RNA Hybridization to DNA Coupled with Cyanogen‐Bromide‐Activated Sephadex

Siddell, Stuart G.

doi:10.1111/j.1432-1033.1978.tb12785.x

Cited by 22 publications

(10 citation statements)

References 35 publications

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“…LSU and its mRNA are both detectable in dark-grown plants, as studies on protein synthesis in isolated etioplasts would indicate [38]. However, whereas the mRNA level increases markedly in the first 12 hr of illumination, the LSU itself remains at a low level for about 24 hr and only then accumulates rapidly.…”

Section: Resultsmentioning

confidence: 98%

Differential regulation of the accumulation of the light‐harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

Bennett

Jenkins

Hartley

1984

J of Cellular Biochemistry

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The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.

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Section: Resultsmentioning

confidence: 98%

Differential regulation of the accumulation of the light‐harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

Bennett

Jenkins

Hartley

1984

J of Cellular Biochemistry

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“…Hybridized nucleic acids can easily be eluted from DPTE-materials by water or 99% formamide in analogy to DBM-cellulose (6). In contrast, supports synthesized by cyanogen bromide activation carry positively charged groups, requiring elution in the presence of moderate salt concentration (5). A couple of hybridization selection reactions with macroporous DPTE S-500 have shown that unspecific adsorption of nucleic acids is even lower than for G-25 materials.…”

Section: Discussionmentioning

confidence: 99%

“…E.coli-DNA sheared or sonicated (see above) was either dissolved in 0.1 ml 0.2 M MES pH 8.0, boiled, cooled, and mixed with 0.9 ml formamide (5) or dissolved in 1 ml 10 mM potassium phosphate pH 8.0, boiled and stored on ice immediately before use (6). The reaction mixture was maintained at 4°C (5) or at room temperature (6) overnight. Between 1 and 2 mg/ml of DNA was used for coupling to Cl-2B, Cl-6B, S-500 and S-1000 in contrast to about 0.1-0.3 mg/ml in the case of G-25 superfine.…”

Section: Methodsmentioning

confidence: 99%

“…A widespread application is the coupling via diazotized aromatic amines bound to finely divided cellulose or cellulose filters (3,4). Less customary is the fixation of DNA to Sephadex G-10 or Sepharoses via activatin by the poisonous cyanogen bromide (5,6). Nevertheless, all methods were applied for hybrid selection of mRNAs from complex RNA mixtures, although with variable success.…”

Section: Introductionmentioning

confidence: 99%

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Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures

Bûnemann¹,

Westhoff

Herrmann

1982

Nucl Acids Res

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Methods commonly used for covalent immobilization of single stranded DNA have been applied to several solid supports (Sephadex G-25 and Cellex 410) as well as to a number of macroporous materials (Sepharose C1-6B, C1-2B; Sephacryl S-500 and S-1000). Coupling efficiencies and stability of covalently bound DNA are compared for both classes of materials. The yields of the immobilization reaction for sonicated DNA are only 10-40% for G-25 and Cellex 410 in contrast to 60-80% for C1-6B and S-500. Under optimal conditions, up to 0.5 mg of DNA can be coupled initially per g of wet macroporous material. The immobilized DNAs are lost from the supports in a biphasic manner, with about 10-20% loss per day during the first 2-3 days at 45 degrees C, followed by only about 1% loss per day at the same temperature thereafter. The influence of the coupling procedure on the generation of mismatch effects has been studied in 2.4 M tetraethylammonium chloride solution for the hybrid formation between immobilized and mobile DNA. The degree of mismatch ranged from 0-3% and depended on the method of immobilization. The unspecific absorption of DNA on macroporous materials is sufficiently low to allow efficient hybrid selection. No size limitations have been observed when plastid mRNAs are selected by cloned fragments of plastid DNA immobilized to macroporous Sephacryl S-500.

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“…Two-dimensional mapping was performed as Coronavirus tryptic peptide fingerprints o~. described by Siddell (1978) with the separation of peptides on thin layer cellulose plates (Eastman Kodak Sheet 13255) by electrophoresis in pH 2.1 buffer and ascending chromatography in butanol-acetic acid-water-pyridine. The plates were dried, in some cases after dipping in 7 % (w/v) PPO (2,5-diphenyloxazole) dissolved in ether, and exposed to Fuji RX film at room temperature or at -7 0 °C as appropriate.…”

Section: Methodsmentioning

confidence: 99%

Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides

Siddell¹

1982

Journal of General Virology

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SUMMARYThe virion proteins and intracellular polypeptides of the murine coronavirus MHV-JHM have been analysed by two-dimensional fingerprinting of their [a~S]methionine-containing tryptic peptides. The analysis shows that the virion proteins gp98, gp65, pp60 and p23 are distinct. Virion protein gp25 has the same polypeptide component as p23, and virion protein gpl70 has a polypeptide component related to gp98. The six virus polypeptides synthesized in infected cells, I50K, 65K, 60K, 30K, 23K and 14K are also distinct. The 170K and 98K species, which are produced by processing, are related to 150K. The 25K species is a processed form of 23K. The identity of corresponding species in the cell and in the virion has been shown and a model describing the genesis of coronavirus JHM proteins can now be proposed.

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RNA Hybridization to DNA Coupled with Cyanogen‐Bromide‐Activated Sephadex

Cited by 22 publications

References 35 publications

Differential regulation of the accumulation of the light‐harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

Differential regulation of the accumulation of the light‐harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

Immobilization of denatured DNA to macroporous supports: I. Efficiency of different coupling procedures

Coronavirus JHM: Tryptic Peptide Fingerprinting of Virion Proteins and Intracellular Polypeptides

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