A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.
Numerous synthetic RNA-based controls for integrating sensing switches with function devices have been demonstrated in a variety of organisms for gene regulation. Although potential advantages of RNA-based genetic control strategies have been shown in clinical applications, successfully extending these engineered systems into medical applications has seldom been reported. Here, a synthetic RNA-based ribozyme system and its application in advancing rationally designed cellular therapy were described. The theophylline-responsive, ribozyme-based device provided a powerful platform for suicide gene expression regulation in tumor cells. Moreover, we demonstrate the ability of our synthetic controller to modulate effectively the viability of the cells in response to drug input. Our RNA-based regulatory system could dose-dependently fine-tune transgene expression in mammalian cells and address urgent limitations in existing genetic control strategies for gene- and cell-based therapies in the future.
Long Interspersed Nuclear Element (LINE) retrotransposons play an important role in genomic innovation as well as genomic instability in many eukaryotes including human. Random insertions and extinction through mutational inactivation make them perfectly time-stamped "DNA fossils". Here, we investigated the origin of a self-cleaving ribozyme in 5' UTR of LINE-1. We showed that this ribozyme only requires 35 nucleotides for self-cleavage with a simple but previously unknown secondary-structure motif that was determined by deep mutational scanning and covariation analysis. Structure-based homology search revealed the existence of this mini-ribozyme in anthropoids but not in prosimians. In human, the most homologs of this mini-ribozyme were found in lineage L1PA6-10 but essential none in more recent L1PA1-2 or more ancient L1PA13-15. We resurrected mini-ribozymes according to consensus sequences and confirmed that mini-ribozymes were active in L1PA10 and L1PA8 but not in L1PA7 and more recent lineages. The result paints a consistent picture for the emergence of the active ribozyme around 40 million years ago, just before the divergence of the new world monkeys (Platyrrhini) and old-world monkeys (Catarrhini). The ribozyme, however, subsequently went extinct after L1PA7 emerged around 30 million years ago with a deleterious mutation. This work uncovers the rise and fall of the mini-LINE-1 ribozyme recorded in the "DNA fossils" of our own genome. More importantly, this ancient, naturally trans-cleaving ribozyme (after removing the non-functional stem loop) may find its modern usage in bioengineering and RNA-targeting therapeutics.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.