Two leaky termination codons in AMV RNA 1

Tol, Ruud G.L. Van; Gemeren, Ron Van; Vloten-Doting, Lous Van

doi:10.1016/0014-5793(80)81220-8

Cited by 22 publications

(10 citation statements)

References 40 publications

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“…A possible explanation for this may be the deficiency of the endogenous tKNA complement o f t h e reticulocyte lysate with respect to the requirements of the Limulus mRNA for efficient translation. Such effects have been observed for other mRNA species [11,21,22].…”

Section: Clzaracterisation Of Antibodiessupporting

confidence: 58%

Translation of mRNA for Limulus polyphemus Haemocyanin Polypeptides in vitro: Studies on Subunit Heterogeneity

Siggens¹,

Wood²

1983

Eur J Biochem

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The haemocyanin of Limulus polyphemus is composed of a munber (possibly 10–15) of polypeptides and is believed to be synthesised in cells called cyanoblasts. In vitro translation in the rabbit reticulocyte heamolysate system and in Xenopusoocytes, of mRNA isolated from cyanoblast‐contanining tissue, allowed the detection of several haemocyanin polypeptides amongst the products of translation. At least seven polypeptides with molecular weights in the range 68000–71000 were identified by an immunological method followed by electrophoretic characterisation on two‐dimensional polyacrylamide gels. Comparison of the polypeptide patterns of authentic haemocyanin, reticulocyte lysate translation products and Xenopus oocyte translation products led tothe conclusion that the polypeptides are unlikely to undergo significant post‐translational modification or to possess cleavable signal sequences. It is proposed that release of haemocyanin into the haemolymph in vivo may involve bursting of the cyanoblasts.

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Section: Clzaracterisation Of Antibodiessupporting

confidence: 58%

Translation of mRNA for Limulus polyphemus Haemocyanin Polypeptides in vitro: Studies on Subunit Heterogeneity

Siggens¹,

Wood²

1983

Eur J Biochem

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“…RNA samples were translated in a mRNAdependent rabbit reticulocyte cell-free system and a cell-free protein-synthesizing system derived from wheat-germ. The reticulocyte lysate was kindly provided by the Plant Virus Group (University of Leiden) and used as described by Van Tol & van Vloten-Doting (1979). The wheat-germ extract was prepared as described by Marcu & Dudock (1974), and had an average E260 = 150 litre mol-I cm-', the protein content being 70mg/ml.…”

Section: Translation Ofrna In Vitromentioning

confidence: 99%

Phytoferritin is synthesized in vitro as a high-molecular-weight precursor. Studies on the synthesis and the uptake in vitro of the precursors of ferritin and ferredoxin by intact chloroplasts

Mark¹,

Briel²,

Huisman³

1983

Biochemical Journal

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Evidence is presented that French-bean (Phaseolus vulgaris) seed ferritin is composed of one type of subunit with an apparent Mr of 26500. In normal and iron-loaded leaf tissues it is detected immunologically with an antiserum raised against purified bean seed ferritin and migrates in SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis with the same mobility as the bean seed ferritin subunit. The biosynthetic pathway of ferritin in normal and iron-loaded leaves was investigated. RNA was extracted, fractionated into polyadenylated RNA and translated in a cell-free rabbit reticulocyte lysate and a wheat-germ-extract system. The products were identified by SDS/polyacrylamide-gel electrophoresis after indirect immunoprecipitation. In all cases the ferritin product had an Mr 5000 higher than that of the native subunit. Uptake and processing of the precursor form of ferritin from iron-loaded leaves by intact chloroplasts was demonstrated. This indicates that, in iron-loaded leaves, ferritin acts as a chloroplast protein. We propose that the ferritin precursor in normal leaves follows the same biosynthetic pathway. This suggests that the iron-buffering function of ferritin in plants takes place in the chloroplast and that non-functional cellular iron will accumulate in this cell organelle.

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“…Butler box sequences found at the 5' end of TMV RNA and the coat protein and Mr 29,987 protein genes, and at several other internal sites, may be signal sequences in this process. Many plant viruses are known to have similar readthrough products of their early genes (26) and it remains to be seen whether they also share the mechanism now described for generating COOH-terminally overlapped proteins.…”

Section: Commentsmentioning

confidence: 99%

Nucleotide sequence of tobacco mosaic virus RNA.

Goelet¹,

Lomonossoff²,

Butler³

et al. 1982

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

594

281

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Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of (7,8), their sequences were determined by the dideoxy chain-termination method (7), and overlaps were determined by computer methods (9). TMV proved to be a poor template for the synthesis of cDNA longer than 2,000 nucleotides, and we found that the efficiency of the second-strand reaction was highly sequence dependent. These problems were circumvented by using a series of synthetic oligodeoxynucleotides to prime synthesis at either random or specific sites along the molecule. Efficient cloning of molecules that were rendered double-stranded was achieved by cleaving the synthetic DNA with restriction endonucleases. The use of several restriction enzymes ensured that overlapping sequences were cloned.Priming with Synthetic Oligonucleotides. Mixtures of fourto seven-residue oligonucleotides, synthesized by phosphodiester chemistry (10), were used as nonspecific primers on TMV RNA or on cDNA. Double-stranded cDNA to most of TMV genome could be synthesized by using these primer "cocktails." To direct the synthesis of double-stranded cDNA to the termini and other poorly sampled regions of TMV RNA, oligonucleotide primers of 13 to 17 residues were synthesized by the solid-phase phosphotriester method (11 1,813-1,829 and 3,159-3,172) were synthesized to obtain clones to the 5' sides of regions of TMV RNA well represented by cDNA clones from previous "shotgun" experiments. cDNA priming was with 10-to 100-fold molar excess of oligonucleotide over TMV RNA template (usually 5 ,ug of TMV RNA in a 25-,u reaction mixture) and standard incubation conditions for reverse transcription were used (12): 42°C and 60 min, with a 30°C and 15-min preincubation for the short oligonucleotide cocktails.Second-Strand Synthesis, Cloning, and Assembly of the Sequence. cDNA freed from RNA by alkaline hydrolysis (100 mM NaOH, 1 mM EDTA for 15 min at 70°C) was used as a template for second-strand synthesis primed by "flip back" or added oligonucleotide primers. The standard reaction used Klenow DNA polymerase and incubation was in 10 mM Tris HCI, pH 7.4/ 10 mM MgCl2/10 mM dithiothreitol/100 mM NaCI/50 AM each deoxynucleoside triphosphate at 37°C for 30 min. In some experiments no attempt was made to purify the cDNA. Oligonucleotides generated in the first-strand reaction were used to prime second-strand synthesis on TMV cDNA by using the conditions of Wickens et al. (12) or the conditions described above after melting and annealing desalted products ofthe firststrand reaction (alkali treatment of the first-strand reaction product was shown by these approaches not to cause deamination of cytidine residues). These double-stranded cDNAs were digested by restriction endonu...

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Two leaky termination codons in AMV RNA 1

Cited by 22 publications

References 40 publications

Translation of mRNA for Limulus polyphemus Haemocyanin Polypeptides in vitro: Studies on Subunit Heterogeneity

Translation of mRNA for Limulus polyphemus Haemocyanin Polypeptides in vitro: Studies on Subunit Heterogeneity

Phytoferritin is synthesized in vitro as a high-molecular-weight precursor. Studies on the synthesis and the uptake in vitro of the precursors of ferritin and ferredoxin by intact chloroplasts

Nucleotide sequence of tobacco mosaic virus RNA.

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