A modified DNA isolation protocol for obtaining pure RT-PCR grade RNA

Klassen, Roland; Pfeiffer, Annika; Meinhardt, Friedhelm

doi:10.1007/s10529-008-9648-y

Cited by 16 publications

(9 citation statements)

References 9 publications

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“…tRNA was isolated as described previously (Klassen et al ., 2008). Quantification and quality control of RNA was performed as described (Jeske et al ., 2006) by spectrophotometry using nano‐drop (NanoDrop Technologies, Wilmington, USA).…”

Section: Methodsmentioning

confidence: 99%

The primary target of the killer toxin from Pichia acaciae is tRNA^Gln

Klassen

Paluszynski

Wemhoff

et al. 2008

Molecular Microbiology

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113

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Section: Methodsmentioning

confidence: 99%

The primary target of the killer toxin from Pichia acaciae is tRNA^Gln

Klassen

Paluszynski

Wemhoff

et al. 2008

Molecular Microbiology

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113

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“…Transcription of k1ORF2 in the parental strain S. cerevisiae 301, the k1⌬ORF4 mutant, and the PADH1::ORF4 complemented strain was verified by reverse transcription-PCR (RT-PCR). Total RNA was isolated as described previously (34), and DNase I digestion was achieved employing the RNase-Free DNase I set (New England BioLabs GmbH, Frankfurt am Main, Germany). Reverse transcription was accomplished using the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany), following the manufacturer's recommendations.…”

Section: Methodsmentioning

confidence: 99%

Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

Wemhoff

Klassen

Meinhardt

2014

Appl Environ Microbiol

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Zymocin is a Kluyveromyces lactis protein toxin composed of ␣␤␥ subunits encoded by the cytoplasmic virus-like element k1 and functions by ␣␤-assisted delivery of the anticodon nuclease (ACNase) ␥ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during ␥ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either ␣␤ (k1ORF2) or ␥ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of ␣'s chitinase domain and the sole cysteine residue of ␤ (Cys250). Since ␤␥ are reportedly disulfide linked, the requirement for the conserved ␥ C231 was probed. Toxicity of intracellularly expressed ␥ C231A indicated no major defect in ACNase activity, while complementation of k1⌬ORF4 by ␥ C231A was lost, consistent with a role of ␤ C250 and ␥ C231 in zymocin assembly. To test the capability of ␣␤ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1⌬ORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1⌬ORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying ␣␤'s capability to deliver other cargo proteins into target cells.T he protein toxin zymocin, produced by the yeast Kluyveromyces lactis, was identified as the first known anticodon nuclease toxin from a eukaryote, selectively cleaving tRNA within the anticodon loop due to highly specific anticodon nuclease (ACNase) activity (1). Zymocin production is correlated with the presence of a pair of linear cytoplasmic genetic double-stranded DNA (ds-DNA) elements, termed pGKL1 (k1 in short) and pGKL2 (k2 in short) (2). The larger k2 is required for cytoplasmic maintenance of k1, which encodes the toxin as well as an immunity determinant (2, 3). k2 provides essential functions for cytoplasmic replication, transcription, and transcript processing, and several of these components show phylogenetic proximity to viruses (4, 5). Since the proposed mode of replication via protein priming is typically found in viruses, the cytoplasmic linear dsDNA elements were termed virus-like elements (VLE) (6). The zymocin toxin is a heterotrimeric ␣␤␥ complex, the smallest subunit of which (␥) exhibits the cytotoxic ACNase activity (1, 7-9). The ␣ and ␤ subunits are generated from the 128-kDa k1ORF2 gene product, which is first translocated to the endoplasmic reticulum (ER), where it becomes glycosylated and cleaved by signal peptidase and then travels to the Golgi apparatus, where it is processed by the Kex1/2 endopeptidase internally at the N-terminal end to produce mature ␣ (99-kDa) and ␤ (30-kDa) subunits (7, ...

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“…tRNA was isolated from wild-type yeast cells (S. cerevisiae CEN.PK2-1c) (van Dijken et al 2000) as described (Klassen et al 2008b). Five mg of tRNA was incubated at 30°C in a total volume of 100 mL in 10 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 50 mM NaCl, 1 mM dithiothreitol, containing 50 ng of PaOrf2His 6 .…”

Section: Paorf2 Toxicity Assaymentioning

confidence: 99%

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

Meineke

Alene

Schwer

et al. 2012

RNA

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PaOrf2 and g-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or g-toxin in S. cerevisiae. Mutational analysis of g-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of g-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and g-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 39 from the mcm 5 s 2 U wobble nucleoside and depends on mcm 5 . A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm 5 wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.

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A modified DNA isolation protocol for obtaining pure RT-PCR grade RNA

Cited by 16 publications

References 9 publications

The primary target of the killer toxin from Pichia acaciae is tRNA^Gln

The primary target of the killer toxin from Pichia acaciae is tRNA^Gln

Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

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A modified DNA isolation protocol for obtaining pure RT-PCR grade RNA

Cited by 16 publications

References 9 publications

The primary target of the killer toxin from Pichia acaciae is tRNAGln

The primary target of the killer toxin from Pichia acaciae is tRNAGln

Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

Contact Info

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The primary target of the killer toxin from Pichia acaciae is tRNA^Gln

The primary target of the killer toxin from Pichia acaciae is tRNA^Gln