An extracellular nuclease from suspension cultures of tobacco

Oleson, Arland E.; Janski, Alvin M.; Clark, Edward T.

doi:10.1016/0005-2787(74)90321-9

Cited by 45 publications

(17 citation statements)

References 31 publications

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“…In plant PCD, increased activities of several kinds of nuclease have also been reported (for review, see Sugiyama et al, 2000). They are categorized into at least four classes depending on their requirement for divalent cations: Zn 2 ϩ -dependent nucleases Ho, 1986, 1987;Thelen and Northcote, 1989;Pérez-Amador et al, 2000), Ca 2 ϩ -dependent nucleases (Oleson et al, 1974(Oleson et al, , 1982, Mg 2 ϩ -dependent nucleases (Marchetti et al, 2001), and Ca 2 ϩ /Mg 2 ϩ -dependent nucleases (Xu and Hanson, 2000). However, there is no direct evidence implicating these PCDrelated nucleases in the degradation of nuclear DNA during the process of PCD.…”

Section: Introductionmentioning

confidence: 99%

ZEN1 Is a Key Enzyme in the Degradation of Nuclear DNA during Programmed Cell Death of Tracheary Elements

2002

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Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the beginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in animal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease, Zinnia endonuclease 1 (ZEN1), functions directly in nuclear DNA degradation during programmed cell death (PCD) of TEs. In-gel DNase assay demonstrated the presence of a 24-kD Ca 2 ؉ /Mg 2 ؉ -dependent nuclease and a 40-kD Zn 2 ؉ -dependent nuclease as well as ZEN1 in 60-h-cultured cells that included differentiating TEs. Such cell extracts possessed the ability to degrade the nuclear DNA isolated from Zinnia elegans cells in the presence of Zn 2 ؉ , and its activity was suppressed by an anti-ZEN1 antibody, indicating that ZEN1 is a central DNase responsible for nuclear DNA degradation. The introduction of the antisense ZEN1 gene into Zinnia cells cultured for 40 h specifically suppressed the degradation of nuclear DNA in TEs undergoing PCD but did not affect vacuole collapse. Based on these results, a common mechanism between animal and plant PCD is discussed.

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Section: Introductionmentioning

confidence: 99%

ZEN1 Is a Key Enzyme in the Degradation of Nuclear DNA during Programmed Cell Death of Tracheary Elements

2002

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“…Exogenous DNase activity has been described previously (3,14) but no effort has been successful in eliminating this activity. This nuclease activity may account in part for the lack of reproducible DNA transformation in plant cell systems.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Deoxyribonuclease Activity in the Medium Surrounding Plant Protoplasts

Slavik¹,

Widholm²

1978

Plant Physiol.

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After 1 hor, exogenous deoxyribonlei acid was degraded within a culture medim at 25 C (pH 6) co ti protots of Daucus careots L. var. sativa Low te eature nc ton (1 C) or the additi of 45 m1-molar sodm citrate to the medim eliminated DNase activity for at least 4.5 hours. Tis DNase actiity was not reduced at pH 7 or 9, nor by addition of 200 mimlar adenosine 5T-triphosphate.Techniques were deveIoped to ensure hb protoplast platag efficiencies and high regenerative capabies after low temperature treatment and the addition of sodim citrate to the medium. Results ndicated citrate concentratons to 45 mm and 1 C te rtures revealed lttle or no effect on protoplast regeneration capaces Protoplast viabilty was 90 to 95% at the time of platig as deteried by phenosafranin stainig and an estimated 50 to 60% of these undergo cell division in the sold agar meum.Although genetic transformation with bacteria is a well documented phenomenon (4), demonstration ofgenetic transformation in eucaryotic organisms has been limited to fungi (12), algae (17), and cultured animal cells (10). There have been reports of DNA uptake and subsequent genetic transformation in higher plants (6,7). However, due to the irreproducibility of the results or the methods used to ascertain DNA uptake and expression, reported DNA transformation in higher plants or plant cell cultures now appears controversial (8).The action of exogenous nuclease on foreign DNA added to cultured plant cells or protoplasts presents a major problem in the uptake of undegraded transforming DNA. This problem was first delineated by Bendich and Filner (3) were grown in liquid suspension in the medium described previously (20) were treated with I mg/ml lysozyme for 1 hr at 37 C. SDS was added to a final concentration of 1% and the mixture was incubated at 60 C for 10 min. Nuclease-free pronase (Sigma) (2 mg/ml) was added and the incubation continued for an additional hr at 37 C. CsCl (Harshaw Chemical) was added to a refractive index of 1.400 and then centrifuged to equilibrium at 218,000g, 21 hr, in a Spinco type 65 rotor. The viscous DNA fraction was collected, diluted 1:5 with 1 x SSC (0.15 M NaCl, 0.015 M Na-citrate, pH 7) and centrifuged at 300,000g, 12 hr, 5 C in a Spinco SW 50.1 rotor.The DNA pellet was resuspended in 0.1 x SSC, and the A260/A590 was between 1.8 and 2.Protoplast Isolation. One g fresh weight of mid-log phase suspension cultured D. carota cells was harvested by centrifugation (75g, 4 min) and resuspended in 20 ml of a solution consisting of 4% Cellulysin (Calbiochem), 2% Macerase (Calbiochem), and 0.4 M sorbitol (pH 6). The cells were incubated at 30 C with gentle shaking in a 250-ml flask for 3.5 to 4 hr. Complete cell wall removal was indicated by complete cell lysis upon addition of SDS to a representative sample (5

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“…To this can be added the degree of activity on native DNA, which can be very low, as in wheat and mung bean, or considerable, as in potato (NOMURA et al 1971). Avena (WYEN et al 1971), tobacco suspension cultures (OLESON et al 1974) and Tradescantia (present work). Other interesting vanations include (1) the absence of 3'-nucleotidase activity from BJORK'S (1965) potato enzyme (but not from that isolated from potato by NOMURA et al (1971) (2) the exonuclease action on RNA, but not DNA, of the wheat enzyme, in contrast to the purely endonuclease action of all the other enzymes studied (KROEKER et al 1975) and (3) the release of (d)GMP rather than (d)AMP as the predominant mononucleotide with the Tradescanria enzyme and also probably with Bjork's potato enzyme.…”

Section: Discussionmentioning

confidence: 99%

Properties of plant nuclease 1 purified from Tradescantia leaves by binding to Concanavalin A-Sepharose

Clapham

2009

Hereditas

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A nuclease with 3'-nucleotidase activity was purifed at least 2,500-fold from Tradescantia paludosa leaves by a process that included chromatography on Concanavalin A-Sepharose. The preparation contains several active isozymes without impurities as judged by gel electrophoresis, but immunological tests revealed one or two mJor and a few minor inactive components. Phosphatase, phosphodiesterase and 5'-nucleotidase were absent or minimal, as were the known glycoproteins, peroxidase and esterase. The nuclease was highly active on RNA and denatured DNA, and less active on native DNA; it hydrolyzed the substrates in endonuclease manner. The mononucleotides had 5'-phosphate ends. Nuclease activity was optimal at pH 5.5-6.5 in tris-acetate buffers, 0.05-0.1 M. DNase activity was insensitive to EDTA and not stimulated by Me" or Ca". Poly A and poly U were good and poly C and poly G poor substrates for the nuclease. but Id)GMP was the predominant mononucleotide from digests of nucleic acids. 3'-AMP, GMP and UMP were about equally good substrates for the nucleotidase at pH 6 but 3'-CMP was degraded at 1/5th the rate with 3'-AMP, and any activity on deoxynucleotides was very slight.

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An extracellular nuclease from suspension cultures of tobacco

Cited by 45 publications

References 31 publications

ZEN1 Is a Key Enzyme in the Degradation of Nuclear DNA during Programmed Cell Death of Tracheary Elements

ZEN1 Is a Key Enzyme in the Degradation of Nuclear DNA during Programmed Cell Death of Tracheary Elements

Inhibition of Deoxyribonuclease Activity in the Medium Surrounding Plant Protoplasts

Properties of plant nuclease 1 purified from Tradescantia leaves by binding to Concanavalin A-Sepharose

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