Preparation and partial characterization of cell-free protein-synthesizing extracts from <i>Tetrahymena pyriformis</i>

David, E T; Smith, Kelvin E.

doi:10.1042/bj1940761

Cited by 13 publications

(6 citation statements)

References 9 publications

(5 reference statements)

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“…Exponential-phase cultures of Tetrahymena were harvested and used for preparing the postmitochondrial extracts as described previously (David & Smith, 1981, in the section on 'Final procedures'), except that the preparation buffer used comprised 25 mM-KCl, 7 mM-magnesium acetate, 1 mM-dithiothreitol, 0.lmM-EDTA, 1.5mM-EGTA and 10% (v/v) glycerol made up in 20mM-triethanolamine/ HCl buffer, pH 7.4.…”

Section: Preparation Of Tetrahymena Postmitochondrial Extracts and Cementioning

confidence: 99%

“…Protein-synthesis assays were performed exactly as described previously (David & Smith, 1981), except that the final incubation volume was decreased to 0.05 ml and the labelled amino acid mixture used was composed of [3Hlphenylalanine plus [3H]leucine.…”

Section: Preparation Of Tetrahymena Postmitochondrial Extracts and Cementioning

confidence: 99%

“…pared from the protozoan Tetrahymena pyriformis have been reported in the literature for some years, critical analysis of the results shows that the protein synthesis obtained is almost entirely due to chain elongation rather than initiation events de novo, because inefficient 'run-off' systems or extracts primed with artificial mRNA, such as poly(U), were used (Mager & Lipmann, 1958;Conklin & Chou, 1971;Allen & Suyama, 1972). However, we have described the preparation and partial characterization of a highly active cell-free protein-synthesizing system from this organism (David & Smith, 1981). It is important to establish whether these cell-free preparations are capable of initiating new rounds of protein synthesis, because of the potentially important use of Tetrahymena extracts as model systems for work on pathogenic protozoa.…”

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Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis

Smith¹,

David²

1981

Biochemical Journal

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Saturating concentrations of the initiation-specific inhibitors poly(I) and T-2 toxin inhibit protein synthesis by over 35% and cause ribosome 'run-off' from the polyribosomes. The elongation-specific inhibitor cycloheximide totally prevents protein synthesis and 'freezes' the ribosomes in the pattern of unincubated controls. These results prove that our Tetrahymena extracts are capable of protein-synthesis initiation, a conclusion which is confirmed by a 30% inhibition of synthesis by the mRNA cap analogue, 7-methylguanosine 5'-monophosphate.

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Section: Preparation Of Tetrahymena Postmitochondrial Extracts and Cementioning

confidence: 99%

Section: Preparation Of Tetrahymena Postmitochondrial Extracts and Cementioning

confidence: 99%

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confidence: 99%

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Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis

Smith¹,

David²

1981

Biochemical Journal

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“…(1) Theoretically, the most satisfying solution is a homologous message-dependent translation system. A Tetrahymena system for translation in vitro has been described (David & Smith, 1981), but it is dependent on endogenous mRNA and has a poor initiation capacity.…”

Section: Introductionmentioning

confidence: 99%

Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena

Andreasen¹,

Dreisig²,

Kristiansen³

1987

Biochemical Journal

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The codon usage of Tetrahymena thermophila and other ciliates deviates from the 'universal genetic code' in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.

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“…It appears that hypotrichs use the unusual codons rarely, Tetrahymena often, and Paramecium prefers them over the normal ones. These observations account for the poor behavior of Tetrahymena messengers in heterologous systems of in vitro protein synthesis (5) and the almost complete lack of synthesis observed when Paramecium messengers are used ( 1 9).…”

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Molecular Biology of the Genes for Immobilization Antigens in Paramecium1

Preer

Rudman

et al. 1987

The Journal of Protozoology

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Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition to known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51C and 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.

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Preparation and partial characterization of cell-free protein-synthesizing extracts from Tetrahymena pyriformis

Cited by 13 publications

References 9 publications

Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis

Initiation of protein synthesis in cell-free extracts of Tetrahymena pyriformis

Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena

Molecular Biology of the Genes for Immobilization Antigens in Paramecium1

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