target the canonical R2 site. Here we extend our earlier studies to show that a separate R2-A clade element, which targets a site other than the canonical R2 site, does so by using the N-terminal zinc fingers and Myb motifs. We further extend our targeting studies beyond R2 clade elements by investigating the ability of the N-terminal zinc fingers from the nematode NeSL-1 element to target its integration site. Our data are consistent with the use of an N-terminal DNA binding domain as one of the major targeting determinants used by RLE-bearing non-LTR retrotransposons to secure a protein subunit near the insertion site. This N-terminal DNA binding domain can undergo modifications, allowing the element to target novel sites. The binding orientation of the N-terminal domain relative to the insertion site is quite variable.
A Novel, Conditional, Lesion Mimic Phenotype in Cotton Cotyledons Due to the Expression of an Endochitinase Gene from Trichoderma Virens
We have observed a novel, lesion mimic phenotype (LMP) in the cotyledons of cotton seedlings expressing an endochitinase gene from Trichoderma virens. This phenotype, however, is conditional and is elicited only when the transgenic seedlings are germinating on a medium that is devoid of mineral nutrients. The LMP manifests itself around the 5th day in the form of scattered, dry necrotic lesions on the cotyledons. The severity of the LMP is correlated with the level of transgene activity. Production of reactive oxygen species and activities of certain defense related enzymes and genes were substantially higher in the cotyledons of seedlings that were growing under mineral nutrient stress. Molecular and biochemical analyses indicated significantly higher-level activities of certain defense-related genes/enzymes at the onset of the phenotype. Treatment with methyl jasmonate can induce LMP in the cotyledons of wild-type (WT) seedlings similar to that observed in the endochitinase-expressing seedlings grown on nutrient-free medium. On the other hand, salicylic acid (SA), its functional analog, benzo(1,2,3) thiadiazole-7-carbothioic acid (BTH), and ibuprofen can rescue the LMP induced by the seedling-growth on nutrient-deficient medium. Nutrient deficiency-induced activation of a defense response appears to be the contributing factor in the development of LMP in endochitinase-expressing cotton seedlings.
