[64] Assay of ovalbumin mRNA in reticulocyte lysate

Schimke, Robert T.; Rhoads, Robert E.; McKnight, G. Stanley

doi:10.1016/0076-6879(74)30066-3

Cited by 54 publications

(14 citation statements)

References 3 publications

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“…RNA Extraction. Each RNA preparation was obtained from 5 to 15 petri dishes or 30 to 70 X 106 cells treated in the same manner. The medium was removed and saved for hormone determination.…”

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confidence: 99%

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Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells.

Seo¹,

Vassart²,

Brocas³

et al. 1977

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

147

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In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone throug induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control. The demonstration of triiodothyronine (T3) binding to nuclear proteins raises the possibility that thyroid hormone may regulate gene expression. Earlier work from Tata's laboratory showed that thyroid hormone-induced protein synthesis was preceded by formation of new RNA (1). Later, DeGroot et al. and Dilman et al. showed increase in the poly(A)-rich fraction of RNA (2, 3). Demonstrations of stimulation of a specific mRNA by thyroid hormones were recently provided by Kurtz et al. (4) and by Roy et al. (5) for a2u-globulin in the rat. However, because a number of hormones are known to stimulate the synthesis of this protein (6) and because thyroid hormone profoundly alters the level of such hormones (7), experiments done in the whole animal do not provide sufficient evidence for the direct induction of -d2u-globulin mRNA by thyroid hormone. Samuels et al. have reported a quantitative correlation between nuclear T3 receptor occupancy and stimulation of growth hormone (GH, somatotropin) synthesis in cultured rat pituitary cells (8). We have adopted a similar experimental model to study more directly the action of thyroid hormone on the induction of a specific mRNA. In a recent report, we have shown that GH and prolactin mRNA activities in the total RNA extracted from a rat pituitary cell line (GH3) that actively synthesizes both hormones can be quantitated using a rabbit reticulocyte lysate cell-free system (9). In this report, we show that in the normal rat serum were 5.4 ,ug/100 ml and 60 ng/100 ml, respectively, and in the Tx rat serum were 0.6 ,g/100 ml and less than 20 ng/100 ml, respectively. RNA Extraction. Each RNA preparation was obta...

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“…RNA Extraction. Each RNA preparation was obtained from 5 to 15 petri dishes or 30 to 70 X 106 cells treated in the same manner. The medium was removed and saved for hormone determination.…”

mentioning

confidence: 99%

“…Following centrifugation for 10 min at 9000 X g the aqueous phase was removed, and the organic phase containing the denatured protein interphase was re-extracted after addition of 8 Trichloroacetic Acid Precipitation. Five microliter aliquots of the centrifuged lysate were spotted onto Whatman GF/A filter disks and processed as described by Schimke et al (15).…”

mentioning

confidence: 99%

Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells.

Seo¹,

Vassart²,

Brocas³

et al. 1977

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

147

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“…A rabbit reticulocyte lysate for cell-free translation prepared as described by Schimke et al (21) was pretreated with micrococcal nuclease before use, according to the procedure of Pelham and Jackson (16) was not added to the reaction mixture since MgCh inhibited the protein synthesis. Five-jzl samples of the reaction mixture were examined for acid-insoluble radioactivity in the same way as described previously (12).…”

Section: Methodsmentioning

confidence: 99%

Effect of High Salt Treatment on Influenza B Viral Protein Synthesis in MDCK Cells

Tsurumi

Aoki

Nishiyama

et al. 1983

Microbiology and Immunology

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Based on the information that high salt inhibits the initiation of cellular mRNA translation which depends on the function of the 5'-terminal structure of mRNA, we compared the effect of high salt on translation of host cellular mRNAs and influenza viral mRNAs, both of which are of 5 '-terminal structure. Brief exposure of influenza B virus-infected MDCK cells to high salt medium resulted in a dosedependent inhibition of viral polypeptide synthesis as well as of cellular polypeptide synthesis, but it had less effect on synthesis of viral polypeptides, particularly nonstructural protein (NS). Under these conditions the Nar content of the infected cells was significantly increased. A similar salt effect on in vitro translation of viral and cellular mRNAs extracted from infected cells was also observed. There was no significant difference in sensitivity to hypertonic block of in vivo translation of influenza viral mRNAs and vesicular stomatitis virus mRNAs, the latter of which possess a virus-directed structure at the 5 '-terminus.

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“…(11) after precipitation with cold 10% trichloroacetic acid and resolubilization with 1 M NaOH. a5S-Labeled protein samples were precipitated by hot trichloroacetic acid (12). After the final trichloroacetic acid rinse, the filters were washed twice with 100% ethanol, dried, and placed in mini-scintillation vials.…”

Section: Methodsmentioning

confidence: 99%

Mutants altering coordinate synthesis of specific myosins during nematode muscle development.

Zengel¹,

Epstein²

1980

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

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Mutations in the unc-52 gene on linkage group II retard the construction of body-wall muscle sarcomeres during larval development in the nematode Caenorhabditis elegans. Unc-52 mutants show decreased accumulation of myosin heavy chains relative to other polypeptides during larval development, correlating with the structural retardation. Pulse radiolabeling experiments show that decreased synthesis of specific body-wall myosin heavy chains that are encoded by the unc-54 gene on linkage group I is responsible for the defective myosin accumulation. In the wild type, a constant ratio of the synthesis of the unc-54-coded myosin B to myosin A, about 2:1, is maintained during the larval stages in which the synthesis of both myosins increases exponentially and rapid sarcomere growth and addition ensues. During the first 26 hr of larval development, before any structural or behavioral effects of unc-52 mutations are apparent, the synthesis of myosin heavy chains is also normal. By 38 hr, decreased synthesis of myosin B is detected in the unc-52 mutant SU200, when sarcomere growth slows considerably. The effects of mutation in the une-52 locus are trans acting upon the synthesis of unc-54-coded myosin in a specific set of muscle cells during a defined period of larval development.The nematode Caenorhabditis elegans synthesizes at least three forms of myosin heavy chains (1). The A form, with a Mr of 210,000 in wild-type and mutant nematodes, is found in both body-wall and pharyngeal muscles. The B form, which also has a Mr of 210,000 in wild-type nematodes, is structurally altered with a Mr of 203,000 in strain E675 arising from mutation in the unc-54 gene on linkage group I. B myosin heavy chains are located only in body-wall muscles. The C form, with a Mr of 206,000 in wild-type and mutant nematodes, is found only in pharyngeal muscles. The A and B myosin heavy chains have different peptide structures and are the products of at least two structural genes (2-6). The A and B chains assemble into myosin molecules that are homodimeric; no hybrid myosin has been detected (3, 7). In body-wall muscles, these two forms of myosin molecules, which may be termed A and B on the basis of their heavy-chain composition, are located within the same cells and in the same sarcomeres (8). A and B myosin heavy chains and myosin molecules are synthesized at a constant ratio of about 1:2 throughout larval development and early adult maturation. The relative amounts of A and B myosins that accumulate are identical to these synthetic ratios, suggesting that synthesis is the primary determinant of the relative accumulation rates of the two myosins in nematode body-wall muscle cells (9).Two lines of evidence suggest that the synthesis of myosin heavy chains in C. elegans is regulated by specific genetic control mechanisms. The unique location of B myosin in the body-wall muscle cells and of C myosin in the pharynx-implies differential gene expression between the two types of muscle cell. The differential synthesis of A and B myosins su...

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[64] Assay of ovalbumin mRNA in reticulocyte lysate

Cited by 54 publications

References 3 publications

Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells.

Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells.

Effect of High Salt Treatment on Influenza B Viral Protein Synthesis in MDCK Cells

Mutants altering coordinate synthesis of specific myosins during nematode muscle development.

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