Purification and Restriction Endonuclease Analysis of Plant Nuclear DNA

Watson, John C.; Thompson, William F.

doi:10.1016/b978-0-12-743655-5.50009-9

Cited by 101 publications

(49 citation statements)

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“…Nuclei were prepared by a modification of the method of Watson and Thompson [23]. Approximately 20 g of fronds were harvested by rinsing with ice-cold distilled water.…”

Section: Methodsmentioning

confidence: 99%

Cytokinin modulation of LHCP mRNA levels: the involvement of post-transcriptional regulation

1988

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When white-light-grown Lemna gibba plants are placed in the dark, the levels of mRNAs for two nuclear-encoded chloroplast proteins, the small subunit of ribulose-1,5-bisphosphate carboxylase (SSU) and the major chlorophyll a/b-binding protein of light-harvesting complex II (LHCP), decline to a small fraction of their previous level. We have reported [4] that red light (R), acting through phytochrome, and benzyladenine (BA), a synthetic cytokinin, independently stimulate accumulation of both mRNAs in the dark. Here, we have analyzed the products of transcription in isolated nuclei to determine if cytokinins act primarily through stimulation of transcription or if post-transcriptional processes are involved. We find that BA pretreatment may slightly stimulate transcription of LHCP RNA either with or without a red-light treatment. However, the effects of BA on the LHCP RNA accumulation were much greater than on transcription. Two naturally occurring cytokinins are also effective in increasing the mRNA abundance. We therefore conclude that, in Lemna, post-transcriptional processes are important in regulation of the LHCP RNA by cytokinins.

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“…Nuclei were prepared by a modification of the method of Watson and Thompson [23]. Approximately 20 g of fronds were harvested by rinsing with ice-cold distilled water.…”

Section: Methodsmentioning

confidence: 99%

Cytokinin modulation of LHCP mRNA levels: the involvement of post-transcriptional regulation

1988

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“…Nuclei were prepared according to Green et al [15] as adapted from the method of Watson and Thompson [49]. About 130 g of 9-12-day old pea shoots were homogenised in 500 ml homogenisation buffer (1 M 2-methyl-2,4-pentanediol, 0.5 Yo (v/v) Triton X-100, 5 mM 2-mercaptoethanol, 0.8 mM PMSF) with a Polytron PTA20SM homogeniser at setting 4 for 60 s. The homogenate was filtered through 6 layers of muslin, a 20 #m nylon mesh and the nuclei were sedimented by centrifugation at 3000 x g for 10 min.…”

Section: Preparation Of Nuclear Extractmentioning

confidence: 99%

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

1994

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Gel retardation assays using pea nuclear extracts have detected specific binding to regions of the promoter of the pea plastocyanin gene (petE). Several complexes which differ in sensitivity to competition with unlabelled promoter fragments and various DNA alternating copolymers, to heat treatment and to digestion with proteinase K have been detected. A protein factor, PCF1, forming one of these complexes was heat-stable and most sensitive to competition with poly(dAdT).poly(dAdT) compared to other alternating copolymers. DNase I footprinting assays showed that tracts of A/T-rich sequence within the -444 to -177 positive regulatory region of the petE promoter were protected in the presence of the pea nuclear extract. The factor PCF1 copurified with a high-mobility-group (HMG) protein preparation from pea chromatin. DNase I footprinting with the HMG protein preparation demonstrated that similar tracts of A/T-rich sequences within the promoter were protected. Southwestern-blot analysis of pea HMG proteins purified by gel filtration through Superose 12 detected a single DNA-binding species of 21 kDa. The properties of the factor PCF1 suggest that it is likely to be an HMG I protein.

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“…The extraction of nuclei was performed according to a method adapted from Watson and Thompson [39]. Frozen tissues (500 g) were homogenized with mortar and pestle under liquid nitrogen with 2 1 of cold homogenization buffer (1 M 2-methyl-2,4-pentanediol (hexylene glycol), 10 mM PIPES/KOH pH 7.0, 10 mM MgC12, 0.5~o v/v Triton X-100, 5 mM 2-mercaptoethanol, 0.8 mM PMSF).…”

Section: Extraction Of Nuclei From Tobacco Leavesmentioning

confidence: 99%

DNA-protein interactions on a cis-DNA element essential for ethylene regulation

et al. 1993

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The PRB-1b gene encodes for a basic-type component of the pathogenesis-related PR-1 protein family. In leaves of tobacco plants, PRB-1b mRNA accumulation is rapidly induced by the application of exogenous ethylene. Promoter deletion analysis was performed in transgenic tobacco plants to delineate cis-acting elements necessary for ethylene responsiveness of the PRB-1b gene. The promoter sequence from position -213 was sufficient to enhance a 20 fold increase of beta-glucuronidase reporter gene expression in transgenic tobacco leaves exposed to 20 microliters/l of ethylene, however -141 bp were not. The functional study was correlated with in vitro analysis of the nuclear protein-DNA complexes formed on the promoter element identified as necessary for ethylene induction. Gel-shift analysis using restriction fragments spanning the sequence between position -237 and -143 revealed two distinct nuclear protein-DNA interactions. The protein-binding sequences were mapped to the contiguous regions G (-200 to -178) and Y (-179 to -154) by gel-shift analysis using oligonucleotides. Fractionation of crude nuclear extract by heparin-agarose chromatography resulted in the differential elution of the two binding activities. The DNA-nuclear protein interactions characterized in vitro can be part of the molecular events which mediate the transcriptional regulation of the PRB-1b gene by ethylene.

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Purification and Restriction Endonuclease Analysis of Plant Nuclear DNA

Cited by 101 publications

References 0 publications

Cytokinin modulation of LHCP mRNA levels: the involvement of post-transcriptional regulation

Cytokinin modulation of LHCP mRNA levels: the involvement of post-transcriptional regulation

HMG protein binding to an A/T-rich positive regulatory region of the pea plastocyanin gene promoter

DNA-protein interactions on a cis-DNA element essential for ethylene regulation

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