TPases are enzymes responsible for transposition activity. ORF is 1000–1500 bp long and encodes for the transposase (TPase) gene. Their TIRs are generally 10–40 bp long and contain protein-binding elements. MLEs have a relatively simple structure, consisting mainly of terminal inverted repeats (TIRs) and an open reading frame (ORF). These hyperactive elements are found to increase their transposition activities when expressed in different host genomes such as bacteria by 200–800 times, and by 10–50 times when mutated. Among the MLEs, two important members, mosaic element 1 ( Mos1) and Haematobia irritans mariner 1 ( Himar1) are widely studied for their cross genome transportability and used as tools in genetic studies. ITm transposons are further classified, among which three major families are, Tc1-like elements ( TLEs), mariner-like elements (MLEs) and Pogo-like elements. Because of their versatile nature, they are used in genetic studies, as a tool in gene tagging, transgenesis and insertional mutagenesis. In nature, they show a widespread distribution, frequent and total random insertions, and have a high frequency of heterologous transposition. ITm transposons are characterized by self-driven mobility of its members and are generally independent of host factors to mediate transposition.
Because of its near-identical sequence similarity to the bacterial insertion sequence, IS630, Tc1/mariner superfamily is expanded to include IS630 elements and is renamed as ITm ( IS630-Tc1-mariner) superfamily. One of the most prevalent DNA transposon families in eukaryotic genomes is the Tc1/mariner superfamily, which plays a significant role in genome evolution. There are several variants within each of the two types. There are two distinctive types, (a) DNA transposons (class II) that transpose by a DNA-mediated “cut-paste” mechanism and (b) retrotransposons (class I) that act through the “copy-paste” mechanism involving an RNA intermediate. Transposons are ubiquitous in plant and animal genomes in abundance. Transposable elements (TEs), or ‘jumping genes’ or transposons, are DNA sequences that have the ability to move within the genome. We speculate that in moso bamboo, NESs regulates the transposition activity of MLEs to maintain the genome integrity. The results suggested that NES is an important regulator of nuclear export of transposase in Ppmar elements and the mutation of the NES domains can either increase or decrease the export signalling. NES-2 and NES-3 elements showed, up to three times increase in transposon excision than the wild types.
In both the MLEs, NES-1 had the highest excision suppression, which was less than half of the excision frequency of the wild type. Co-transformation of the vector together with non-autonomous Ppmar transposons and NES-lacking transposases was used to assess the differential excision frequencies of the mutants NES domains. In the transposon excision assay, the mutant and wild type NES of both the Ppmar elements were integrated into an Ade2 vector, and within the Ade2 gene. The NES-1 domain was related to the comparatively high spread of ECFP, while NES-2 and NES-3 indicated a low spread, implying that NES activity on nuclear export increased when the NES is made leucine-rich, while the signalling activity was reduced when the leucine content was lowered in the NES domain. Further, ECFP had a wider localisation in the cellular matrix, but EYFP was largely located in the nucleus. Fluorescence assay revealed that blue fluorescence of ECFP was more intense than the red fluorescence of the EYFP in the yeast cell matrix. To differentiate protein localisation under the NES influence, ECFP alone was fused to wild and mutant NES domains either on N- or C-terminal and not to EYFP. In the fluorescence assay, the mutants, NES-1, 2 and 3 along with the wild types ( NES-0) were fused with fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were co-transformed into yeast system.
For this, by site-directed mutagenesis, three NES mutants were developed from each of the MLE. To understand the functions of NES in transposon activity, we have conducted two experiments, fluorescence and excision frequency assays in the yeast system. Houz) genome possessing transposases that harbour nuclear export signal (NES) domain, but not any nuclear localization signal (NLS) domain. Ppmar1 and Ppmar2 are two active mariner-like elements (MLEs) cloned from moso bamboo ( Phyllostachys edulis (Carrière) J.
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