One of three nuclear localization signals of maize <i>Activator</i> (<i>Ac</i>) transposase overlaps the DNA‐binding domain

Boehm, Ulrich; Heinlein, Manfred; Behrens, Ute; Kunze, Reinhard

doi:10.1046/j.1365-313x.1995.7030441.x

Cited by 34 publications

(28 citation statements)

References 43 publications

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“…The NoNLS-TPase-EGFP localized mainly to the cytoplasm, whereas the NLSTPase-EGFP and NLS K5E -TPase-EGFP proteins were predominantly nuclear. Both NoNLS-TPase-EGFP and NLS-TPase-EGFP showed a strong tendency to form aggregates in the cytoplasm and nucleus, respectively (Figure 3, A-C), that resembled the Activator TPase aggregates reported in plants (Heinlein et al 1994;Boehm et al 1995). In contrast, the NLS K5E rarely formed aggregates even at visibly higher expression levels.…”

Section: Resultsmentioning

confidence: 63%

“…Effects of nuclear localization on TPase functions: In plants, the Ac TPase is nuclear localized and a truncation of the first 102 N-terminal amino acids, which contains a strong nuclear localization signal, severely reduces nuclear transport (Heinlein et al 1994;Boehm et al 1995). However, the truncated TPase 103-807 reportedly produced even higher Ds excision rates compared to full-length Ac TPase in a transient Ds excision assay in Petunia protoplasts cotransfected with Ds and TPase constructs (Houba-Herin et al 1990;Becker et al 1992;Kunze et al 1993).…”

Section: Discussionmentioning

confidence: 99%

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Trans-Kingdom Transposition of the Maize Dissociation Element

et al. 2006

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Transposons are very valuable tools for genetic manipulation. However, the number of transposable elements that have been suitably adapted for experimental use is insufficient and the spectrum of heterologous hosts in which they have been deployed is restricted. To date, only transposons from animal hosts have been utilized in heterologous animal species and transposons of plant origin have been used in plant genetics. There has been no experimental evidence that any of the known elements could transpose in hosts belonging to both kingdoms. Here we demonstrate that the maize Dissociation (Ds) element is capable of effective Activator (Ac) transposase-mediated transposition in the zebrafish Danio rerio, yielding remarkable germline transmission rates. In addition, mammalian cells were also found to be conducive to Ds transposition. Furthermore, we demonstrate that nuclear localization of Ac transposase is essential for genomic Ds transposition. Our results support the hypothesis that Ac/Ds elements do not rely on hostspecific factors for transposition and that host factors involved in their mobility mechanism are widely conserved. Finally, even in vertebrate cells, the Ac/Ds system displays accurate transposition, largefragment carrying capacity, high transposition frequencies, efficient germline transmission, and reporter gene expression, all of which are advantageous for various genetic applications and animal biotechnology.

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Section: Resultsmentioning

confidence: 63%

Section: Discussionmentioning

confidence: 99%

Trans-Kingdom Transposition of the Maize Dissociation Element

et al. 2006

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“…Secondly, the truncated mRNA may be translated to produce an Ac transposase protein of only 200-250 amino acids (Figure 4c). Within this region of the protein are three nuclear localization signals (NLS) and a DNA binding motif (Boehm et al, 1995;Heinlein et al, 1994;Kunze et al, 1993). Earlier work in Petunia protoplasts has demonstrated that fusion of the N-terminal 189 amino acids of Ac TPase to GUS protein is sufficient to direct GUS activity to the protoplast nucleus (Boehm et al, 1995).…”

Section: Is the Alternative Processing Of The Ac Transcript Inmentioning

confidence: 99%

Alternative processing of the maize Ac transcript in Arabidopsis

et al. 1997

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SummaryThe successful application of the maize transposable element system AciDs as a genome mutagen in heterologous plant species has recently proved the versatility and power of this technique in plant molecular biology. However, the frequency of Ac/Ds transposition is considerably lower in Arabidopsis thaliana than in most other dicot plant species that have been studied. Since previous research has established that transcripts derived from monocot genes can be alternatively processed in dicot plants, we have investigated both the efficiency of intron splicing and poiyadenylation of the maize Ac transposase pre-mRNA in Arabidopsis thaliana, Nicotiana tabacum, Nicotiana plumbaginifolia and Zea mays. In this paper, we demonstrate that intron 4 is alternatively spliced within Arabidopsis, using cryptic 5' and 3' splice sites within the intron sequence, leading to a heterogeneous population of full length transposase transcript. Furthermore, analysis of transposase transcript polyadenylation revealed that at least four alternative poly(A) sites were utilized between introns 2 and 3, resulting in truncated transposase transcripts. Finally, by Northern blotting, we established that the truncated transposase transcript was the most abundant form of transposase message in Arabidopsis. In contrast to these findings, the alternative splicing and premature polyadenylation of Ac message in Arabidopsis was unparalleled in the other species examined. We suggest that the poor frequency of transposition of Ac in Arabidopsis may be in part due to the low quantity of correctly processed transposase transcript available in this species.

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“…NLSs have been best characterized in animal cells and yeast and more recently in plants. In particular, functional plant cell NLSs have been identified in maize in both the anthocyanin regulatory proteins Opaque2 and R (Varagona et al, 1992;Shieh et al, 1993) and the Ac transposase (Boehm et al, 1995), in auxininducible proteins in pea (Abel and Theologis, 1995), and in the GT box-binding transcription factor GT-2 from Arabidopsis (Dehesh et al, 1995). They have also been described in karyophilic proteins encoded by plant pathogens, namely, the NIa protein encoded by tobacco etch virus (Carrington et al, 1991) and the VirD2 and VirE2 proteins encoded by the Ti plasmid of Agrobucterium tumefuciens Howard et al, 1992).…”

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confidence: 99%

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

1996

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For the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, BR1 and BL1. These act in a cooperative manner to transport the viral singlestranded DNA genome from its site of replication in the nucleus to the cell periphery (A.A. Sanderfoot, S.C. Lazarowitz [1995] Plant Cell 7: 1185-1194). We have proposed that BR1 functions as a nuclear shuttle protein, transporting the viral single-stranded DNA to and from the nucleus as a complex that is recognized by BL1 for movement to adjacent cells. To further investigate this, we expressed BR1 mutants known to affect viral infectivity in Spodoptera frugiperda insect cells and Nicotiana tabacum L. cv Xanthi protoplasts and found these to be defective in either their nuclear targeting or their ability to be redirected to the cell periphery when co-expressed with BL1. Translational fusions to p-glucuronidase and alanine-scanning mutagenesis further demonstrated that the C-terminal 86 amino acids of BR1 contains a domain(s) essential for i t s interaction with B L I and identified two nuclear localization signals within the N-terminal 113 residues of BR1. These nuclear localization signals were precisely located within distinct 16-and 22-peptide segments of BR1. These studies support and extend our model for squash leaf curl virus movement, showing that BR1 has a domain structure, with an N-terminal region required for nuclear targeting and a C-terminal region required for i t s interaction with BL1. Department of Microbiology, University of lllinois 23Regulated trafficking across the nuclear membrane is an essential component of many signal transduction pathways in eukaryotic cells and a fundamental aspect of infection by animal and plant viruses that replicate in the nucleus. The transport of proteins across the nuclear membrane is an energy-requiring process mediated by proteins associated with nuclear pore complexes (Newmeyer and Forbes, 1988). Although nuclear pores have an effective size-exclusion limit of approximately 60 kD, proteins smaller than this appear not to diffuse efficiently across the nuclear membrane (reviewed by Silver, 1991). Karyophilic proteins contain NLSs, short basic regions of approximately 10 to 20 amino acids (reviewed

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One of three nuclear localization signals of maize Activator (Ac) transposase overlaps the DNA‐binding domain

Cited by 34 publications

References 43 publications

Trans-Kingdom Transposition of the Maize Dissociation Element

Trans-Kingdom Transposition of the Maize Dissociation Element

Alternative processing of the maize Ac transcript in Arabidopsis

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

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