ABSTRACT
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.
Subject(s)
DNA Transposable Elements , DNA-Binding Proteins/metabolism , Insect Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Base Sequence , Cell Extracts , Cell Line , Cell Nucleus/metabolism , Molecular Sequence Data , Moths/cytology , Spodoptera/cytology , Terminal Repeat SequencesABSTRACT
The terminal DNA sequence requirements for piggyBac transposable element excision were explored using a plasmid-based assay in transfected, cultured insect cells. A donor plasmid containing duplicate 3' piggyBac terminal inverted repeats was constructed that allowed individual nucleotides or groups of nucleotides within one of the 3' repeats to be mutated. The relative extent of excision using the mutated end versus the wild-type end was then assayed. Removal of even one of the terminal 3' G nucleotides from the piggyBac inverted repeat, or removal of the dinucleotide AA from the flanking TTAA target site prevents excision of piggyBac at the mutated terminus. Incorporation of an asymmetric TTAC target site at the 3' end does not prevent excision from the mutated end. Thus, both piggyBac DNA and flanking host DNA appear to play crucial roles in the excision process.
Subject(s)
DNA Transposable Elements , DNA/genetics , Animals , Base Sequence , Cell Line , DNA Primers/genetics , Escherichia coli/genetics , Mutagenesis, Insertional , Nucleopolyhedroviruses/genetics , Plasmids/genetics , Restriction Mapping , Spodoptera , TransfectionABSTRACT
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.
Subject(s)
DNA Nucleotidyltransferases/genetics , DNA Transposable Elements/genetics , Lepidoptera/genetics , Animals , Base Sequence , Cell Line , DNA/genetics , Escherichia coli/genetics , Gene Expression/genetics , Genes, Suppressor/genetics , Models, Genetic , Molecular Sequence Data , Plasmids/genetics , Spodoptera/genetics , Transfection , TransposasesABSTRACT
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.
Subject(s)
DNA Transposable Elements , Genes , Lepidoptera/genetics , Transcription, Genetic , Animals , Base Sequence , Blotting, Southern , Cell Line , Cloning, Molecular , DNA, Complementary , Genome , Lepidoptera/embryology , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Nucleic AcidABSTRACT
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.
Subject(s)
DNA Transposable Elements/genetics , Nucleopolyhedroviruses/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , DNA, Viral/genetics , Molecular Sequence Data , Nucleopolyhedroviruses/metabolism , Serial Passage , Spodoptera/cytologyABSTRACT
A new member of the beta-chemokine family, macrophage inflammatory protein (MIP)-related protein-2 (MRP-2) was isolated from a murine macrophage cell line, RAW 264.7. MRP-2 is composed of 122 amino acids of which the first 21 residues constitute a putative signal sequence. The putative mature protein is composed of 101 amino acids with a molecular weight of 11,600. MRP-2 is structurally similar to MIP-related protein-1 (MRP-1) (C10) and MIP-1 alpha. MRP-2 shows a 50.8% sequence identity at the protein level to MRP-1 and 46.3% identity to MIP-1 alpha. MRP-2 detects approximately 1.3 kilobase mRNA from monocyte and macrophage cell lines but does not detect the mRNA from T and B cells. The MRP-2 gene termed Scya9 was mapped to the central region of mouse chromosome 11 near the Scya1 and Scya2 genes, which are also members of the beta-chemokine superfamily. The Scya gene cluster was located between neurofibromatosis type 1 (Nf1) and myeloperoxidase (Mpo). rMRP-2 significantly suppressed colony formation by murine and human bone marrow granulocyte-macrophage (CFU-granulocyte-macrophage), erythroid (burst-forming unit-E), and multipotential (CFU-granulocyte-erythroid-macrophage-megakaryocyte) progenitor cells stimulated by combinations of growth factors.