884 resultados para Addition silicone
Resumo:
Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.
Resumo:
In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants. Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites. In eukaryotic cells, the addition of multiple adenosine residues to the 3′ end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation. In bacteria, the adenylation of several RNAs greatly accelerates their decay. The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides. Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added. Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay.
Resumo:
The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i6A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i6A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified ARC1, whose product interacts with aminoacyl synthetases. Interestingly, we identified ERG20. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i6A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.
Resumo:
V(D)J recombination generates a remarkably diverse repertoire of antigen receptors through the rearrangement of germline DNA. Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds random nucleotides (N regions) to recombination junctions, is a key enzyme contributing to this diversity. The current model is that TdT adds N regions during V(D)J recombination by random collision with the DNA ends, without a dependence on other cellular factors. We previously demonstrated, however, that V(D)J junctions from Ku80-deficient mice unexpectedly lack N regions, although the mechanism responsible for this effect remains undefined in the mouse system. One possibility is that junctions are formed in these mice during a stage in development when TdT is not expressed. Alternatively, Ku80 may be required for the expression, nuclear localization or enzymatic activity of TdT. Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts are devoid of N regions, as were junctions in Ku80-deficient mice. In these cells TdT protein is abundant at the time of recombination, localizes properly to the nucleus and is enzymatically active. Based on these data, we propose that TdT does not add to recombination junctions through random collision but is actively recruited to the V(D)J recombinase complex by Ku80.
Resumo:
A search of databases with the sequence from the 5′ untranslated region of a Hydra cDNA clone encoding a receptor protein-tyrosine kinase revealed that a number of Hydra cDNAs contain one of two different sequences at their 5′ ends. This finding suggested the possibility that mRNAs in Hydra receive leader sequences by trans-splicing. This hypothesis was confirmed by the finding that the leader sequences are transcribed as parts of small RNAs encoded by genes located in the 5S rRNA clusters of Hydra. The two spliced leader (SL) RNAs (SL-A and -B) contain splice donor dinucleotides at the predicted positions, and genes that receive SLs contain splice acceptor dinucleotides at the predicted positions. Both of the SL RNAs are bound by antibody against trimethylguanosine, suggesting that they contain a trimethylguanosine cap. The predicted secondary structures of the Hydra SL RNAs show significant differences from the structures predicted for the SLs of other organisms. Messenger RNAs have been identified that can receive either SL-A or -B, although the impact of the two different SLs on the function of the mRNA is unknown. The presence and features of SL addition in the phylum Cnidaria raise interesting questions regarding the evolution of this process.