PubMed · 4890671
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S F Ryan. 1969. Artifact emphysema.. https://doi.org/10.1164/arrd.1969.99.5.801
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Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.
Minicells from Escherichia coli P678-54 are refractory towards procedures known to induce bacteriolysis of DNA-containing E. coli cells. Although still engaged in murein synthesis, minicells could not be lysed by penicillin G. Likewise, endogenous overproduction of the cloned soluble lytic transglycosylase, the predominant murein hydrolytic activity in E. coli, failed to lyse minicells. Furthermore, induction of the phage MS2 lysis protein, a hydrophobic protein assumed to trigger the autolytic system of the host, did not result in bacteriolysis. It is concluded that the murein hydrolases present in minicells are under a tight cellular control.
Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.