Poly(A)-associated RNA in plants

Sagher, Daphna; Edelman, Marvin; Jakob, Karl M.

doi:10.1016/0005-2787(74)90005-7

Cited by 48 publications

(15 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our estimate of the size of sycamore poly(A) from electrophoresis under denaturing conditions is an average of 160 nucleotides with molecules ranging in size from 50-250 nucleotides in length. This estimate of poly(A) size is in reasonable agreement with similar estimates from other plants [3]. We find that poly(A)-containing RNA from a variety of sources shows a tendency to aggregate and in order to overcome this RNA is denatured by heating to 60 "C in the presence of 8 M urea before gel electrophoresis.…”

Section: Discussionsupporting

confidence: 89%

“…Plant RNA has been shown to contain poly(A) sequences 150-250 nucleotides long [3] and oligo(dT)-cellulose, which selectively binds poly(A)-containing RNA, has been used to purify presumptive mRNA from polyribosomes of Phuseolus aureus [4]. An RNA fraction purified in this way from Gjyycine rnctx has been shown to direct the synthesis of the plant protein leg-haemoglobin in vitro [5] and there seems little doubt that at least some plant mRNAs contain poly(A).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Subcellular Distribution and Properties of Poly(A)-Containing RNA from Cultured Plant Cells

Covey

Grierson

1976

Eur J Biochem

View full text Add to dashboard Cite

Cultured sycamore cells rapidly incorporate ["Hluridine or [jLP]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. I n consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40 % of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average M , = lo6. Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41 AMP. Roughly 6 % of this RNA is resistant to digestion by ribonuclease A and T,. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 "C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.The demonstration that a number of mammalian mRNAs contain sequences of poly(adeny1ic acid) [l, 21 has stimulated the search for poly(A)-associated RNA in other organisms. Plant RNA has been shown to contain poly(A) sequences 150-250 nucleotides long [3] and oligo(dT)-cellulose, which selectively binds poly(A)-containing RNA, has been used to purify presumptive mRNA from polyribosomes of Phuseolus aureus [4]. An RNA fraction purified in this way from Gjyycine rnctx has been shown to direct the synthesis of the plant protein leg-haemoglobin in vitro [5] and there seems little doubt that at least some plant mRNAs contain poly(A). We are interested in the possibility of exploiting the presence of poly(A) to study the maturation and transport of plant mRNA. In order to establish whether this approach is valid it is necessary to know what proportion of mRNA molecules contain poly(A) and to learn more about the distribution of poly(A)-containing RNA in various sub-cellular fractions. This paper describes the results of preliminary experi-_ _~ ments carried out with these aims in mind. A crude nuclear fraction was obtained by grinding (3.0-6.0) x lo7 mid-log-phase cells in a mortar at 0-4 "C with 5 ml of buffer (0.1 M sucrose, 0.2 M Tris-HC1 pH 8.4, 2 mM calcium chloride, 5 mM magnesium chloride, 0.1 %, bovine serum albumin). The slurry was filtered through 4 layers of muslin and nuclei were pelleted by centrifugation at 1500 x g for 3 min at 4 "C. The supernatant was removed and the pellet was resuspended in buffer containing 0.25 % Triton X-100. Nuclei were pelleted by centrifugation at 1500 x g for 3 min. Total nucleic acids

show abstract

Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

Subcellular Distribution and Properties of Poly(A)-Containing RNA from Cultured Plant Cells

Covey

Grierson

1976

Eur J Biochem

View full text Add to dashboard Cite

show abstract

“…1 shows that RNA extracted from our' purified chloroplast fraction has <5% cytoplasmic ribosomal RNA contamination. Localization of LS-carboxylase messenger activity in the poly(Aflacking RNA fraction Many eukaryotic messenger RNAs are characterized by the presence of a covalently linked poly(A) segment (7,(19)(20)(21)23). This group includes mitochondrial messenger RNAs from animals (24,25) and fungi (22).…”

Section: Resultsmentioning

confidence: 99%

“…A similar wheat germ system has been described by Marcus et from Spriodela oligorrhiza. Gels were processed as described (7).…”

Section: Methodsmentioning

confidence: 99%

“…We purified and partially characterized ribulosebisphosphate carboxylase from phototrophic Euglena cells, de-termining the size, quaternary organization, amino-acid composition, and immunochemical reactions of the native enzyme and individual subunits (6). Concurrently, we investigated the presence of poly(A)-associated messenger RNA in Euglena and in a higher plant, determining the size of the polyadenylate segments and their covalent linkage to a specific class of RNA molecules (7). These studies formed the basis for the present work in which poly(A)-containing and poly(A)-lacking RNA fractions were isolated from purified Euglena chloroplasts, and from wild-type and mutant cells.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Large subunit ribulosebisphosphate carboxylase messenger RNA from Euglena chloroplasts.

Sagher¹,

Grosfeld²,

Edelman³

1976

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

View full text Add to dashboard Cite

RNA from wild-type Euglena, aplastidic mutant cells, and purified chloroplasts was chromatographed on oligo(dT).cellulose at 4°. The poly(A)containing and poly(Alacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abilities to specifically stimulate synthesis of large subunit ribulosebisphosphate carboxylase [EC 4.1 (7). These studies formed the basis for the present work in which poly(A)-containing and poly(A)-lacking RNA fractions were isolated from purified Euglena chloroplasts, and from wild-type and mutant cells. These RNAs were then tested for their specific abilities to act as templates in the syntheses of the large subunit of the carboxylase (LS-carboxylase), using a cell-free proteinsynthesizing system from wheat germ. METHODSCell Growth and Chloroplast Preparation. Wild-type Euglena gracilis var. bacillaris was cultured phototrophically and harvested as described (6). The aplastidic mutants, WsBUL and WjoBSmL (courtesy of J. A. Schiff) were grown heterotrophically (5). Chloroplasts were prepared from freshly harvested phototrophic cells according to the procedure of Eisenstadt and Brawerman (8) except that cells were broken in a cooled Yeda press (9) under argon at 1500 lbs./ inch2 (10.4 MPa) and a flow rate of 6-8 ml/min. RNA Extraction. Packed chloroplasts, isolated from 40 g of packed cells, were lysed with 20-30 ml of 2% sodium triisopropylnaphthalene sulfonate, 1% NaCl, 2.5 mM MgCl2, and 0.05 M Tris-HCI, pH 7.6. RNA was extracted with phenol and chloroform as described by Penman (10) and precipitated with 0.2 M NaCl and 2.5 volumes of cold ethanol. All preparations were treated with 10 ,ug/ml of DNase I (RNase-free) for 15 min at 00 (11). The phenol-chloroform and alcohol precipitation steps were then repeated. The above procedure was also used for extraction of RNA from whole cells. All extractions were performed at 220.

show abstract

The Genetic Mechanism: II The Cell’s Employment of DNA

Dillon

1978

The Genetic Mechanism and the Origin of Life

View full text Add to dashboard Cite

Poly(A)-associated RNA in plants

Cited by 48 publications

References 24 publications

Subcellular Distribution and Properties of Poly(A)-Containing RNA from Cultured Plant Cells

Subcellular Distribution and Properties of Poly(A)-Containing RNA from Cultured Plant Cells

Large subunit ribulosebisphosphate carboxylase messenger RNA from Euglena chloroplasts.

The Genetic Mechanism: II The Cell’s Employment of DNA

Contact Info

Product

Resources

About