RESUMO
The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.
Assuntos
Elementos de DNA Transponíveis/genética , DNA/genética , Vetores Genéticos/genética , Mutagênese Insercional/genética , Animais , Sequência de Bases , Células Eucarióticas/metabolismo , Dados de Sequência Molecular , Plasmídeos/genética , Deleção de Sequência , Transformação GenéticaRESUMO
The transposons piggyBac and tagalong are Lepidopteran transposons that exhibit extreme site-specificity for the tetranucleotide TTAA upon insertion and excise in a characteristic precise fashion, regenerating a single TTAA target site. The precise excision of both piggyBac and tagalong can occur in the absence of factors encoded by either transposon, possibly through the recruitment of host proteins and/or cross-mobilizing transposons. In this report, we utilize mobility shift assays and exonuclease III protection analyses to identify DNA binding activities from IPLB-SF21AE and TN-368 cells that are specific for piggyBac and tagalong-terminal repeats.
Assuntos
Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Extratos Celulares , Linhagem Celular , Núcleo Celular/metabolismo , Dados de Sequência Molecular , Mariposas/citologia , Spodoptera/citologia , Sequências Repetidas TerminaisRESUMO
The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells. We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line. Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase. The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well. This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells.
Assuntos
DNA Nucleotidiltransferases/genética , Elementos de DNA Transponíveis/genética , Lepidópteros/genética , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Escherichia coli/genética , Expressão Gênica/genética , Genes Supressores/genética , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/genética , Spodoptera/genética , Transfecção , TransposasesRESUMO
Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short-inverted-repeat elements are characterized by their extreme specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggyBac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN-368 cells or IPLB-SF21 AE cells. The precise excision of these elements in infected IPLB-SF21 AE cells occurs in the absence of either tagalong or piggyBac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion in a single family of unique transposons.
Assuntos
Baculoviridae/genética , Elementos de DNA Transponíveis , Mariposas/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Óperon Lac , Dados de Sequência Molecular , Recombinação Genética , Spodoptera/genéticaRESUMO
The IFP2 element is a unique Lepidopteran transposon that has been associated with spontaneous Baculovirus mutants isolated following passage of the virus in the TN-368 cell line. Independent genomic representatives of IFP2 from TN-368 cells show little sequence divergence, suggesting that IFP2 was recently introduced into this genome and is highly stable. IFP2 is inserted within AT-rich regions of the TN-368 genome and targets TTAA sites. The specificity for TTAA target sites during transposition is not limited to the movement of IFP2 during an active Baculovirus infection, but is a property of its movement in uninfected cells as well. The exact origin of IFP2 remains obscure since it is found in two independently established Trichoplusia ni cell lines but not in three others, and we have not yet identified any IFP2 sequences in either field collected larvae or laboratory colonies.
Assuntos
Elementos de DNA Transponíveis , Genes , Lepidópteros/genética , Transcrição Gênica , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , Clonagem Molecular , DNA Complementar , Genoma , Lepidópteros/embriologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
We characterize a new Lepidopteran transposon associated with FP mutations of baculoviruses. This transposon, designated hitchhiker, is 579 bp long with 39-bp imperfect inverted terminal repeats. hitchhiker inserts with extreme specificity for a single trinucleotide target site, TTA, within the 25K gene. This transposon is the most frequently identified insertion in serial passage baculovirus mutants isolated from TN-368 cells.