Isolation of intact individual species of single- and double-stranded RNA after fractionation by polyacrylamide gel electrophoresis

Schuerch, Alfred R.; Mitchell, Wayne R.; Joklik, Wolfgang K.

doi:10.1016/0003-2697(75)90517-5

Cited by 55 publications

(20 citation statements)

References 31 publications

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“…Electrophoresis of DNA was in 4% polyacrylamide gels containing 8 M urea and 0.1%SDS (urea-PAGE) [4], and DNA components were extracted from gels as described [5]. DNA M r was determined after glyoxalation [6].…”

Section: Electrophoresis Methodsmentioning

confidence: 99%

Characterisation of tomato golden mosaic virus as a geminivirus

Hamilton

1981

FEMS Microbiology Letters

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Section: Electrophoresis Methodsmentioning

confidence: 99%

Characterisation of tomato golden mosaic virus as a geminivirus

Hamilton

1981

FEMS Microbiology Letters

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“…Bands appearing as shadows on the TLC plate were cut out. RNA, extracted from the gel slices by the method of Schuerch et al (1975), was stored in 0.1 x TNE buffer (0-005 M-Tris-HC1, 0-015 M-NaCI, 0.01 mM-Na2EDTA, adjusted to pH 7.9 with HC1) at -20 °C.…”

Section: Methodsmentioning

confidence: 99%

Relationships between the RNA Components of Chronic Bee-Paralysis Virus and those of Chronic Bee-Paralysis Virus Associate

Overton

Buck

Bailey

et al. 1982

Journal of General Virology

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SUMMARYThe RNA components of chronic bee-paralysis virus (CPV) and those of chronic bee-paralysis virus associate (CPVA) have been compared. CPV has five singlcstranded RNA components, two larger RNAs dcsignatcd l (tool. wt. 1.35 × 106; 4200 nucleotidcs) and 2 (mol. wt. 0.9 × 106; 2800 nuclcotides), and three smaUcr RNAs designated 3a, 3b and 3c, each with mol. wt. 0-35 × I06 (I I00 nuclcotidcs). CPVA has three single-stranded RNA components dcsignatcd A, B and C, each with tool. wt. 0.35 × 106 (I 100 nuclcotides). Gcl clcctrophorctic and T~ fingerprint analyses have shown that RNAs 3a, 3b and 3c are very similar to, and probably identical to, RNAs A, B and C respectively, i.e. thcse thrcc RNAs appear to bc cncapsidated in both CPV and CPVA particles. A positive correlation was observed between the proportion of RNAs 3a, 3b and 3c in CPV particles and the amount of CPVA (and hence RNAs A, B and C) which had replicated. Fingerprint analysis has shown that RNAs A, B and C arc distinct but related and also that about 50 ~ of the sequence of each thcse three RNAs is homologous with RNA 2. It is therefore possible that, although CPV and CPVA are scrologically unrelated, the RNAs of CPVA have evolved from CPV RNA 2. INTRODUCTIONChronic bee-paralysis virus (CPV), which causes a trembling condition soon followed by death in the adult honey bee, Apis mellifera (Bailey et al., 1963), consists of ellipsoidal particles falling into four size classes with modal lengths of 30, 40, 55 and 65 nm, each approximately 20 nm wide. These components have sedimentation coefficients of 82S, 97S to 106S, 110S to 124S and 125S to 136S respectively and are found in the same relative proportions in preparations from naturally infected bees, from artificially infected bees and from bees injected with terminal dilutions of bottom component only. In caesium chloride gradients, all size classes have a buoyant density of 1.33 g/ml. The particles contain single-stranded RNA with a base composition of 20~ G, 24~ o A, 28 ~ C and 28 ~o U. Serologically, the components are indistinguishable (Bailey, 1976;Bailey et al., 1968).Chronic bee-paralysis virus associate (CPVA) is frequently found in bees infected with CPV (Bailey, 1976). It consists of 17 nm isometric particles which are serologicaUy unrelated to those of CPV. The virus particles have a buoyant density of 1.38 g/ml in caesium chloride, a sedimentation coefficient of 41S, and contain single-stranded RNA. CPVA cannot multiply when injected into bees unless CPV is included in the inoculum, which suggests that CPVA and CPV are related as satellite and helper viruses (Bailey et al., 1980)~ However, CPVA sometimes multiplies in bees injected with CPV preparations which contain no detectable CPVA. In this paper, we report the identification of several RNA components from CPV and CPVA and the results of comparison of these RNAs by T~ oligonucleotide fingerprinting.

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“…The dsRNA was obtained from virus particles by phenol extraction and ethanol precipitation, and, after denaturation by heating for 1 to 5 min in 0-1 mM-sodium EDTA, pH 7.0, used as a template for synthesis of 32p-labelled cDNA by reverse transcription using random oligonucleotide primers (Taylor et al, 1976). When cDNA probes from individual RNA components were needed, RNA was first separated by PAGE and then electroeluted and purified by precipitation with cetyltrimethylammonium bromide (Schuerch et al, 1975). Gel transfer hybridization.…”

Section: Assignment Of Jungal Isolates To V-c Groupsmentioning

confidence: 99%

Sequence Relationships between Virus Double-stranded RNA from Isolates of Gaeumannomyces graminis in Different Vegetative Compatibility Groups

Jamil¹,

Buck²,

Carlile³

1984

Journal of General Virology

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SUMMARYDouble-stranded RNA, from isolates of Gaeumannomyces graminis var. tritici in nine vegetative compatibility groups, was separated by polyacrylamide gel electrophoresis and transferred to aminophenylthioether-paper. Hybridization between these blots and cDNA probes prepared to double-stranded RNA from viruses obtained from three of the isolates, revealed several close relationships between double-stranded RNA from isolates in different vegetative compatibility groups. The implications of this finding for virus transmission in G. graminis are discussed.

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Isolation of intact individual species of single- and double-stranded RNA after fractionation by polyacrylamide gel electrophoresis

Cited by 55 publications

References 31 publications

Characterisation of tomato golden mosaic virus as a geminivirus

Characterisation of tomato golden mosaic virus as a geminivirus

Relationships between the RNA Components of Chronic Bee-Paralysis Virus and those of Chronic Bee-Paralysis Virus Associate

Sequence Relationships between Virus Double-stranded RNA from Isolates of Gaeumannomyces graminis in Different Vegetative Compatibility Groups

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