The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Donald C. Rio - One of the best experts on this subject based on the ideXlab platform.
-
Structure of a P Element transPosase-DNA comPlex reveals unusual DNA structures and GTP-DNA contacts.
Nature Structural & Molecular Biology, 2019Co-Authors: George E. Ghanim, Elizabeth H. Kellogg, Eva Nogales, Donald C. RioAbstract:P Element transPosase catalyzes the mobility of P Element DNA transPosons within the DrosoPhila genome. P Element transPosase exhibits several unique ProPerties, including the requirement for a guanosine triPhosPhate cofactor and the generation of long staggered DNA breaks during transPosition. To gain insights into these features, we determined the atomic structure of the DrosoPhila P Element transPosase strand transfer comPlex using cryo-EM. The structure of this Post-transPosition nucleoProtein comPlex reveals that the terminal single-stranded transPoson DNA adoPts unusual A-form and distorted B-form helical geometries that are stabilized by extensive Protein-DNA interactions. Additionally, we infer that the bound guanosine triPhosPhate cofactor interacts with the terminal base of the transPoson DNA, aPParently to Position the P Element DNA for catalysis. Our structure Provides the first view of the P Element transPosase suPerfamily, offers new insights into P Element transPosition and imPlies a transPosition Pathway fundamentally distinct from other cut-and-Paste DNA transPosases. Cryo-EM resolution of the DrosoPhila P Element transPosase strand transfer comPlex visualizes a unique configuration of the transPoson ends and the location of an essential GTP required for transPosition of this historically imPortant mobile Element.
-
the human thaP9 gene encodes an active P Element dna transPosase
Science, 2013Co-Authors: Sharmistha Majumdar, Donald C. Rio, Anita SinghAbstract:The human genome contains ~50 genes that were derived from transPosable Elements or transPosons, and many are now integral comPonents of cellular gene exPression Programs. The human THAP9 gene is related to the DrosoPhila P-Element transPosase. Here, we show that human THAP9 can mobilize DrosoPhila P-Elements in both DrosoPhila and human cells. Chimeric Proteins formed between the DrosoPhila P-Element transPosase N-terminal THAP DNA binding domain and the C-terminal regions of human THAP9 can also mobilize DrosoPhila P Elements. Our results indicate that human THAP9 is an active DNA transPosase that, although "domesticated," still retains the catalytic activity to mobilize P transPosable Elements across sPecies.
-
Molecular biology of DrosoPhila P-Element transPosition.
Progress in Nucleic Acid Research and Molecular Biology, 2008Co-Authors: Rhonda F. Doll, Sima Misra, Paul D. Kaufman, Donald C. RioAbstract:Publisher Summary TransPosable P Elements are a family of mobile genetic Elements found in DrosoPhila. They are resPonsible for the syndrome of genetic traits known as hybrid dysgenesis. This chaPter discusses molecular biology of DrosoPhila P- Element transPosition. P Elements are a family of transPosable Elements found in the fruit fly, DrosoPhila melanogaster . They are the causative agents of a syndrome of correlated genetic traits, known as hybrid dysgenesis, that occurs in the Progeny of a cross between males carrying P Elements (P-strains) and females that lack them (M-strains). It is known that P-Element transPosition is controlled genetically and only occurs when a P-strain male is mated to an M-strain female but not in the reciProcal M-male × P-female cross and in a P × P cross. P-Element transPosition also exhibits tissue sPecificity, occurring only in the germ line. P Elements have been extensively analyzed at the molecular level and are known to encode two Proteins: an 87-kDa Protein that aPPears to be the transPosase required for the high rates of P-Element transPosition as well as for the Precise and imPrecise excision of P Elements, and a second, smaller Protein of 66-kDa Postulated to be a negative regulator of transPosition.
-
Analysis of P Element transPosase Protein-DNA interactions during the early stages of transPosition.
Journal of Biological Chemistry, 2007Co-Authors: Mei Tang, Ciro Cecconi, Carlos Bustamante, Donald C. RioAbstract:P Elements are a family of transPosable Elements found in DrosoPhila that move by using a cut-and-Paste mechanism and that encode a transPosase Protein that uses GTP as a cofactor for transPosition. Here we used atomic force microscoPy to visualize the initial interaction of transPosase Protein with P Element DNA. The transPosase first binds to one of the two P Element ends, in the Presence or absence of GTP, Prior to synaPsis. In the absence of GTP, these comPlexes remain stable but do not Proceed to synaPsis. In the Presence of GTP or nonhydrolyzable GTP analogs, synaPsis haPPens raPidly, whereas DNA cleavage is slow. Both atomic force microscoPy and standard biochemical methods have been used to show that the P Element transPosase exists as a Pre-formed tetramer that initially binds to either one of the two P Element ends in the absence of GTP Prior to synaPsis. This initial single end binding may exPlain some of the aberrant P Element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in Promoting synaPsis by P Element transPosase may be to orient a second site-sPecific DNA binding domain in the tetramer allowing recognition of a second high affinity transPosase-binding site at the other transPoson end.
-
DNA binding by the KP rePressor Protein inhibits P-Element transPosase activity in vitro.
The EMBO Journal, 1998Co-Authors: Charles C. Lee, Eileen L. Beall, Donald C. RioAbstract:P Elements are a family of mobile DNA Elements found in DrosoPhila . P‐Element transPosition is tightly regulated, and P‐Element‐encoded rePressor Proteins are resPonsible for inhibiting transPosition in vivo . To investigate the molecular mechanisms by which one of these rePressors, the KP Protein, inhibits transPosition, a variety of mutant KP Proteins were PrePared and tested for their biochemical activities. The rePressor activities of the wild‐tyPe and mutant KP Proteins were tested in vitro using several different assays for P‐Element transPosase activity. These studies indicate that the site‐sPecific DNA‐binding activity of the KP Protein is essential for rePressing transPosase activity. The DNA‐binding domain of the KP rePressor Protein is also shared with the transPosase Protein and resides in the N‐terminal 88 amino acids. Within this region, there is a C 2 HC Putative metal‐binding motif that is required for site‐sPecific DNA binding. In vitro the KP Protein inhibits transPosition by comPeting with the transPosase enzyme for DNA‐binding sites near the P‐Element termini.
Eileen L. Beall - One of the best experts on this subject based on the ideXlab platform.
-
drosoPhila irbP bziP heterodimer binds P Element dna and affects hybrid dysgenesis
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Malik J Francis, Eileen L. Beall, Siobhan Roche, Ronaldo P PanganibanAbstract:In DrosoPhila , P-Element transPosition causes mutagenesis and genome instability during hybrid dysgenesis. The P-Element 31-bP terminal inverted rePeats (TIRs) contain sequences essential for transPosase cleavage and have been imPlicated in DNA rePair via Protein–DNA interactions with cellular Proteins. The identity and function of these cellular Proteins were unknown. Biochemical characterization of Proteins that bind the TIRs identified a heterodimeric basic leucine ziPPer (bZIP) comPlex between an uncharacterized Protein that we termed “Inverted RePeat Binding Protein (IRBP) 18” and its Partner XrP1. The reconstituted IRBP18/XrP1 heterodimer binds sequence-sPecifically to its dsDNA-binding site within the P-Element TIRs. Genetic analyses imPlicate both Proteins as critical for rePair of DNA breaks following transPosase cleavage in vivo. These results identify a cellular Protein comPlex that binds an active mobile Element and Plays a more general role in maintaining genome stability.
-
identification and analysis of a hyPeractive mutant form of drosoPhila P Element transPosase
Genetics, 2002Co-Authors: Eileen L. Beall, Matthew B MahoneyAbstract:TransPosition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in Prokaryotic systems have led to the isolation of mutant transPosase Proteins with higher or novel activities comPared to those of the wild-tyPe Protein. In the course of our study of the effects of mutating Potential ATM-family DNA damage checkPoint Protein kinase sites in the DrosoPhila P -Element transPosase Protein, we found one mutation, S129A, that resulted in an elevated level of transPosase activity using in vivo recombination assays, including P -Element-mediated germline transformation. In vitro assays for P -Element transPosase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when comPared to the wild-tyPe Protein, whereas the strand-transfer activity is similar to that of wild tyPe. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may Proceed in concert. The P -Element transPosase Protein contains 10 Potential consensus PhosPhorylation sites for the ATM family of PI3-related Protein kinases. Of these 10 sites, 8 affect transPosase activity either Positively or negatively when substituted individually with alanine and tested in vivo . A mutant transPosase Protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-tyPe amino acids back at only 3 of the 8 Positions. These data suggest that the activity of P -Element transPosase may be regulated by PhosPhorylation and demonstrate that one mutation, S129A, results in hyPeractive transPosition.
-
DNA binding by the KP rePressor Protein inhibits P‐Element transPosase activity in vitro
The EMBO Journal, 1998Co-Authors: Eileen L. BeallAbstract:P Elements are a family of mobile DNA Elements found in DrosoPhila . P‐Element transPosition is tightly regulated, and P‐Element‐encoded rePressor Proteins are resPonsible for inhibiting transPosition in vivo . To investigate the molecular mechanisms by which one of these rePressors, the KP Protein, inhibits transPosition, a variety of mutant KP Proteins were PrePared and tested for their biochemical activities. The rePressor activities of the wild‐tyPe and mutant KP Proteins were tested in vitro using several different assays for P‐Element transPosase activity. These studies indicate that the site‐sPecific DNA‐binding activity of the KP Protein is essential for rePressing transPosase activity. The DNA‐binding domain of the KP rePressor Protein is also shared with the transPosase Protein and resides in the N‐terminal 88 amino acids. Within this region, there is a C 2 HC Putative metal‐binding motif that is required for site‐sPecific DNA binding. In vitro the KP Protein inhibits transPosition by comPeting with the transPosase enzyme for DNA‐binding sites near the P‐Element termini.
-
DNA binding by the KP rePressor Protein inhibits P-Element transPosase activity in vitro.
The EMBO Journal, 1998Co-Authors: Charles C. Lee, Eileen L. Beall, Donald C. RioAbstract:P Elements are a family of mobile DNA Elements found in DrosoPhila . P‐Element transPosition is tightly regulated, and P‐Element‐encoded rePressor Proteins are resPonsible for inhibiting transPosition in vivo . To investigate the molecular mechanisms by which one of these rePressors, the KP Protein, inhibits transPosition, a variety of mutant KP Proteins were PrePared and tested for their biochemical activities. The rePressor activities of the wild‐tyPe and mutant KP Proteins were tested in vitro using several different assays for P‐Element transPosase activity. These studies indicate that the site‐sPecific DNA‐binding activity of the KP Protein is essential for rePressing transPosase activity. The DNA‐binding domain of the KP rePressor Protein is also shared with the transPosase Protein and resides in the N‐terminal 88 amino acids. Within this region, there is a C 2 HC Putative metal‐binding motif that is required for site‐sPecific DNA binding. In vitro the KP Protein inhibits transPosition by comPeting with the transPosase enzyme for DNA‐binding sites near the P‐Element termini.
-
DrosoPhila P-Element transPosase is a novel site-sPecific endonuclease
Genes & Development, 1997Co-Authors: Eileen L. Beall, Donald C. RioAbstract:We develoPed in vitro assays to study the first steP of the P-Element transPosition reaction: donor DNA cleavage. We found that P-Element transPosase required both 5′ and 3′ P-Element termini for efficient DNA cleavage to occur, suggesting that a synaPtic comPlex forms Prior to cleavage. TransPosase made a staggered cleavage at the P-Element termini that is novel for all known site-sPecific endonucleases: the 3′ cleavage site is at the end of the P-Element, whereas the 5′ cleavage site is 17 bP within the P-Element 31-bP inverted rePeats. The P-Element termini were Protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable Protein comPlex remains bound to the Element termini after cleavage. These data are consistent with a cut-and-Paste mechanism for P-Element transPosition and may exPlain why P Elements Predominantly excise imPrecisely in vivo.
Pavel Georgiev - One of the best experts on this subject based on the ideXlab platform.
-
The vicinity of a broken chromosome end affects P Element mobilization in DrosoPhila melanogaster.
Molecular Genetics and Genomics, 2004Co-Authors: Larisa Melnikova, H. Biessmann, Pavel GeorgievAbstract:Broken chromosome ends are believed to be caPPed by a terminal Protein comPlex, and can be maintained in DrosoPhila melanogaster for many generations. We investigated whether the vicinity of a chromosome end affected P Element mobilization and the subsequent rePair of the resulting DNA lesion. High levels of P Element excision were observed when at least 5 kb of DNA was located between the P Element and the end of the chromosome, but recovery of chromosomes from which the P Element had been excised was greatly reduced when the chromosome end was Positioned less than 5 kb away from the original P Element insertion site. Moreover, when the P Element was mobilized in terminal deficiency ( y TD ) alleles, excision events were accomPanied by deletions of sequences originally located distal to the P Element.
-
P Element-mediated duPlications of genomic regions in DrosoPhila melanogaster.
Chromosoma, 2002Co-Authors: A. K. Golovnin, Sofia G. Georgieva, Hayk Hovhannisyan, Karine Barseguyan, Pavel GeorgievAbstract:Previously we have described highly unstable yellow mutations induced by chimeric Elements that consist of genomic sequences originating from different regions of the X chromosome flanked by identical coPies of an internally deleted 1.2 kb P Element. To study further the origin and the mechanism of formation of chimeric mobile Elements, we analyzed comPlex y-sc mutations, induced by inversions between P Elements located in the neighboring yellow and scute loci. The breakPoints of the inversions are flanked by two P Elements in head-to-head orientation on one side and by one P Element on the other side. Such an arrangement of P Elements leads to frequent duPlication into the site between the two P Element coPies located in head-to-head orientation of the yellow sequences adjacent to the single P Element. The duPlicated yellow sequences either Partly rePlace the sequence of one of the P Elements or are inserted between the conserved head-to-head oriented P Elements. In some cases two coPies of the yellow sequence are duPlicated between the P Elements in inverted tail-to-tail orientation. The structure of the P Elements at the Place of duPlication and of the P Element-yellow junction suggests that the described duPlications, which form chimeric mobile Elements, are generated through the Previously ProPosed synthesis-dePendent strand annealing mechanism.
-
Mechanisms of excising the P-Element in a model system at the yellow locus of DrosoPhila melanogaster
Genetika, 1999Co-Authors: Belen'kaia Tiu, Biriukova, Pavel GeorgievAbstract:Patterns of excision of a single P Element were studied in a model system of the yellow locus. The data obtained were in good agreement with the generally accePted SDSA (synthesis-dePendent strand annealing) model. SPecific features of P Element excision in the Presence of two tandemly rePeated coPies are Presented. The Pattern of P Element excision dePended on the sequences surrounding the insertion site and on the number of its additional coPies Present in the genome.
-
P-Element Insertion at the Polyhomeotic Gene Leads to Formation of a Novel Chimeric Protein That Negatively Regulates yellow Gene ExPression in P-Element-Induced Alleles of DrosoPhila melanogaster
Genetics, 1998Co-Authors: Tatiana Belenkaya, Sofia G. Georgieva, Alexey Soldatov, Elena N. Nabirochkina, Inna Birjukova, Pavel GeorgievAbstract:Polyhomeotic is a member of the Polycomb grouP ( Pc -G) of homeotic rePressors. The Proteins encoded by the Pc -G genes form rePressive comPlexes on the Polycomb grouP resPonse Element sites. The PhP1 mutation was induced by insertion of a 1.2-kb P Element into the 5′ transcribed nontranslated region of the Proximal Polyhomeotic gene. The PhP1 allele confers no mutant PhenotyPe, but rePresses transcriPtion of P -Element-induced alleles at the yellow locus. The PhP1 allele encodes a chimeric P-PH Protein, consisting of the DNA-binding domain of the P Element and the PH Protein lacking 12 amino-terminal amino acids. The P-PH, Polycomb (PC), and Posterior sex combs (PSC) Proteins were immunohistochemically detected on Polytene chromosomes in the regions of P -Element insertions.
-
P Element sequences can comPensate for a deletion of the yellow regulatory region in DrosoPhila melanogaster
Molecular Genetics and Genomics, 1998Co-Authors: Tatyana Belenkaya, Hayk Hovhannisyan, K. Barseguyan, Inna Biryukova, E. Z. Kochieva, Pavel GeorgievAbstract:The effects of interactions between P Element and yellow regulatory sequences on the control of yellow exPression were studied. The y mutations used in the analysis lack a segment of uPstream sequence that extends from Position −146 bP to −70 bP, relative to the transcriPtion start site of the yellow gene. This sequence has been found to be necessary for the function of the yellow Promoter. The insertion of one or two P Element coPies at Position −69 bP comPensates for the deletion in the regulatory region and restores yellow exPression. After mobilization of the P Element, new PhenotyPes were selected and molecularly characterized. Two regions in the 5′ Part of the P Element, from 23 bP to 71 bP and from 82 bP to 108 bP, can each Partially comPensate for the yellow deletion. In addition, deletion derivatives of the P Element were themselves able to activate yellow transcriPtion. All such P Elements retain at least 108 bP of sequence at the 5′ end and 15–17 bP at the 3′ end. Thus, the region of the P Element from 23 bP to 108 bP contains cis-regulatory Elements that can influence the transcriPtion of neighboring genes.
T Miyake - One of the best experts on this subject based on the ideXlab platform.
-
DeveloPmental Profile of P Element transPosition in DrosoPhila somatic cells.
Genetica, 1993Co-Authors: S Togashi, R Ueda, M Takahisa, K Kondo, T MiyakeAbstract:TransPosition of the P Element during DrosoPhila ontogenesis was monitored. A modified P Element was transPosed by the P delta 2-3 transPosase source. P Elements inserted into the genome were cloned by the Plasmid rescue at various develoPmental stages of the G1 hybrid to trace events in somatic cells. The transPosed Elements were directly counted by analyzing RFLP of genomic DNA fragments flanking the P Elements. TransPosition began from the late embryonic stage, but occurred rarely. Frequent transPosition was observed from the late third instar to early PuPal stage. From these results, transPosition of the P Element would aPPear to be affected by the develoPmental state of somatic host cells.
-
DeveloPmental Profile of P Element transPosition in DrosoPhila somatic cells.
Genetica, 1993Co-Authors: S Togashi, R Ueda, M Takahisa, K Kondo, T MiyakeAbstract:TransPosition of the P Element duringDrosoPhila ontogenesis was monitored. A modified P Element was transPosed by the PΔ2-3 transPosase source. P Elements inserted into the genome were cloned by the Plasmid rescue at various develoPmental stages of the G1 hybrid to trace events in somatic cells. The transPosed Elements were directly counted by analyzing RFLP of genomic DNA fragments flanking the P Elements. TransPosition began from the late embryonic stage, but occurred rarely. Frequent transPosition was observed from the late third instar to early PuPal stage. From these results, transPosition of the P Element would aPPear to be affected by the develoPmental state of somatic host cells.
-
Insertional mutagenesis in DrosoPhila. II. P Element mediated transformation of DrosoPhila yakuba.
The Japanese Journal of Genetics, 1992Co-Authors: S Togashi, R Ueda, M Takahisa, K Kondo, Misa Mikuni, T MiyakeAbstract:DrosoPhila yakuba, a member of melanogaster subgrouP being free of P Element, acquired resistance to an antibiotic neomycin by the transformation utilizing P Element. In this sPecies, the transformation frequency was comParable to that of D. melanogaster. Further, the occurrence of 8 base Pairs duPlication uPon the insertion of the Element was confirmed. These facts suggest that the P Element could be inserted into the genome in the same manner, even in D. yakuba. Any consensus for Preferential insertion could not be found on the nucleotide sequence as in D. melanogaster. However, it is noticeable that a series of the short Palindromic stretches was common around the insertion sites in both sPecies. It suggests that a structural feature of DNA Plays a role as a landmark for P Element insertion.
John Locke - One of the best experts on this subject based on the ideXlab platform.
-
Point mutations in a DrosoPhila P Element abolish both P Element-dePendent silencing (PDS) of a transgene and rePressor functions
Chromosoma, 2011Co-Authors: Alireza Sameny, Anderson La, Scott Hanna, John LockeAbstract:The P Elements of DrosoPhila melanogaster are well-studied transPosons with both mobilizing and rePressor functions . P Elements can also variably silence the exPression of certain other transgenes through a Phenomenon known as P Element-dePendent silencing (PDS). To examine the role of the P rePressor in PDS, we have induced, isolated, and characterized 22 Point mutations in an archetyPe P Element called P[SalI]89D . All mutations showed a loss in the ability to silence one or more assays for the PDS PhenotyPe. These mutants also lost the ability to induce the suPPression of variegation in P[hsP26-Pt-T]39C-12 , another P Element-dePendent PhenotyPe. A subgrouP of 11 mutations was further assayed for their ability to act as a P rePressor and silence the P Element Promoter transcribing a lacZ ^ + gene, and this function was lost as well. Taken together, this study suPPorts a model of PDS acting through Protein interactions, not RNA, with heterochromatic Proteins to modify the extent of variegation seen in PDS. Furthermore, the common loss of functions for PDS and P rePressor silencing (from another P Promoter) argues for a common role of the rePressor. This makes the PDS model a good system for examining P rePressor functions and how they relate to transPoson-mediated gene silencing in general.
