Regulation of tyrosine hydroxylase mRNA stability by oxygen in PC12 cells.
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The sequences of the 3' untranslated region (UTR) of the manganese superoxide dismutase (MnSOD) genes in wheat (Triticum aestivum) were found to be quite variable with different predicted thermostabilities. The degradation rates of the 3' UTR variants and the coding region were measured following exposure to endogenous nucleases. The degradation rates of the 3' UTR variants for 15 min were not significantly different, meaning the degradation rates of the 3' UTR variants were not directly related to the thermostabilities. However, the degradation rate of the coding region was significantly faster than those of the 3' UTR variants. Further investigation revealed the coding region seemed to have specific sites for degradation, indicating a possibility of increasing MnSOD expression by the degradation site alteration.
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During I.13.17. hybridoma batch cultivation, the levels of cytoplasmic mRNAs specifying immunoglobulin heavy (H) and light (L) chains peaked during the exponential phase and decreased significantly during the decline phase. This variation was found to parallel the rate of DNA synthesis and the total cytoplasmic RNA content. Moreover, we have investigated the possibility that the stability of H- and L-chain mRNAs could account for the observed difference in their levels throughout the batch culture. Using a transcription inhibitor (actinomycin D), we found that the differential accumulation of both mRNAs reflected transcript stability. However, at any growth stage of the culture, the specific monoclonal antibody production rate (pg IgG cell-1 h-1) was not reflected by the levels of H- or L-chain mRNAs, but could be partially correlated to the specific metabolic activity of the cells.
The facilitated diffusion of D-glucose across the plasma membrane is carried out by a set of stereospecific transport proteins known as the glucose transporters. These integral membrane proteins are members of a gene family where tissue-specific expression of one or more members will determine in part the net rate of glucose entry into the cell. The regulation of glucose transporter gene expression is a critical feature of cellular homeostasis, as defects in specific transporter expression can lead to profound alterations in cellular physiology. In this review, we provide a brief descriptive background on the family of glucose transporters and examine in depth the regulation of the two transporters expressed in adipose tissue, GLUTI, a basal growth-related transporter and GLUT4, the insulin-responsive glucose transporter.
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Detailed analysis of cell-free translation, coupled transcription-translation in static conditions and a continuous flow system based on E. coli S30 extracts was performed. Degradation of template mRNA was the predominant trigger to terminate the protein synthesis. In a coupled system, mRNA was preserved by repeated transcription whereas the starvation of nucleotide triphosphates led to the termination of protein synthesis in less than 1 h. In the CFCF system, NTP was held at the level of initial concentration and therefore did not arrest the translation for 15 h. The accurate coupling of transcriptional rate and translational rate was also crucial to enhance the efficiency of protein synthesis.
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Alternative splicing of the sarco/endoplasmic reticulum (SERCA2) Ca2+ pump transcript generates the two isoforms: SERCA2a in left ventricular myocytes (LVM) and SERCA2b in most tissues. Nuclear protein extracts from left ventricular myocytes can cause a decay of the 3'-region of the SERCA2a. To determine if all the domains in the 800 b SERCA2a 3'-end region (3344-4243) are equally stable, we examined in vitro decay of synthetically capped, polyadenylated overlapping RNA fragments 2A1-2A6 from the 3'-end region of SERCA2a. Whereas 2A1-2A5 RNAs were stable, the distal fragment 2A6 (4135-4243 b) decayed rapidly. Deleting the 2A6 sequence from the 800-b 3'-end region increased its stability. In mobility shift assays, 2A6 bound to protein(s) in the LVM nuclear extracts in a specific manner: unlabelled 2A6 or the 800 b 3'-region RNA competed for binding but poly A, poly U, and poly C RNA did not. Secondary structure analysis revealed three hairpin loops in 2A6. Experiments using small synthetic RNA fragments for competition with 2A6 binding to nuclear proteins were consistent with a model involving the three hairpin loops. Thus, the secondary structure of the distal domain of SERCA2a RNA may be important in regulating its stability.
Recent studies have led to the identification of more than 50 small regulatory RNAs in Escherichia coli. Only a subset of these RNAs has been characterized. However, it is clear that many of the RNAs, such as the MicF, OxyS, DsrA, Spot42 and RyhB RNAs, act by basepairing to activate or repress translation or to destabilize mRNAs. Basepairing between these regulatory RNAs and their target mRNAs requires the Sm-like Hfq protein which most likely functions as an RNA chaperone to increase RNA unfolding or local target RNA concentration. Here we summarize the physiological roles of the basepairing RNAs, examine their prevalence in bacteria and discuss unresolved questions regarding their mechanisms of action.
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20-Hydroxyecdysone induces poly(A) shortening and the subsequent degradation of transcripts encoding the larval glue protein LGP-1 in Drosophila virilis late third larval instar salivary glands. Degradation concurs with the transient increase of ribonucleolytic activities in the gland cells. In vitro nuclease assays using crude cytoplasmic extracts of ecdysone-treated salivary glands demonstrate degradation to be deadenylation-independent and that the induced ribonucleolytic activities initiate the degradation of the Lgp-1 transcripts in putative single-stranded loop regions. The independence of degradation from deadenylation is also found in vivo in transformed D. melanogaster carrying a modified Lgp-1 gene.
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