Mass isolation of plant chromosomes and nuclei

Hadlaczky, Gyula; Bisztray, Gy.; Praznovszky, Tünde; Dudits, Dénes

doi:10.1007/bf00405195

Cited by 69 publications

(20 citation statements)

References 18 publications

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“…Cultured Arabidopsis and alfalfa cells harvested by filtration were extracted by grinding in buffer EB (25 mM Tris⅐HCl, pH 7.6͞15 mM MgCl 2 ͞15 mM EGTA͞75 mM NaCl͞60 mM ␤-glycerolphosphate͞1 mM DTT͞0.1% Nonidet P-40͞0.1 mM Na 3 VO 4 ͞20 mM NaF͞1 mM PMSF͞10 mg/liter aprotinin and leupeptin͞5 mg/ liter chymostatin and pepstatin) to prepare whole-cell extracts by centrifugation (45,000 ϫ g for 30 min at 4°C). Cytoplasmic and nuclear protein fractions were isolated as described (46). Purified nuclei were extracted with EB buffer containing 275 mM NaCl, cleared by centrifugation as above, and stored at Ϫ70°C.…”

Section: Methodsmentioning

confidence: 99%

The VirD2 pilot protein of Agrobacterium -transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

Bako

Umeda

Tiburcio

et al. 2003

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

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The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacteriumtransferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclindependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein.VirD2 is found in tight association with the TATA box-binding protein in vivo. These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes.transferred DNA integration ͉ transcription-coupled repair ͉ transcription factor IIH

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Section: Methodsmentioning

confidence: 99%

The VirD2 pilot protein of Agrobacterium -transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

Bako

Umeda

Tiburcio

et al. 2003

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

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“…Nuclear isolation was done as described previously (Hadlaczky et al, 1983). Protoplasts were isolated from 10 mL of suspension-cultured cells by incubating them for 1 h in 2% cellulase (RIO, Onozuka), 0.5% driselase (Sigma), 0.5% macerozyme (Serva, Heidelburg, Germany), 1% pectinase (Serva), and 0.1% pectolyase (Sigma) dissolved in 0.18 M mannitol, 0.18 M sorbitol, 3.5 mM CaCl 2 , 0.4 ITIM NaH 2 PO 4 , and 0.5% Mes (pH 5.6).…”

Section: Cell Fractionationmentioning

confidence: 99%

The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus

et al. 1997

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To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. l h e cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. l h e cdc2Ms kinase is activated at the Cl/S transition when phosphate-starved cells from the CO phase re-enter the cell cycle and remain active as cells transit the S, C2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13""", it was active only in the C 2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse ~2 7~' p ' and the yeast p40""', blocked the purified plant CDK activity.By irradiation of root meristems it was found that the two active phases of the cell cycle, the DNA synthesis (S) phase and segregation of chromosomes (M) phase, are each preceded by a regulatory phase called the G1 and G2 gap phases, respectively (Howard and Pelc, 1953). Within the gap phases the cell cycle can be interrupted at specific points at which developmental signals, the accomplishment of previous events in the cell cycle, as well as DNA

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“…However, with the 8-HQ solution, better metaphases were seen in relation to those with colchicine, since chromosomes pretreated with this drug were more compacted (Figure 3) than those treated with 8-HQ (Figure 1). Although colchicine is extensively utilized in cytogenetic studies (e.g., Hadlaczky et al, 1983;Sharma and Sharma, 1999), the drug made the identification of the chromosomes' secondary constrictions difficult. According to Singh (1993), 8-HQ is very useful in chromosomal analyses because it allows a good visualization of the secondary constrictions.…”

Section: Resultsmentioning

confidence: 99%

Chromosome number and cytogenetics of Euphorbia heterophylla L.

Aarestrup¹,

Karam²,

Fernandes³

2008

Genet. Mol. Res.

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AbstRACt. Euphorbia heterophylla L. (Euphorbiaceae) is a herbaceous species of great economic importance due to its invasive potential and consequent damage to agriculture and pasture land. For the first time, we provide information on its chromosome number, morphology, and behavior of mitotic chromosomes. Seeds were germinated and submitted to four treatments to obtain metaphases: 0.5% colchicine for 2 to 5 h, at ambient temperature; 0.5% colchicine for 16 to 24 h; 0.0029 M 8-hydroxyquinoline (8-HQ) for 2 to 5 h at ambient temperature, and 0.0029 M 8-HQ for 16 to 24 h at 4°C. The material was then fixed in methanol:acetic acid (3:1) and kept at -20°C for 24 h. Roots were macerated in the enzyme solution of Flaxzyme™ (NOVO FERMENT™)-distilled water (1:40) at 34°C for 2 h and later fixed again. Chromosome preparations were obtained by the dissociation of the apical meristems. The best chromosome preparations were obtained with the use of 8-HQ for 21 h 30 min at 4°C. E. heterophylla showed 2n = 28 chromosomes. The short arm of the largest pair of chromosomes of the complement (pair number 1) displayed a secondary constriction while the nucleolus was observed in the interphasic cell. Structural rearrangements were also observed in the E. heterophylla L. genome. The genomic instability associated with polyploidy may be the result of selection shaped by environmental adaptations and/or human-induced manipulation through agricultural practices.

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Mass isolation of plant chromosomes and nuclei

Cited by 69 publications

References 18 publications

The VirD2 pilot protein of Agrobacterium -transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

The VirD2 pilot protein of Agrobacterium -transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus

Chromosome number and cytogenetics of Euphorbia heterophylla L.

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