Conformational studies on transfer ribonucleic acid. Fluorescence lifetime and nanosecond depolarization measurements on bound ethidium bromide

Tao, Terence; Nelson, James H.; Cantor, Charles R.

doi:10.1021/bi00820a004

Cited by 118 publications

(83 citation statements)

References 27 publications

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“…The 263K strain of scrapie agent (9) was used at the fourth passage level as described (1 20,10,5, 2.5, and 0.25 units/ml diluted in buffer recommended by the supplier; and (ix) diluent alone. Grids were immersed under 0.5 ml of solution and incubated in a moist chamber.…”

Section: Methodsmentioning

confidence: 99%

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

Narang

Asher

Gajdusek

1988

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

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Abnormal tubulofilamentous structures have been identified in electron micrographs of thin sections and negatively stained impression grids prepared from brains of animals with scrapie and other spongiform encephalopathies, and we showed that such tubules contain a core of rdamentous structures resembling scrapie-associated fibrils (SAF). We treated impression grids from brains of scrapie-infected hamsters with several substances that bind to or cleave proteins and nucleic acids to see if they had any effect on the abnormal tubuloframentous structures. Treatment with three proteolytic enzymes reduced the caliber of the tubules from about 50 nm to 30 nm; subsequent treatment of the 30-nm tubules with DNase I left many typical SAF as well as transitional forms in which twisted SAF emerged from tubules. DNase treatment of the original thicker tubules had no effect, and no SAF were seen on grids. Treatment of the 30-nm tubules with any of three other nucleases (micrococcal, mung bean, and BAL-31) also produced SAF. However, treatment with RNase A had no effect either on the original 50-nm tubules or on the 30-nm tubules produced by proteolysis. Detergent treatment of any of the preparations produced SAF. Treatment with ethidium bromide resulted in staining of the tubules that was inhibited by magnesium ions. The data suggest that the abnormal tubulofilamentous particles found in spongiform encephalopathies may consist of an outer cylinder of protein, an inner cylinder of DNA, and an innermost core of SAF.We recently described a simple method of negative-stain transmission electron microscopy using grids touched to the freshly cut surface of brain tissue. In brains infected with the agents ofthe spongiform encephalopathies, we found abnormal tubular structures (1) that generally resembled tubulovesicular particles often seen in thin sections of plastic-embedded brain specimens (2,3). We demonstrated that treatment of those tubules with detergent released inner filaments that were morphologically identical to scrapie-associated fibrils (SAF) (4) and reacted with antisera prepared against the proteaseresistant sialoglycoprotein "prion protein [27][28][29][30] (5, 6) of which SAF are composed (7,8). We report here a study of the effects of several proteases and nucleases and of ethidium bromide on the abnormal tubules. The abnormal tubules appear to consist of at least three layers: (i) an outer coat of protease-sensitive material, (ii) an intermediate layer that is digested by a DNase and several other nucleases but not by an RNase, and (iii) an inner core of SAF. MATERIALS AND METHODSScrapie Virus. The 263K strain of scrapie agent (9) was used at the fourth passage level as described (1 20,10,5, 2.5, and 0.25 units/ml diluted in buffer recommended by the supplier; and (ix) diluent alone. Grids were immersed under 0.5 ml of solution and incubated in a moist chamber. Grids were removed at intervals from 30 min to 120 min. After a 30-min incubation, grids treated with collagenase, trypsin, or proteinase K were...

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Section: Methodsmentioning

confidence: 99%

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

Narang

Asher

Gajdusek

1988

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

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“…The good correlation of observed vs. calculated binding constants strongly suggests that competitive binding of tetracycline to tRNA inhibits codon-anticodon inter~tctions rather than intercalating drug molecules. Supporting this theory is the difference in strength and in ,the number of binding sites seen between tetracyc~ne-tRNA and ethidium bromide-tRNA interactions [16]. Ethidium bromide is known to be intercalated into helical regions of tRNA.…”

Section: Resultsmentioning

confidence: 99%

The effects of streptomycin and tetracycline on codon—anticodon interactions

Högenauer

Turnowsky

1972

FEBS Letters

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“…Because the RNA helix must elongate or alter its conformation to accept an intercalator (Berman & Young 1981, Waring 1981, changes in tertiary structure which alter the flexibility of RNA also affect intercalation (Reinhardt et al 1982, White & Draper 1989. For example, intercalation of EthBr with phenylalanine specific tRNA (tRNAPhe) has been shown to b e limited to one or several binding sites under conditions which stabilize the tRNAPhe tertiary structure (Tao et al 1970, Urbanke et al 1973, Kean et al 1985. Therefore, if RNA tertiary structure was modified with increasing RNA concentration, either the number of EthDi binding sites in RNA or the affinity of EthDi at a particular binding site may have been altered.…”

Section: Discussionmentioning

confidence: 99%

An evaluation of fluorescence technigues for measuring DNA and RNA in marine microorganisms

Mordy¹,

Carlson²

1991

Mar. Ecol. Prog. Ser.

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The DNA-specific fluorochrome Hoechst 33258 (H33258) and the DNA-and RNAsensitive fluorochrome ethidium homodlmer (EthDi) were used to detect n g ml-' concentrations of nuclelc acids in marine microorganisms. These fluorochromes were 4 to 5 times more sensitive to DNA than ethidium bromide, and their use significantly reduced the amount of sample necessary to measure nuclelc acids in seawater. Both fluorochromes responded in a linear fashion to standard DNA, but the response of EthDi to RNA was non-linear for the majority of RNA types tested. Chlorophyll a quenched fluorescence of both fluorochromes, especially of EthDi, at concentrations that could be encountered in sample homogenates. Two techniques were used to measure DNA and RNA on cultures of marine bacteria and phytoplankton; H33258 and EthDi together (double fluorochrome technique), and EthDi with and without RNase (RNase digestion method). In general, DNA and RNA were measured equally well using either technique; discrepancies between techniques were attributed to EthDi quenching or the specificity of H33258 for DNA rich in dA-dT base pairs. The double fluorochrome technique was used to determine depth profiles of DNA and RNA/DNA in size-fractionated natural seawater These properties showed significant changes between the surface and 100 m and may be related to changes In biomass or metabolic activity. Although consideration must be given to the RNA standard used and the presence of naturally occuring pigments, the combination of H33258 and EthDi can provide a rapid and sensitive measure of nucleic acids in seawater without the use of nucleases.

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Conformational studies on transfer ribonucleic acid. Fluorescence lifetime and nanosecond depolarization measurements on bound ethidium bromide

Cited by 118 publications

References 27 publications

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie.

The effects of streptomycin and tetracycline on codon—anticodon interactions

An evaluation of fluorescence technigues for measuring DNA and RNA in marine microorganisms

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