Chapter 9 Isolation of Nuclei Suitable for in Vitro Transcriptional Studies

Cushman, John C.

doi:10.1016/s0091-679x(08)61026-2

Cited by 15 publications

(18 citation statements)

References 24 publications

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“…One-week-old wild-type (C24) and cpl1 seedlings grown in vitro were cold-treated (0°C, 15 h). Nuclei were isolated, and the reaction was performed as described by Cushman (34). Labeled transcripts were purified with TRIzol (Invitrogen) according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

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C-terminal domain phosphatase-like family members (AtCPLs) differentially regulateArabidopsis thalianaabiotic stress signaling, growth, and development

Koiwa

Barb

Xiong

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

146

184

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Cold, hyperosmolarity, and abscisic acid (ABA) signaling induce RD29A expression, which is an indicator of the plant stress adaptation response. Two nonallelic Arabidopsis thaliana (ecotype C24) T-DNA insertional mutations, cpl1 and cpl3, were identified based on hyperinduction of RD29A expression that was monitored by using the luciferase (LUC) reporter gene (RD29A::LUC) imaging system. Genetic linkage analysis and complementation data established that the recessive cpl1 and cpl3 mutations are caused by T-DNA insertions in AtCPL1 (Arabidopsis C-terminal domain phosphatase-like) and AtCPL3, respectively. Gel assays using recombinant AtCPL1 and AtCPL3 detected innate phosphatase activity like other members of the phylogenetically conserved family that dephosphorylate the C-terminal domain of RNA polymerase II (RNAP II). cpl1 mutation causes RD29A::LUC hyperexpression and transcript accumulation in response to cold, ABA, and NaCl treatments, whereas the cpl3 mutation mediates hyperresponsiveness only to ABA. Northern analysis confirmed that LUC transcript accumulation also occurs in response to these stimuli. cpl1 plants accumulate biomass more rapidly and exhibit delayed flowering relative to wild type whereas cpl3 plants grow more slowly and flower earlier than wild-type plants. Hence AtCPL1 and AtCPL3 are negative regulators of stress responsive gene transcription and modulators of growth and development. These results suggest that C-terminal domain phosphatase regulation of RNAP II phosphorylation status is a focal control point of complex processes like plant stress responses and development. AtCPL family members apparently have both unique and overlapping transcriptional regulatory functions that differentiate the signal output that determines the plant response. P lants tolerate environmental stress because of numerous physiological adaptations, which have been attributed to the function of various determinant genes (1). In Arabidopsis thaliana, transcription of RD (Responsive to Dehydration) (2) and COR (Cold Responsive) (3) genes is activated by cold or hyperosmotic stress. The plant hormone abscisic acid (ABA) activates transcription of some RD and COR genes through an interaction involving the cis element ABRE (ABA-responsive element) and basic leucine zipper transcription factors such as ABFs͞AREBs (4, 5). However, expression of some RD or COR genes is activated by low temperature or desiccation, independent of ABA, by the interaction of CBF͞DREB DNA-binding proteins with another cis element, DRE (6, 7). Overexpression of CBF͞DREB in transgenic Arabidopsis plants induces ectopic expression of RD͞COR genes and confers desiccation and cold tolerance (7,8).Recent genetic dissection of cold-, hyperosmolarity-, and ABA-induced signaling that regulates gene expression and adaptation indicates that the cascade signature is modulated by numerous positive and negative regulators (9-14). Signaling control of plant gene expression is known now to include components that function at various stages in mRNA metaboli...

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Section: Methodsmentioning

confidence: 99%

“…Eight-day-old seedlings growing in vitro were incubated either in 25°C or 0°C for 15 h before isolation of nuclei. Nuclear isolation, run-on reaction, and hybridization were performed as described by Cushman (34). Transcripts hybridized to immobilized probe were detected by Typhoon phosphorimager after 16 h of exposure.…”

Section: Isolation Of Cpl Mutantsmentioning

confidence: 99%

C-terminal domain phosphatase-like family members (AtCPLs) differentially regulateArabidopsis thalianaabiotic stress signaling, growth, and development

Koiwa

Barb

Xiong

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

146

184

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“…Subcellular localization studies using GFP fusion may not reflect a biologically relevant subcellular location due to the large size of the GFP, which may influence the final destination of the fusion protein (Barkla et al, 1995;Vera-Estrella et al, 1999) or into a nuclear fraction by isolating intact nuclei (Cushman, 1995). Protein fractions were resolved by SDS-PAGE and analyzed by western blotting using a monoclonal antibody raised against the 63 His tag.…”

Section: Subcellular Fractionationmentioning

confidence: 99%

“…The final nuclear pellet was resuspended in 50 mL of nuclei storage buffer, aliquoted, frozen and stored at 280°C. Nuclear storage buffer consisted of 20% (v/v) glycerol, 20 mM (HEPES)-KOH, pH 7.2, 5 mM MgCl 2 , 1 mM DTT, and 10% Triton X-100 (Cushman, 1995).…”

Section: Cell Fractionationmentioning

confidence: 99%

Autophosphorylation and Subcellular Localization Dynamics of a Salt- and Water Deficit-Induced Calcium-Dependent Protein Kinase from Ice Plant

et al. 2004

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A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K 0.5 5 0.15 mM). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 63 His-tagged McCPK1.Calcium is a ubiquitous and pivotal second messenger in the signal transduction networks that plants use to respond to a wide variety of physiological stimuli. Cytosolic Ca 21 fluctuations have been observed in response to a number of stimuli, including red light, abscisic acid (ABA), GA, drought, hyperand hypo-osmotic stress, ionic stress, touch, cold, heat shock, oxidative stress, fungal elicitors, and nodulation factors (Sanders et al., 2002 (Harmon et al., , 2001Hrabak, 2000;Hrabak et al., 2003).CDPKs are composed of a single polypeptide chain with a catalytic kinase domain at the N terminus, an intervening junction domain, and a calmodulin-like domain at the C terminus containing up to four functional Ca 21 -binding EF hands. The junction domain acts as an autoinhibitor in a pseudosubstrate fashion (Harper et al., 1994;Vitart et al., 2000;Weljie et al., 2000). Binding of Ca 21 to the calmodulin-like domain results in a conformational change leading to the release of the autoinhibitor domain from the active site and kinase activation (Harmon et al., 1994;Harper et al., 1994). Many CDPKs also have additional Nterminal leader and C-terminal domains composed of highly variable amino acid sequences. AtCPK25 and some CDPK-related kinases possess degenerate calmodulin domains (Lindzen and Choi, 1995;Zhang and Lu, 2003) and appa...

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“…Isolation of nuclei was carried out according to method I1 as recently described by Cushman (1995). In vitro transcription assays were conducted as previously described (Cushman et al, 1989).…”

Section: In Vitro Transcription Assaysmentioning

confidence: 99%

Posttranscriptional and Posttranslational Control of Enolase Expression in the Facultative Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L

1995

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Chapter 9 Isolation of Nuclei Suitable for in Vitro Transcriptional Studies

Cited by 15 publications

References 24 publications

C-terminal domain phosphatase-like family members (AtCPLs) differentially regulateArabidopsis thalianaabiotic stress signaling, growth, and development

C-terminal domain phosphatase-like family members (AtCPLs) differentially regulateArabidopsis thalianaabiotic stress signaling, growth, and development

Autophosphorylation and Subcellular Localization Dynamics of a Salt- and Water Deficit-Induced Calcium-Dependent Protein Kinase from Ice Plant

Posttranscriptional and Posttranslational Control of Enolase Expression in the Facultative Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum L

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