In vitro 32P-labelling of viroid RNA for hybridization studies

Negruk, V. I.; Grill, Laurence K.; Semancik, J. S.

doi:10.1016/0166-0934(80)90063-4

Cited by 18 publications

(8 citation statements)

References 5 publications

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“…Nucleic acid blot hybridizations were performed as described by Alwine et al (16) by using methylmercuric hydroxide gels (17) to separate the nucleic acids prior to transfer to the diazobenzyloxymethyl cellulose paper. The radioactive probe for the hybridization studies was prepared from electrophoretically purified dsRNA sequences by the 32P end-labeling technique on the hot formamide-cleaved RNA molecules (18 to a subcellular fractionation procedure to identify the location of specific nucleic acids associated with male sterility. The mitochondrial DNA was initially analyzed in V.faba because this appears to be the location of the genetic material for the trait in maize.…”

Section: Methodsmentioning

confidence: 99%

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba

Grill

Garger

1981

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

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The cytoplasmic male sterility (CMS) trait of at least one line of Viea faba plants is always associated with the presence of high molecular weight double-stranded RNA in the leaf tissue extracts. Subcellular fractions of leaf tissue from CMS and fertile maintainer plants were initially analyzed in an attempt to locate, identify, and characterize the genetic material involved with the sterility trait. This CMS-associated high molecular weight RNA was found only in the cytosol of the "447" male sterile line ofV. faba plants and could not be isolated from the recurrent parent (maintainer), from lines that had been fertility-restored, or from lines that had reverted from the sterile condition. We have been able to move the CMS-associated RNA from donor to fertile host plants through a dodder bridge. These hosts not only contain the RNA but now exhibit a male sterile phenotype, as detected by visual examination of the flower, the pollen, morphological characteristics, and pollen staining ability.Male sterility is most useful for the production of commercial amounts of hybrid seed if the factors responsible for the trait are maternally inherited. Sources of such a cytoplasmic male sterility (CMS) have been found in many different species of plants (1). In the maize and sorghum cytoplasmic male sterile lines, it has been demonstrated that the genetic information responsible for the CMS trait resides in the mitochondria (2-4) apparently as episomal DNA (5). Research on these monocotyledonous plants has led to a transposon-like model for the mitochondrial CMS DNA (6). However, there has been very little research on the causes of CMS in the dicotyledonous plants. There have been suggestions that viruses or virus-like particles (VLPs) are responsible for the sterility in the broad bean plants (Vidafaba L.) and petunias (7-9).An electron microscopic study of "447" male sterile, maintainer, restored, and reverted V.faba lines has shown the presence of cytoplasmic spherical bodies (=70 nm in diameter) in only the male sterile plants (8). In a short report we have indicated that there are particles in the CMS plants that contain a family of double-stranded RNA (dsRNA) molecules (10); we now further characterize these nucleic acids and identify one of them as consistently being associated with the sterility trait.The dsRNA particle appears to have characteristics similar to a virus, leading us to help substantiate a mechanism of sterility which is quite different from that involving the episomal DNA in maize mitochondria. These results and the eventual transfer of the dsRNA agent and the CMS biological trait provide compelling biochemical evidence that cytoplasmic male sterility in the broad bean plant is caused by the presence of at least one dsRNA species. MATERIALS AND METHODSPlant Material. Seeds of V.faba L. were provided by D. A. Bond (Plant Breeding Institute, Cambridge, England) and P. Berthelem (Institut National de la Recherche Agronomique Station for Plant Improvement, LeRheu, France) or were purchased from...

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Section: Methodsmentioning

confidence: 99%

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba

Grill

Garger

1981

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

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“…3, these inserts were further subcloned into the pKS vector (Stratagene) to derive strand-specific RNA probes of the desired polarity (lanes 140 to 141, probe polarity + or -). The other probes used were dsRNA that had been submitted to random hydrolysis and post-labelled with [y-~2P]ATP, as described by Negruk et al (1980), using T4-induced polynucleotide kinase (Fig. 3, lane dsRNA +/-), or the series of PstI inserts excised from pZO 140 to pZO143 which were labelled by random priming (Fig.…”

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confidence: 99%

Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba

Pfeiffer

Jung

Heitzler

et al. 1993

Journal of General Virology

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“…The highest yield (180 ~tg dsRNA/g of lyophilized mycelium) was obtained from the French-derived H strain EP 713 and the lowest (16 ~tg dsRNA) was from the American H strain EP 868. For preparation of hybridization probes, the dsRNA was further purified by centrifugation in linear-log density gradients (Brakke & Van Pelt, 1970), and partially hydrolysed using deionized formamide at 100 °C (Negruk et al, 1980). The denatured dsRNA fragments were labelled to a specific activity of 106 c.p.m./p.g with [~32p]ATP using polynucleotide kinase (Maniatis et al, 1982;Jordan & Dodds, 1983).…”

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Lack of Sequence Homology between Double-stranded RNA from European and American Hypovirulent Strains of Endothia parasitica

L'Hostis¹,

Hiremath

Rhoads

et al. 1985

Journal of General Virology

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SUMMARYDot blots of dsRNA from European and American hypovirulent (H) strains of the chestnut blight fungus, Endothia parasitica, were hybridized with 32p-5'-end-labelled fragments of denatured dsRNA of French, Italian and American origins. Although dsRNA from European or American strains reacted well with labelled RNA probes from strains from the same continent there was little or no cross-hybridization between RNA from strains from different continents.

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In vitro 32P-labelling of viroid RNA for hybridization studies

Cited by 18 publications

References 5 publications

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba

Identification and characterization of double-stranded RNA associated with cytoplasmic male sterility in Vicia faba

Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba

Lack of Sequence Homology between Double-stranded RNA from European and American Hypovirulent Strains of Endothia parasitica

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