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N Craig

Publications and source records attributed to N Craig.

30 records · Page 2Linked to original sources
PubMed1986

Treatment of Chinese hamster ovary cells with the transcriptional inhibitor actinomycin D inhibits binding of messenger RNA to ribosomes.

Inhibitors of RNA synthesis such as actinomycin D, MPB, and cordycepin progressively inhibit the initiation of protein synthesis in intact, nucleated mammalian cells. This inhibition is not dependent on the levels of mRNA, ribosomes, or tRNA. Lysates prepared from CHO cells treated with actinomycin D do not incorporate labeled globin mRNA or ovalbumin mRNA into 80S initiation complexes at the rates of untreated control extract. The ability of the extracts to produce and accumulate 48S preinitiation complexes was assessed using the 60S subunit joining inhibitors edeine and 5'-guanylyl imidodiphosphate. Control extracts were able to accumulate both the 48S preinitiation complexes and the migration-related intermediates in the presence of both inhibitors. However, lysates derived from CHO cells treated with actinomycin D were unable to produce these complexes. This was also true at low temperature, a condition that does not inhibit mRNA binding but prevents migration of the 43S complex along the mRNA. Mixing experiments with extracts from untreated control or AMD-treated CHO cells provided no evidence for a translational inhibitor. Thus, our data are consistent with the hypothesis that treatment of whole cells with actinomycin D inhibits protein synthesis initiation at the level of mRNA binding and not at migration or 60S subunit joining.

Animals↗
PubMed1983

Inhibition of protein synthesis in CHO cells by actinomycin D: lesion occurs after 40S initiation complex formation.

Inhibitors of RNA synthesis such as actinomycin D, 2-mercapto-1-(beta-4-pyridylethyl)benzimidazole, and cordycepin progressively inhibit the initiation of protein synthesis in intact nucleated mammalian cells independent of their effect on mRNA synthesis. The mechanism of this effect is unknown. The activity of cell-free lysates is not directly affected by these inhibitors, suggesting that their effect is indirect and requires an intact cell. However, lysates prepared from L-cells or CHO cells treated with the inhibitors do exhibit a decrease in initiation activity corresponding in magnitude to the effect seen in intact cells. Mixing experiments with lysates isolated from untreated or treated cells provide no evidence for a translational inhibitor. However, experiments analyzing the incorporation of [35S]methionine and [35S]Met-tRNAf into initiation complexes showed that while the level of labeled 40S initiation complex in lysates from treated cells was the same or higher than in control lysates, the rate or efficiency of formation of the 80S initiation complex was inhibited. These results imply that the transcriptional inhibitors do not affect the level or charging of the initiation tRNAMet, the activity of the eIF-2 initiation factor needed for ternary complex formation, and the availability of active 40S ribosomal subunits. Thus, this site of action is different from that observed in other translational control systems such as the hemin response in reticulocytes and the interferon-induced translation inhibition in virally infected cells. This effect may reflect the cell's coordination of nuclear transcription and cytoplasmic translation.

Adenosine Triphosphate↗
PubMed1979

Effect of temperature on protein and immunoglobulin synthesis and secretion in two mouse myeloma cell lines.

Protein synthesis in differentiated MOPC-21 and MPC-11 mouse myeloma cells was studied to determine the basis for the differences in the temperature and actinomycin D sensitivity of translation between non-differentiated mouse L-cells and differentiated rabbit reticulocytes. The temperature dependence of total protein synthesis was similar to that of L-cells and reticulocytes, being biphasic in Arrhenius plots with apparent activation energies of approximately 25 and 42 kcal/mol, above and below 25 degress C. The dependence of the secretion process was different since it was not biphasic, having a single activation energy of about 22 kcal/mol. Myeloma polysomes were like L-cell polysomes in their response to lower temperature and reached a minimum level of 50% at 15 degress C. This response was also found for the specific polysomes synthesizing the IgG H- and L-chains. In the presence of actinomycin D, myeloma polysomes declined exponentially with a half-life of approximately 6 hours. These two L-cell-like responses were not found in reticulocytes. Translation of both the IgG mRNAs and the non-IgG mRNAs was reduced by lower temperatures and actinomycin D, even though the L-chain mRNA was slightly more resistant, suggesting that this mRNA is slightly more efficient. The results of these experiments suggest that the translational differences between L-cells and reticulocytes are not mRNA dependent, but are cell type differences.

Animals↗
PubMed1975

Regulation of translation in rabbit reticulocytes and mouse L-cells; comparison of the effects of temperature.

Various parameters of protein synthesis were analyzed in rabbit reticulocytes exposed to various temperatures for up to five hours. Between 10 degrees C and 40 degrees C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10-24 degrees C; 22 kcal/mole, 24-40 degrees C), as did protein elongation and release (35 kcal/mole, 10-25 degrees C; 12 kcal/mole, 25-40 degrees C). However, the level of polysomes remained essentially unchanged between 0 degrees C and 42 degrees C which implies that the activation energy for polypeptide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that in cultured mouse L-cells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar in their temperature dependence of initiation and in their rate of elongation (5-6 amino acids/second at 36 degrees C.

Animals↗
PubMed1975

Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.

Animals↗
PubMed1969

Studies on the origin of ribosomes in Amoeba proteus.

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).

Amoeba↗
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Communication↗